Jiří Stulík

Outer membrane vesicles secreted by gram-negative bacteria play an important role in bacterial physiology as well as in virulence and host–pathogen interaction. Isolated vesicles of some bacteria have also been studied for their immunomodulatory potential in the vaccine development. However, the production of vesicles in sufficient amount, purity and reproducibility remains a critical challenge for subsequent analyses in most bacteria. In the present review methods of production, isolation, purification and quantification of outer membrane vesicles are summarized and discussed.

ABSTRACT The tetratricopeptide repeat (TPR) structural motif is known to occur in a wide variety of proteins present in prokaryotic and eukaryotic organisms. The TPR motif represents an elegant module for the assembly of various multiprotein complexes, and thus, TPR-containing proteins often play roles in vital cell processes. As the TPR profile is well defined, the complete TPR protein repertoire of a bacterium with a known genomic sequence can be predicted. This provides a tremendous opportunity for investigators to identify new TPR-containing proteins and study them in detail. In the past decade, TPR-containing proteins of bacterial pathogens have been reported to be directly related to virulence-associated functions. In this minireview, we summarize the current knowledge of the TPR-containing proteins involved in virulence mechanisms of bacterial pathogens while highlighting the importance of TPR motifs for the proper functioning of class II chaperones of a type III secretion system in the pathogenesis of Yersinia , Pseudomonas , and Shigella .

Nucleoid-associated proteins belong to a group of small but abundant proteins in bacterial cells. These transcription regulators are responsible for many important cellular processes and also are involved in pathogenesis of bacteria. The best-known nucleoid-associated proteins, such as HU, FIS, H-NS, and IHF, are often discussed. The most important findings in research concerning HU protein are described in this mini review. Its roles in DNA compaction, shape modulation, and negative supercoiling induction have been studied intensively. HU protein regulates bacteria survival, growth, SOS response, virulence genes expression, cell division, and many other cell processes. Elucidating the mechanism of HU protein action has been the subject of many research projects. This mini review provides a comprehensive overview of the HU protein.

Intracellular bacterial pathogens generally express chaperones such as Hsp100s during multiplication in host cells, allowing them to survive potentially hostile conditions. Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularaemia. The ability of F. tularensis to multiply and survive in macrophages is considered essential for its virulence. Although previous mutant screens in Francisella have identified the Hsp100 chaperone ClpB as important for intracellular survival, no detailed study has been performed. We demonstrate here that ClpB of F. tularensis live vaccine strain (LVS) is important for resistance to cellular stress. Promoter analysis shows that the transcriptional start is preceded by a sigma32-like promoter sequence and we demonstrate that expression of clpB is induced by heat shock. This indicates that expression of clpB is dependent on the heat-shock response mediated by sigma32, the only alternative sigma-factor present in Francisella. Our studies demonstrate that ClpB contributes to intracellular multiplication in vitro, but is not essential. However, ClpB is absolutely required for Francisella to replicate in target organs and induce disease in mice. Proteomic analysis of membrane-enriched fractions shows that five proteins are recovered at lower levels in the mutant strain. The crucial role of ClpB for in vivo persistence of Francisella may be linked to its assumed function in reactivation of aggregated proteins under in vivo stress conditions.

The expression of calcium-binding protein S100A9 was investigated in 23 matched sets of colorectal carcinoma and normal colon mucosa using two-dimensional gel electrophoresis. We found that, from a group of 23 patients, the level of S100A9 protein, in comparison with matched normal colon mucosa, was significantly increased in malignant tissues of 16 patients (70%). Furthermore, an additional protein, identified by matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) as S100A8, exhibited an increased expression in the same specimens of malignant tissues as the S100A9 protein. The immunohistological analysis revealed the accumulation of S100A9 positive cells, macrophages and polymorphonuclear leukocytes along the invasive margin of colorectal carcinoma. The S100A8 protein was found to be produced in the same location. The possible participation of both proteins and, especially, its heterodimeric complex calprotectin in colorectal carcinoma regression could be taken into account.

Francisella tularensis subsp. holarctica is the common causal agent of tularemia in Europe. Besides clinical signs, the diagnosis of the disease mostly depends on serological tests. To date, there is a lack of information about the F. tularensis antigens that induce antibody response. Therefore, we have started comprehensive mapping of immunoreactive antigens using the attenuated live vaccine strain of F. tularensis LVS originating from the European virulent strain. For this purpose, the immunoreactivity of sera collected from patients suffering from tularemia, together with the control sera of patients with Lyme disease and healthy blood donors, were examined by means of one-dimensional and two-dimensional immunoblotting. Furthermore, whole cell bacterial lysates, isolated integral membrane proteins and basic proteins were exploited as antigens. By this approach more than 80 different immunorelevant antigens were detected. Most of them came from whole cell bacterial lysate and integral membrane proteins. Conversely, only a negligible reaction was found in the case of basic proteins. Forty-five spots were further selected for mass spectrometric analyses and 22 of them were annotated. Among the spots that provided characteristic reactions with sera from patients with tularemia, 60 kDa and 10 kDa chaperonins that occurred in several charge and mass variants, predominated.

PROTEOMICSVolume 5, Issue 8 p. 2090-2103 Regular Article Proteomic analysis of anti-Francisella tularensis LVS antibody response in murine model of tularemia Jana Havlasová, Jana Havlasová Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicSearch for more papers by this authorLenka Hernychová, Lenka Hernychová Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicSearch for more papers by this authorMartin Brychta, Martin Brychta Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicSearch for more papers by this authorMartin Hubálek, Martin Hubálek Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicSearch for more papers by this authorJurai Lenco, Jurai Lenco Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicSearch for more papers by this authorPär Larsson, Pär Larsson Swedish Defence Research Agency, Umeå, Sweden Department of Clinical Microbiology, Clinical Bacteriology, Umeå University, Umeå, SwedenSearch for more papers by this authorMargaretha Lundqvist, Margaretha Lundqvist Swedish Defence Research Agency, Umeå, SwedenSearch for more papers by this authorMats Forsman, Mats Forsman Swedish Defence Research Agency, Umeå, SwedenSearch for more papers by this authorZulana Kročová, Zulana Kročová Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicSearch for more papers by this authorJiri Stulík, Corresponding Author Jiri Stulík jstulik@pmfhk.cz Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicInstitute of Molecular Pathology, Purkyne Military Medical Academy, Třebešská 1575, 500 01 Hradec Králové, Czech Republic Fax: +420 495512451===Search for more papers by this authorAks Macela, Aks Macela Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicSearch for more papers by this author Jana Havlasová, Jana Havlasová Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicSearch for more papers by this authorLenka Hernychová, Lenka Hernychová Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicSearch for more papers by this authorMartin Brychta, Martin Brychta Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicSearch for more papers by this authorMartin Hubálek, Martin Hubálek Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicSearch for more papers by this authorJurai Lenco, Jurai Lenco Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicSearch for more papers by this authorPär Larsson, Pär Larsson Swedish Defence Research Agency, Umeå, Sweden Department of Clinical Microbiology, Clinical Bacteriology, Umeå University, Umeå, SwedenSearch for more papers by this authorMargaretha Lundqvist, Margaretha Lundqvist Swedish Defence Research Agency, Umeå, SwedenSearch for more papers by this authorMats Forsman, Mats Forsman Swedish Defence Research Agency, Umeå, SwedenSearch for more papers by this authorZulana Kročová, Zulana Kročová Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicSearch for more papers by this authorJiri Stulík, Corresponding Author Jiri Stulík jstulik@pmfhk.cz Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicInstitute of Molecular Pathology, Purkyne Military Medical Academy, Třebešská 1575, 500 01 Hradec Králové, Czech Republic Fax: +420 495512451===Search for more papers by this authorAks Macela, Aks Macela Proteome Center for the Study of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy, Hradec Králové, Czech RepublicSearch for more papers by this author First published: 13 May 2005 https://doi.org/10.1002/pmic.200401123Citations: 65AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Francisella tularensis live vaccine strain infection of mice has been established as an experimental model of tularemia that is suitable for studies of immune mechanisms against the intracellular pathogen. In this study, the model was used to explore immunogenic repertoire of F. tularensis with the aim of identifying new molecules able to activate the host immune system, potential bacterial markers with vaccine, and diagnostic applications. Immunoproteomic approach based on the combination of two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry was applied. Globally, 36 different proteins were identified, which strongly reacted with sera from experimentally infected mice, including several putative virulence markers of intracellular pathogens as nucleoside diphosphate kinase, isocitrate dehydrogenase, RNA-binding protein Hfq, and molecular chaperone ClpB. Of them, 27 proteins are described for the first time as immunorelevant Francisella proteins. When comparing murine immunoproteome of F. tularensis with our previous data from human patients, 25 of the total of 50 identified murine sera immunoreactive spots were recognized by human sera collected from patients suffering from tularemia, as well. Immune sera from two Lps gene congenic strains of mice, C3H/HeN (Lpsn) and C3H/HeJ (Lpsd), represented murine immunoproteome in this study. The spectrum of immunoreactive spots detected by two-dimensional immunoblotting varied throughout the course of infection depending on murine strain. Nevertheless, the antibody patterns of the two strains showed significant homogeneity in being directed against almost identical subset of antigens. Citing Literature Volume5, Issue8No. 8 May 2005Pages 2090-2103 RelatedInformation

Wheat belongs to six major food allergens inducing IgE-mediated hypersensitivity reaction manifesting as cutaneous, gastrointestinal, and respiratory symptoms. Although cereals are a staple food item in most diets, only a few wheat proteins causing hypersensitivity have been identified. To characterize wheat allergens, salt-soluble wheat extracts were separated by 1-DE and 2-DE and IgE-binding proteins were detected by immunoblotting using sera of patients with allergy to ingested wheat. Proteins, frequently recognized by IgE on 2-DE were analyzed by MALDI-TOF and QTOF and their spectrum was completed by 1-DE and LCQ(DECA) nLC-MS/MS IT technique. Using all three techniques we identified 19 potential wheat allergens such as alpha-amylase inhibitors, beta-amylase, profilin, serpin, beta-D-glucan exohydrolase, and 27K protein. Employing newly developed ELISA, levels of IgE Abs against Sulamit wheat extract and alpha-amylase inhibitors type 1 and 3 were quantified and shown to be significantly elevated in sera of allergic patients compared to those of healthy controls. The level of IgE Abs against alpha-amylase inhibitor type 3 was lower, slightly above the cut-off value in the majority of patients' sera. Our findings contribute to the identification of wheat allergens aimed to increase the specificity of serum IgE and cell activation diagnostic assays.

Chronic anthracycline cardiotoxicity is a feared complication of cancer chemotherapy. However, despite several decades of primarily hypothesis-driven research, the molecular basis of this phenomenon remains poorly understood. The aim of this study was to obtain integrative molecular insights into chronic anthracycline cardiotoxicity and the resulting heart failure. Cardiotoxicity was induced in rabbits (daunorubicin 3 mg/kg, weekly, 10 weeks) and changes in the left ventricular proteome were analyzed by 2D-DIGE. The protein spots with significant changes (p<0.01, >1.5-fold) were identified using MALDI-TOF/TOF. Key data were corroborated by immunohistochemistry, qRT-PCR and enzyme activity determination and compared with functional, morphological and biochemical data. The most important alterations were found in mitochondria — especially in proteins crucial for oxidative phosphorylation, energy channeling, antioxidant defense and mitochondrial stress. Furthermore, the intermediate filament desmin, which interacts with mitochondria, was determined to be distinctly up-regulated and disorganized in its expression pattern. Interestingly, the latter changes reflected the intensity of toxic damage in whole hearts as well as in individual cells. In addition, a marked drop in myosin light chain isoforms, activation of proteolytic machinery (including the proteasome system), increased abundance of chaperones and proteins involved in chaperone-mediated autophagy, membrane repair as well as apoptosis were found. In addition, dramatic changes in proteins of basement membrane and extracellular matrix were documented. In conclusion, for the first time, the complex proteomic signature of chronic anthracycline cardiotoxicity was revealed which enhances our understanding of the basis for this phenomenon and it may enhance efforts in targeting its reduction.

Determining disease-associated changes in protein glycosylation provides a better understanding of pathogenesis. This work focuses on human immunoglobulin A1 (IgA1), where aberrant O-glycosylation plays a key role in the pathogenesis of IgA nephropathy (IgAN). Normal IgA1 hinge region carries 3 to 6 O-glycans consisting of N-acetylgalactosamine (GalNAc) and galactose (Gal); both sugars may be sialylated. In IgAN patients, some O-glycans on a fraction of IgA1 molecules are Gal-deficient. Here we describe a sample preparation protocol with optimized cysteine alkylation of a Gal-deficient polymeric IgA1 myeloma protein prior to in-gel digestion and analysis of the digest by MALDI-TOF/TOF mass spectrometry (MS). Following a novel strategy, IgA1 hinge-region O-glycopeptides were fractionated by reversed-phase liquid chromatography using a microgradient device and identified by MALDI-TOF/TOF tandem MS (MS/MS). The acquired MS/MS spectra were interpreted manually and by means of our own software. This allowed assigning up to six O-glycosylation sites and demonstration, for the first time, of the distribution of isomeric O-glycoforms having the same molecular mass, but a different glycosylation pattern. The most abundant Gal-deficient O-glycoforms were GalNAc4Gal3 and GalNAc5Gal4 with one Gal-deficient site and GalNAc5Gal3 and GalNAc4Gal2 with two Gal-deficient sites. The most frequent Gal-deficient sites were at Ser230 and/or Thr236.In this work, we studied the O-glycosylation in the hinge region of human immunoglobulin A1 (IgA1). Aberrant glycosylation of the protein plays a key role in the pathogenesis of IgA nephropathy. Thus identification of the O-glycan composition of IgA1 is important for a deeper understanding of the disease mechanism, biomarker discovery and validation, and implementation and monitoring of disease-specific therapies. We developed a new procedure for elucidating the heterogeneity of IgA1 O-glycosylation. After running a polyacrylamide gel electrophoresis under denaturing conditions, the heavy chain of IgA1 was subjected to in-gel digestion by trypsin. O-glycopeptides were separated from the digest on capillary columns using a microgradient chromatographic device (replacing commonly used liquid chromatographs) and subjected to MALDI-TOF/TOF mass spectrometry (MS) and tandem mass spectrometry (MS/MS) involving post-source decay fragmentation. We show that the complete modification of cysteines by iodoacetamide prior to electrophoresis is critical for successful MS/MS analyses on the way to deciphering the microheterogeneity of O-glycosylation in IgA1. Similarly, the removal of the excess of the reagent is equally important. The acquired MS/MS allowed assigning up to six O-glycosylation sites and identification of isomeric O-glycoforms. We show that our simplified approach is efficient and has a high potential to provide a method for the rapid assessment of IgA1 heterogeneity that is a less expensive and yet corroborating alternative to LC-(high-resolution)-MS protocols. The novelty and biological significance reside in the demonstration, for the first time, of the distribution of the most abundant isoforms of HR O-glycopeptides of IgA1. As another new feature, we introduce a software solution for the interpretation of MS/MS data of O-glycopeptide isoforms, which provides the possibility of fast and easier data processing. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

Bacterial proteins exhibiting two or more unrelated functions, referred to as moonlighting proteins, are suggested to contribute to full virulence manifestation in pathogens. An expanding number of published studies have revealed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to be a multitasking protein with virulence impact in a number of pathogenic bacteria. This protein can be detected on the bacterial surface or outside the bacterial cell, where it interacts with host proteins. In this way, GAPDH is able to modulate various pathogenic processes. Moreover, it has been shown to be involved in non-enzymatic processes inside the bacterial cell. In this mini review, we summarize main findings concerning the multiple localization and protein interactions of GAPDH derived from bacterial pathogens of humans. We also briefly discuss problems associated with using GAPDH as a vaccine antigen and endeavor to inspire further research to fill gaps in the existing knowledge.

Ubiquitination of proteins, like phosphorylation and acetylation, is an important regulatory aspect influencing numerous and various cell processes, such as immune response signaling and autophagy. The study of ubiquitination has become essential to learning about host-pathogen interactions, and a better understanding of the detailed mechanisms through which pathogens affect ubiquitination processes in host cell will contribute to vaccine development and effective treatment of diseases. Pathogenic bacteria (e.g., Salmonella enterica, Legionella pneumophila and Shigella flexneri) encode many effector proteins, such as deubiquitinating enzymes (DUBs), targeting the host ubiquitin machinery and thus disrupting pertinent ubiquitin-dependent anti-bacterial response. We focus here upon the host ubiquitination system as an integral unit, its interconnection with the regulation of inflammation and autophagy, and primarily while examining pathogens manipulating the host ubiquitination system. Many bacterial effector proteins have already been described as being translocated into the host cell, where they directly regulate host defense processes. Due to their importance in pathogenic bacteria progression within the host, they are regarded as virulence factors essential for bacterial evasion. However, in some cases (e.g., Francisella tularensis) the host ubiquitination system is influenced by bacterial infection, although the responsible bacterial effectors are still unknown.

Determination of the prognosis and treatment outcomes of dilated cardiomyopathy is a serious problem due to the lack of valid specific protein markers. Using in-depth proteome discovery analysis, we compared 49 plasma samples from patients suffering from dilated cardiomyopathy with plasma samples from their healthy counterparts. In total, we identified 97 proteins exhibiting statistically significant dysregulation in diseased plasma samples. The functional enrichment analysis of differentially expressed proteins uncovered dysregulation in biological processes like inflammatory response, wound healing, complement cascade, blood coagulation, and lipid metabolism in dilated cardiomyopathy patients. The same proteome approach was employed in order to find protein markers whose expression differs between the patients well-responding to therapy and nonresponders. In this case, 45 plasma proteins revealed statistically significant different expression between these two groups. Of them, fructose-1,6-bisphosphate aldolase seems to be a promising biomarker candidate because it accumulates in plasma samples obtained from patients with insufficient treatment response and with worse or fatal outcome. Data are available via ProteomeXchange with the identifier PXD046288.

Our current results, aimed at the detection of protein abundance alterations that could be associated with the process of colon tumorigenesis, are summarized. The matched sets of macroscopically normal colon mucosa and colorectal carcinoma were examined by a one- or two-dimensional electrophoretic approach and proteins were identified using immunoblotting or mass spectrometry. The following results were observed: The levels of liver fatty acid-binding protein, actin-binding protein/smooth muscle protein 22-alpha and cyclooxygenase 2 were downregulated in colorectal carcinoma compared to normal colon mucosa. Conversely, the expression of a novel variant of heat shock protein70 and several members of the S100 protein family of calcium-binding proteins (two isoforms of S100A9, S100A8, S100A11 and S100A6) were upregulated in transformed colon mucosa. Despite the variations of the levels of expression of given protein among analyzed samples, all quantitative changes were found to be statistically significant (Mann-Whitney test assuming p < or = 0.05). We conclude that the proteomic approach is useful for the study of complex biological events underlying the process of colorectal tumorigenesis.

Development of cancer is a complex process involving multiple changes in gene expression. To unravel these alterations, a proteome approach aimed at the identification of qualitative and quantitative changes in protein composition, including their post-translational modifications, attracts great attention. Our study was focused on the identification of proteins whose amount is altered in the course of malignant transformation of colon mucosa. Proteins extracted from tissue specimens or cell lysates were separated by two-dimensional gel electrophoresis (2-DE). Comparative analyses of 2-DE protein patterns were done using computerized image analysis. Selected proteins exhibiting statistically significant abundance alterations comparing healthy and diseased tissues were identified by mass spectrometry. Globally, we have found 57 proteins that exhibited either a significant decrease or increase in amount in pathological tissues, and 18 of these were annotated by mass spectrometry. The alterations in the expression of nine proteins were common for both precancerous and neoplastic tissues suggesting their role in colon tumorigenesis. The epithelial origin of all identified spots was checked in two cell lines Caco-2 and DLD-1 originating from well-differentiated and poorly differentiated colon carcinoma, respectively.

Ever since proteomics was proven to be capable of characterizing a large number of differences in both protein quality and quantity, it has been applied in various areas of biomedicine, ranging from the deciphering molecular pathogenesis of diseases to the characterization of novel drug targets and the discovery of potential diagnostic biomarkers. Indeed, the biomarker discovery in human plasma is clearly one of the areas with enormous potential. However, without proper planning and implementation of specific techniques, the efforts and expectations may very easily be hampered. Numerous earlier projects aimed at clinical proteomics, characterized by exaggerated enthusiasm, often underestimated some principal obstacles of plasma biomarker discovery. Consequently, ambiguous and insignificant results soon led to a more critical view in this field. In this article, we critically review the current state of proteomic approaches for biomarker discovery and validation, in order to provide basic information and guidelines for both clinicians and researchers. These need to be closely considered prior to initiation of a project aimed at plasma biomarker discovery. We also present a short overview of recent applications of clinical proteomics in biomarker discovery.

Most of the cellular processes are regulated by reversible phosphorylation of proteins, which in turn plays a critical role in the regulation of gene expression, cell division, signal transduction, metabolism, differentiation, and apoptosis. Mass spectrometry of phosphopeptides obtained from tryptic protein digests has become a powerful tool for characterization of phosphoproteins involved in these processes. However, there is a general need to significantly enrich the phosphopeptide content to compensate their low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. This paper aims to give a comprehensive overview on the methods involved in recent phosphoproteomics. It presents a description of contemporary enrichment techniques with references to particular studies and compares different approaches to characterization of phosphoproteome by mass spectrometry.

Shotgun proteomics is capable of characterizing differences in both protein quality and quantity, and has been applied in various biomedical applications. Unfortunately, the high complexity and dynamic range of proteins in studied samples, clinical in particular, often hinders the identification of relevant proteins. Indeed, information-rich, low abundance proteins often remain undetected, whereas repeatedly reported altered concentrations in high abundance proteins are often ambiguous and insignificant. Several techniques have therefore been developed to overcome this obstacle and provide a deeper insight into the proteome. Here we report a novel approach, which enables iTRAQ reagent quantitation of peptides fractionated based on presence of a cysteine residue (thus CysTRAQ). For the first time, we prove that iTRAQ quantitation is fully compatible with cysteinyl peptide enrichment and is not influenced by the fractionation process. Moreover, the employment of the method combined with high-resolution TripleTOF 5600 mass spectrometer for very fast MS/MS acquisition in human amniotic fluid analysis significantly increased the number of identified proteins, which were simultaneously quantified owing to the introduction of iTRAQ labeling. We herein show that CysTRAQ is a robust and straightforward method with potential application in quantitative proteomics experiments, i.e. as an alternative to the ICAT reagent approach.

Francisella tularensis is a Gram-negative, facultative intracellular bacterium, causing a severe disease called tularemia. It secretes unusually shaped nanotubular outer membrane vesicles (OMV) loaded with a number of virulence factors and immunoreactive proteins. In the present study, the vesicles were purified from a clinical isolate of subsp. holarctica strain FSC200. We here provide a comprehensive proteomic characterization of OMV using a novel approach in which a comparison of OMV and membrane fraction is performed in order to find proteins selectively enriched in OMV vs. membrane. Only these proteins were further considered to be really involved in the OMV function and/or their exceptional structure. OMV were also isolated from bacteria cultured under various cultivation conditions simulating the diverse environments of F. tularensis life cycle. These included conditions mimicking the milieu inside the mammalian host during inflammation: oxidative stress, low pH, and high temperature (42°C); and in contrast, low temperature (25°C). We observed several-fold increase in vesiculation rate and significant protein cargo changes for high temperature and low pH. Further proteomic characterization of stress-derived OMV gave us an insight how the bacterium responds to the hostile environment of a mammalian host through the release of differentially loaded OMV. Among the proteins preferentially and selectively packed into OMV during stressful cultivations, the previously described virulence factors connected to the unique intracellular trafficking of Francisella were detected. Considerable changes were also observed in a number of proteins involved in the biosynthesis and metabolism of the bacterial envelope components like O-antigen, lipid A, phospholipids, and fatty acids. Data are available via ProteomeXchange with identifier PXD013074.

Abstract Celiac disease is an autoimmune disorder in which gluten peptides presented by specific HLA‐DQ2‐ and HLA‐DQ8‐positive antigen presenting cells elicit immune response in connective tissue of lamina propria. Immunoglobulin A (IgA) antiendomysial antibodies are specific for celiac disease and are used for screening, diagnosis and follow‐up of this disease with an almost 100% sensitivity and specificity. The major target antigen of IgA antiendomysial antibodies was identified as tissue transglutaminase; nevertheless, the existence of the additional unique celiac disease‐specific autoantigens is anticipated. In this study we have utilized a proteomic approach in order to search out new autoantigens recognized by serum antibodies of patients with active celiac disease. We report the detection of 11 proteins that were immunorecognized with various frequencies by sera of patients with celiac disease. Four autoantigens were identified by mass fingerprinting approach as actin, ATP synthase β chain and two charge variants of enolase α. While production of IgA antibodies against actin molecules were described earlier, the existence of autoantibodies to ATP synthase β chain and enolase α species in sera collected from patients with active celiac disease are described for the first time. These results are suggestive of the existence of additional celiac disease autoantigens with possible diagnostic utility.

Abstract Francisella tularensis is the causative agent of the zoonotic disease tularemia. Four subspecies of this pathogen, namely ssp. tularensis , mediaasiatica , holarctica , and novicida are spread throughout the northern hemisphere. Although there are marked variations in their virulence to mammals, the subspecies are difficult to identify as they are closely genetically related. We carried out the comparative proteome analysis of cellular extracts from isolates representing the highly virulent subspecies tularensis , and the less virulent subspecies mediaasiatica and holarctica in order to identify new diagnostic markers and putative factors of virulence. We identified 27 protein spots that were either specifically present or at significantly higher abundance in ssp. tularensis strains, 22 proteins in ssp. mediaasiatica strains, and 26 proteins in ssp. holarctica strains. Subspecies tularensis ‐specific proteins might represent putative virulence factors. Of 27 identified tularensis ‐specific spots 17 represented charge and mass variants of proteins occurring in other subspecies, 7 spots were found to be present at higher abundance, and 3 spots were specifically present in tularensis strains. Amongst them, PilP protein, as a component neccessary for the biogenesis of the type IV pilus, virulence and adhesion factor for many human pathogen, was identified. Furthermore, the identification of additional 27 proteins common for ssp. tularensis and mediaasiatica , and 19 proteins shared by ssp. mediaasiatica and holarctica documented apparent closer genetic similarity between ssp. tularensis and mediaasiatica .

Francisella tularensis (F. tularensis) is highly infectious for humans via aerosol route and untreated infections with the highly virulent subsp. tularensis can be fatal. Our knowledge regarding key virulence determinants has increased recently but is still somewhat limited. Surface proteins are potential virulence factors and therapeutic targets, and in this study, we decided to target three genes encoding putative membrane lipoproteins in F. tularensis LVS. One of the genes encoded a protein with high homology to the protein family of disulfide oxidoreductases DsbA. The two other genes encoded proteins with homology to the VacJ, a virulence determinant of Shigella flexneri. The gene encoding the DsbA homologue was verified to be required for survival and replication in macrophages and importantly also for in vivo virulence in the mouse infection model for tularemia. Using a combination of classical and shotgun proteome analyses, we were able to identify several proteins that accumulated in fractions enriched for membrane-associated proteins in the dsbA mutant. These proteins are substrate candidates for the DsbA disulfide oxidoreductase as well as being responsible for the virulence attenuation of the dsbA mutant.

It appears that most glycoproteins found in pathogenic bacteria are associated with virulence. Despite the recent identification of novel virulence factors, the mechanisms of virulence in Francisella tularensis are poorly understood. In spite of its importance, questions about glycosylation of proteins in this bacterium and its potential connection with bacterial virulence have not been answered yet. In the present study, several putative Francisella tularensis glycoproteins were characterized through the combination of carbohydrate-specific detection and lectin affinity with highly sensitive mass spectrometry utilizing the bottom-up proteomic approach. The protein PilA that was recently found as being possibly glycosylated, as well as other proteins with designation as novel factors of virulence, were among the proteins identified in this study. The reported data compile the list of potential glycoproteins that may serve as a takeoff platform for a further definition of proteins modified by glycans, faciliting a better understanding of the function of protein glycosylation in pathogenicity of Francisella tularensis.

Preterm prelabor rupture of membranes (PPROM) complicated by microbial invasion of the amniotic cavity (MIAC) leading to histological chorioamnionitis (HCA) significantly impacts perinatal morbidity. Unfortunately, no well-established tool for identifying PPROM patients threatened by these disorders is available.We performed an unbiased exploratory analysis of amniotic fluid proteome changes due to MIAC and HCA. From among the top five proteins that showed the most profound and significant change, we sought to confirm results concerning cathelicidin (P49913, CAMP_HUMAN), since an ELISA kit was readily available for this protein. In our exploratory proteomic study, cathelicidin showed a ∼6-fold higher concentration in PPROM patients with confirmed MIAC and HCA. We verified significantly higher levels of cathelicidin in exploratory samples (women without both MIAC and HCA: median 1.4 ng/ml; women with both conditions confirmed: median 3.6 ng/ml; p = 0.0003). A prospective replication cohort was used for independent validation and for assessment of cathelicidin potential to stratify women with MIAC leading to HCA from women in whom at least one of these conditions was ruled out. We confirmed the association of higher amniotic fluid cathelicidin levels with MIAC leading to HCA (the presence of both MIAC and HCA: median 3.1 ng/ml; other women: median 1.4 ng/ml; p<0.0001). A cathelicidin concentration of 4.0 ng/ml was found to be the best cut-off point for identifying PPROM women with both MIAC and HCA. When tested on the validation cohort, a sensitivity of 48%, a specificity of 90%, a likelihood ratio of 5.0, and an area under receiver-operating characteristic curve of 71% were achieved for identification of women with MIAC leading to HCA.Our multi-stage study suggests cathelicidin as a candidate marker that should be considered for a panel of amniotic fluid proteins permitting identification of PPROM women with MIAC leading to HCA.

ABSTRACT Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. This facultative intracellular bacterium replicates in vivo mainly inside macrophages and therefore has developed strategies to resist this stressful environment. Here, we identified a novel genetic locus that is important for stress resistance and intracellular survival of F. tularensis. In silico and transcriptional analyses suggest that this locus (genes FTL_0200 to FTL_0209 in the live vaccine strain [LVS]) constitutes an operon controlled by the alternative sigma factor σ 32 . The first gene, FTL_0200 , encodes a putative AAA+ ATPase of the MoxR subfamily. Insertion mutagenesis into genes FTL_0200 , FTL_0205 , and FTL_0206 revealed a role for the locus in both intracellular multiplication and in vivo survival of F. tularensis . Deletion of gene FTL_0200 led to a mutant bacterium with increased vulnerability to various stress conditions, including oxidative and pH stresses. Proteomic analyses revealed a pleiotropic impact of the Δ FTL_0200 deletion, supporting a role as a chaperone for FTL_0200. This is the first report of a role for a MoxR family member in bacterial pathogenesis. This class of proteins is remarkably conserved among pathogenic species and may thus constitute a novel player in bacterial virulence.

FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical to that of DsbA glycoprotein, as well as to one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings, we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation, and we further suggest an inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways. FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical to that of DsbA glycoprotein, as well as to one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings, we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation, and we further suggest an inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways. The investigation of protein glycosylation in bacteria has attracted a lot of attention during the last decade, because of an increased knowledge of the involvement of such a co-translational modification in the virulence of particular bacteria (1Arora S.K. Neely A.N. Blair B. Lory S. Ramphal R. Role of motility and flagelin glycosylation in the pathogenesis of Pseudomonas aeruginosa burn wound infections.Infect. Immun. 2005; 73: 4395-4398Crossref PubMed Scopus (138) Google Scholar, 2Karlyshev A.V. Everest P. Linton D. Cawthraw S. Newell D.G. Wren B.W. The Campylobacter jejuni general glycosylation system is important for attachment to human epithelial cells and in the colonization of chicks.Microbiology. 2004; 150: 1957-1964Crossref PubMed Scopus (148) Google Scholar, 3Szymanski C.M. Burr D.H. Guerry P. Campylobacter protein glycosylation affects host cell interactions.Infect. Immun. 2002; 70: 2242-2244Crossref PubMed Scopus (191) Google Scholar). In several pathogens, the loss of glycosylation was found to result in accumulation of unglycosylated flagellins in the cell, providing evidence that glycosylation is essential for the correct assembly of flagella and subsequent motility (4Goon S. Kelly J.F. Logan S.M. Ewing C.P. Guerry P. Pseudaminic acid, the major modification on Campylobacter flagellin, is synthesized via the Cj1293 gene.Mol. Microbiol. 2003; 50: 659-671Crossref PubMed Scopus (149) Google Scholar, 5Josenhans C. Vossebein L. Friedrich S. Suerbaum S. The neuA/flmD gene cluster of Helicobacter pylori is involved in flagellar biosynthesis and flagellin glycosylation.FEMS Microbiol. Lett. 2002; 210: 165-172Crossref PubMed Google Scholar, 6Gryllos I. Shaw J.G. Gavín R. Merino S. Tomás J.M. Role of flm locus in mesophilic Aeromonas species adherence.Infect. Immun. 2001; 69: 65-74Crossref PubMed Scopus (48) Google Scholar). On the other hand, it has been concluded that glycosylation does not play a major role in pilus-mediated adhesion in Neisseria meningitidis (7Marceau M. Forest K. Béretti J.L. Tainer J. Nassif X. Consequences of the loss of O-linked glycosylation of meningococcal type IV pilin on piliation and pilus-mediated adhesion.Mol. Microbiol. 1998; 27: 705-715Crossref PubMed Scopus (101) Google Scholar). Moreover, a general O-glycosylation system is present in the major intestinal symbiont Bacteroides fragilis, indicating that glycosylation is not always associated with virulence properties (8Fletcher C.M. Coyne M.J. Villa O.F. Chatzidaki-Livanis M. Comstock L.E. A general O-glycosylation system important to the physiology of a major human intestinal symbiont.Cell. 2009; 137: 321-331Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Francisella tularensis represents one of the six etiological agents under consideration as a potential biological threat because of its low infectious dose and the ease of its transmission (9Dennis D.T. Inglesby T.V. Henderson D.A. Bartlett J.G. Ascher M.S. Eitzen E. Fine A.D. Friedlander A.M. Hauer J. Layton M. Lillibridge S.R. McDade J.E. Osterholm M.T. O'Toole T. Parker G. Perl T.M. Russell P.K. Tonat K. Tularemia as a biological weapon: Medical and public health management.JAMA. 2001; 285: 2763-2773Crossref PubMed Scopus (1168) Google Scholar). Two F. tularensis subspecies, holarctica and tularensis, are primarily infectious for humans (10Svensson K. Larsson P. Johansson D. Byström M. Forsman M. Johansson A. Evolution of subspecies of Francisella tularensis.J. Bacteriol. 2005; 187: 3903-3908Crossref PubMed Scopus (107) Google Scholar). In the past few years, questions arose regarding the potential role of glycosylation in the virulence of F. tularensis. A previous report indicated that one potential pilin subunit encoded by pilA, which is required for virulence of both subspecies, is post-translationally modified (11Forslund A.L. Kuoppa K. Svensson K. Salomonsson E. Johansson A. Byström M. Oyston P.C. Michell S.L. Titball R.W. Noppa L. Frithz-Lindsten E. Forsman M. Forsberg A. Direct repeat-mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis.Mol. Microbiol. 2006; 59: 1818-1830Crossref PubMed Scopus (80) Google Scholar). Since then, the glycosylation of PilA has been reported in other studies (12Salomonsson E. Forsberg A. Roos N. Holz C. Maier B. Koomey M. Winther-Larsen H.C. Functional analyses of pilin-like proteins from Francisella tularensis: Complementation of type IV pilus phenotypes in Neisseria gonorrhoeae.Microbiology. 2009; 155: 2546-2559Crossref PubMed Scopus (20) Google Scholar, 13Balonova L. Hernychova L. Mann B.F. Link M. Bilkova Z. Novotny M.V. Stulik J. Multimethodological approach to identification of glycoproteins from the proteome of Francisella tularensis, an intracellular microorganism.J. Proteome Res. 2010; 9: 1995-2005Crossref PubMed Scopus (38) Google Scholar, 14Egge-Jacobsen W. Salomonsson E.N. Aas F.E. Forslund A.L. Winther-Larsen H.C. Maier J. Macellaro A. Kuoppa K. Oyston P.C. Titball R.W. Thomas R.M. Forsberg Å. Prior J.L. Koomey M. O-Linked glycosylation of the PilA pilin protein of Francisella tularensis: Identification of the endogenous protein-targeting oligosaccharyltransferase and characterization of the native oligosaccharide.J. Bacteriol. 2011; 193: 5487-5497Crossref PubMed Scopus (35) Google Scholar), and recently the O-linked carbohydrates associated with PilA have been characterized (14Egge-Jacobsen W. Salomonsson E.N. Aas F.E. Forslund A.L. Winther-Larsen H.C. Maier J. Macellaro A. Kuoppa K. Oyston P.C. Titball R.W. Thomas R.M. Forsberg Å. Prior J.L. Koomey M. O-Linked glycosylation of the PilA pilin protein of Francisella tularensis: Identification of the endogenous protein-targeting oligosaccharyltransferase and characterization of the native oligosaccharide.J. Bacteriol. 2011; 193: 5487-5497Crossref PubMed Scopus (35) Google Scholar). In our previous study, using both detection and enrichment methods in combination with liquid chromatography-mass spectrometry, we mapped the glycoproteome in the FSC200 strain of F. tularensis subsp. holarctica. In addition to PilA, we suggested that several additional candidate proteins could be targets for glycosylation including DsbA (FTH_1071), an uncharacterized protein FTH_0069, FopA, Tul4, and LemA (13Balonova L. Hernychova L. Mann B.F. Link M. Bilkova Z. Novotny M.V. Stulik J. Multimethodological approach to identification of glycoproteins from the proteome of Francisella tularensis, an intracellular microorganism.J. Proteome Res. 2010; 9: 1995-2005Crossref PubMed Scopus (38) Google Scholar). However, we were unable to elucidate the repertoire of glycans and their structures because of the failure of both enzymatic and chemical release techniques. Because their role in virulence may be associated with their carbohydrates, DsbA and FTH_0069 are of particular interest for an in-depth analysis of their glycosylation. The protein DsbA has recently been identified to be essential for intracellular survival and replication of F. tularensis in the mouse monocyte macrophage cell line J774.2 and for in vivo virulence in the mouse infection model of tularemia (15Straskova A. Pavkova I. Link M. Forslund A.L. Kuoppa K. Noppa L. Kroca M. Fucikova A. Klimentova J. Krocova Z. Forsberg A. Stulik J. Proteome analysis of an attenuated Francisella tularensis dsbA mutant: Identification of potential DsbA substrate proteins.J. Proteome Res. 2009; 8: 5336-5346Crossref PubMed Scopus (38) Google Scholar). FTL_1096, the DsbA homologue in the live vaccine strain (LVS), was recently found to be recruited into the membrane rafts of J774.2 macrophages upon F. tularensis invasion. 1Härtlova, A., Balounova, J., Benesova, M., Straskova, A., Sobol, M., Krocova, Z., Hozak, P., Filipp, D., and Stulik, J., manuscript in preparation. 1Härtlova, A., Balounova, J., Benesova, M., Straskova, A., Sobol, M., Krocova, Z., Hozak, P., Filipp, D., and Stulik, J., manuscript in preparation. The O-linked glycosylation of DsbA was recently elucidated in F. tularensis subsp. tularensis, holarctica LVS, and F. novicida (16Thomas R.M. Twine S.M. Fulton K.M. Tessier L. Kilmury S.L. Ding W. Harmer N. Michell S.L. Oyston P.C. Titball R.W. Prior J.L. Glycosylation of DsbA in Francisella tularensis subspecies tularensis.J. Bacteriol. 2011; 193: 5498-5509Crossref PubMed Scopus (27) Google Scholar). Here, we additionally confirmed that the DsbA homologue in the FSC200 strain is also glycosylated. Similarly, FTH_0069, an orthologue of FTT1676, which was recently identified as a novel virulence factor of F. tularensis subsp. tularensis SchuS4, is important for both intracellular survival and proliferation in mice (17Wehrly T.D. Chong A. Virtaneva K. Sturdevant D.E. Child R. Edwards J.A. Brouwer D. Nair V. Fischer E.R. Wicke L. Curda A.J. Kupko 3rd, J.J. Martens C. Crane D.D. Bosio C.M. Porcella S.F. Celli J. Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophages.Cell. Microbiol. 2009; 11: 1128-1150Crossref PubMed Scopus (147) Google Scholar). Previously, we demonstrated that FTT1676 induced a long term antibody response in a technician accidentally infected with SchuS4 (18Janovská S. Pávková I. Reichelová M. Hubáleka M. Stulík J. Macela A. Proteomic analysis of antibody response in a case of laboratory-acquired infection with Francisella tularensis subsp. tularensis.Folia Microbiol. 2007; 52: 194-198Crossref PubMed Scopus (21) Google Scholar). The glycosylation status of FTH_0069 has not been previously described, and here we provide the first evidence that FTH_0069 is glycosylated in the subspecies holarctica strain FSC200. Additionally, using bioinformatics, we identified proteins that are likely to be involved in a glycosylation pathway of F. tularensis subsp. holarctica FSC200. Three of these predicted proteins, FTT0791, FTT0798, and PglA (FTT0905), are known to be involved in DsbA and PilA glycosylation in the strains SchuS4 and FSC200, respectively (14Egge-Jacobsen W. Salomonsson E.N. Aas F.E. Forslund A.L. Winther-Larsen H.C. Maier J. Macellaro A. Kuoppa K. Oyston P.C. Titball R.W. Thomas R.M. Forsberg Å. Prior J.L. Koomey M. O-Linked glycosylation of the PilA pilin protein of Francisella tularensis: Identification of the endogenous protein-targeting oligosaccharyltransferase and characterization of the native oligosaccharide.J. Bacteriol. 2011; 193: 5487-5497Crossref PubMed Scopus (35) Google Scholar, 16Thomas R.M. Twine S.M. Fulton K.M. Tessier L. Kilmury S.L. Ding W. Harmer N. Michell S.L. Oyston P.C. Titball R.W. Prior J.L. Glycosylation of DsbA in Francisella tularensis subspecies tularensis.J. Bacteriol. 2011; 193: 5498-5509Crossref PubMed Scopus (27) Google Scholar). The F. tularensis strains and plasmids employed in this study are listed in Table I. The bacteria for all the strains were grown, harvested, and lysed within a BioSafety Level 2 containment facility. F. tularensis was cultured on McLeod agar supplemented with bovine hemoglobin (Becton Dickinson) and IsoVitaleXTM (Becton Dickinson) at 36.8 °C for 24–48 h. The cultivation of F. tularensis in Chamberlain medium was performed as previously described (13Balonova L. Hernychova L. Mann B.F. Link M. Bilkova Z. Novotny M.V. Stulik J. Multimethodological approach to identification of glycoproteins from the proteome of Francisella tularensis, an intracellular microorganism.J. Proteome Res. 2010; 9: 1995-2005Crossref PubMed Scopus (38) Google Scholar).Table IF. tularensis strains and plasmids used in this studyStrain/plasmidCharacteristics/descriptionReference/sourceStrain FSC200F. tularensis subsp. holarctica FSC200Ref. 47Johansson A. Berglund L. Eriksson U. Göransson I. Wollin R. Forsman M. Tärnvik A. Sjöstedt A. Comparative analysis of PCR versus culture for diagnosis of ulceroglandular tularemia.J. Clin. Microbiol. 2000; 38: 22-26Crossref PubMed Google Scholar FSC679 (FSC200/ΔpglA)FSC200/in-frame deletion of codons 10–469 of FTT_0905Ref. 14Egge-Jacobsen W. Salomonsson E.N. Aas F.E. Forslund A.L. Winther-Larsen H.C. Maier J. Macellaro A. Kuoppa K. Oyston P.C. Titball R.W. Thomas R.M. Forsberg Å. Prior J.L. Koomey M. O-Linked glycosylation of the PilA pilin protein of Francisella tularensis: Identification of the endogenous protein-targeting oligosaccharyltransferase and characterization of the native oligosaccharide.J. Bacteriol. 2011; 193: 5487-5497Crossref PubMed Scopus (35) Google Scholar FSC699 (FCS200/Δ1103)FSC200/in-frame deletion of codons 4–369 of FTT_1103Ref. 15Straskova A. Pavkova I. Link M. Forslund A.L. Kuoppa K. Noppa L. Kroca M. Fucikova A. Klimentova J. Krocova Z. Forsberg A. Stulik J. Proteome analysis of an attenuated Francisella tularensis dsbA mutant: Identification of potential DsbA substrate proteins.J. Proteome Res. 2009; 8: 5336-5346Crossref PubMed Scopus (38) Google Scholar FSC200/ΔwbtDEF:CmFSC200 with inactivated production of O-antigenThis study E. coli S17-1thi thr leu tonA supE recA::RP4–2-Tc::Mu Kn::Tn7Ref. 20Simon R. Priefer U. Pühler A. A broad host range mobilization system for in vivo genetic engineering: Transposon mutagenesis in Gram-negative bacteria.Nat. Biotechnol. 1983; 1: 784-791Crossref Scopus (5641) Google ScholarPlasmid pSMP22-RT1Suicide plasmid used to inactivate O-antigen cluster of F. tularensis subsp. tularensisRef. 19Thomas R.M. Titball R.W. Oyston P.C. Griffin K. Waters E. Hitchen P.G. Michell S.L. Grice I.D. Wilson J.C. Prior J.L. The immunologically distinct O antigens from Francisella tularensis subspecies tularensis Francisella novicida are both virulence determinants and protective antigens.Infect. Immun. 2007; 75: 371-378Crossref PubMed Scopus (75) Google Scholar Open table in a new tab Escherichia coli S17-1 was grown on LB agar base or LB broth at 37 °C. When appropriate, antibiotics (all purchased from Sigma-Aldrich) were used at the following concentrations: for the cultivation of the transformed E. coli S17-1, 25 μg/ml of chloramphenicol was added to growth medium; the selection of transconjugants was performed on plates with 75 μg/ml polymyxin B and 2.5 μg/ml chloramphenicol; plates containing 5% sucrose were utilized for the sucrose selection of the mutated F. tularensis strains, and the clones of the mutated F. tularensis were cultured on agar supplemented with 2.5 μg/ml of chloramphenicol. A region of the F. tularensis subsp. holarctica FSC200 O-antigen cluster (wbtDEF) was targeted for deletion and insertion of antibiotic marker to disrupt the entire function of the cluster. For the inactivation of the O-antigen cluster, a suicide vector pSMP22-RT1 was generously provided by R. Thomas. pSMP22-RT1 was originally used for the deletion of the O-antigen encoding genes in F. tularensis subsp. tularensis (19Thomas R.M. Titball R.W. Oyston P.C. Griffin K. Waters E. Hitchen P.G. Michell S.L. Grice I.D. Wilson J.C. Prior J.L. The immunologically distinct O antigens from Francisella tularensis subspecies tularensis Francisella novicida are both virulence determinants and protective antigens.Infect. Immun. 2007; 75: 371-378Crossref PubMed Scopus (75) Google Scholar). The cloned 2.5-kb deletion construct shows a 100% sequence homology with the sequence from FSC200. pSMP22-RT1 was transformed into E. coli S17-1 (20Simon R. Priefer U. Pühler A. A broad host range mobilization system for in vivo genetic engineering: Transposon mutagenesis in Gram-negative bacteria.Nat. Biotechnol. 1983; 1: 784-791Crossref Scopus (5641) Google Scholar) and subsequently introduced into the FSC200 strain by conjugation using essentially the same method as published by Golovliov et al. (21Golovliov I. Sjöstedt A. Mokrievich A. Pavlov V. A method for allelic replacement in Francisella tularensis.FEMS Microbiol. Lett. 2003; 222: 273-280Crossref PubMed Scopus (167) Google Scholar). The F. tularensis subsp. holarctica strain FSC200 double recombinant strain was designated as FSC200/ΔwbtDEF::Cm. The integration of the suicide plasmid and the subsequent generation of the ΔwbtDEF::Cm mutant in FSC200 was verified by PCR. The primers wbtD_1F and wbtF_1R (the primers were designed for the detection of the deletion construct cloned into pSMP22-RT1) resulted in an amplicon with a length of 1780 bp (the length of wild type amplicon is ∼2300 bp). ΔwbtDEF::Cm-positive clones were obtained after sucrose selection and identified using the primers wbtD_1F and wzy_1R (the primer was designed on a chromosomal sequence downstream from the wbtDEF cluster), providing a PCR product having 2470 bp (the length of an amplicon from the wild type is ∼3 kb long), and finally with a combination of the primers wbtD_1F and wbtE_1R (designed on a deleted region of wbtDEF) that does not give rise to any PCR product in FSC200/ΔwbtDEF::Cm mutant. The primer sequences are available on request. Groups of five 6–8-week-old female BALB/c mice were infected subcutaneously with the FSC200/ΔwbtDEF::Cm mutant (using doses of 3 × 102 and 3 × 106 cfu/mouse) and the wild type FSC200 strain (dose 3 × 102 cfu/mouse). The control groups of mice were inoculated with a sterile saline only. The mice were housed under conventional conditions; food and water were given ad libitum, and the mice were allowed to acclimatize for at least 7 days before the beginning of the experiment. The infected mice were observed two or three times per day to properly monitor the progress of the infection. Cell lysis was performed using a French pressure cell as previously described (13Balonova L. Hernychova L. Mann B.F. Link M. Bilkova Z. Novotny M.V. Stulik J. Multimethodological approach to identification of glycoproteins from the proteome of Francisella tularensis, an intracellular microorganism.J. Proteome Res. 2010; 9: 1995-2005Crossref PubMed Scopus (38) Google Scholar). Fractions enriched in membrane proteins were collected by ultracentrifugation of the whole cell lysate at 115,000 × g for 1 h at 4 °C. The supernatant was discarded, and the membrane pellet was resuspended in ice-cold PBS and then collected by centrifugation at 115,000 × g for 30 min at 4 °C. The final membrane protein-containing pellet was resuspended in PBS, and the protein content in the sample was quantified by a bicinchoninic acid assay. LPS 2The abbreviations used are:LPSlipopolysaccharideHexhexoseHexNAcN-acetylhexosamineRTretention time. was purified from F. tularensis subsp. holarctica FSC200, FSC200/ΔwbtDEF::Cm, and FSC200/ΔpglA mutant strains using the hot phenol-water extraction method of Westphal and Jann (22Westphal O. Jann K. Bacterial lipopolysaccharides: Extraction with phenol-water and further applications of the procedure.Methods Carbohydr. Chem. 1965; 5: 83-91Google Scholar) with slight modifications. Briefly, a 1-mg amount of the crude membrane fraction was incubated with 1.4 ml of a 50%/50% phenol/water solution at 65 °C for 30 min. The samples were cooled on ice and centrifuged at 4 °C to separate organic and aqueous layer. The phenol layer was back-extracted three times with hot water. The three aqueous layers containing the LPS were centrifuged at high speed to remove any residual phenol. lipopolysaccharide hexose N-acetylhexosamine retention time. SDS-PAGE was performed on a 30-μg sample of the membrane-proteins enriched fraction and a 10-μl aliquot of LPS from the first aqueous layer according to the protocol employed by Laemmli (23Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207159) Google Scholar). After electrophoresis, the separated proteins and LPS were transferred onto PVDF membranes using a semi-dry Western blotting technique. The primary antibodies used for the immunodetection of DsbA and PilA were a rabbit anti-FTT1103 polyclonal antibody (Apronex, Vestec, Czech Republic) and rabbit anti-PilA serum (kindly provided by Kerstin Kuoppa at FOI, Swedish Defense Research Agency, Umea, Sweden), respectively. The detection of LPS was accomplished using a mouse monoclonal anti-F. tularensis LPS FB11 antibody (Abcam, Cambridge, UK). The secondary antibodies used were swine anti-rabbit IgG/HRP (Dako, Glostrup, Denmark) for both anti-FTT1103 and anti-PilA antibodies, whereas goat anti-mouse IgG/HRP (Dako) was used for anti-LPS. A chemiluminescence detection was performed using a BM chemiluminescence blotting substrate POD according to the manufacturer's instructions (Roche Applied Science). The membrane proteins were repeatedly precipitated with cold acetone prior to two-dimensional electrophoresis. The protein precipitate was resolubilized in a rehydration buffer containing 7 m urea, 2 m thiourea, 1% (w/v) ASB-14, 1% Triton X-100, 0.12% De Streak, 40 mm Tris base, 0.5% bromphenol blue, 1% ampholytes, pH 3–10, and 0.5% PharmalyteTM, pH 8–10.5. The isoelectric focusing, reduction, alkylation, and SDS-PAGE performed herein were described previously (13Balonova L. Hernychova L. Mann B.F. Link M. Bilkova Z. Novotny M.V. Stulik J. Multimethodological approach to identification of glycoproteins from the proteome of Francisella tularensis, an intracellular microorganism.J. Proteome Res. 2010; 9: 1995-2005Crossref PubMed Scopus (38) Google Scholar). Pro-Q Emerald staining was performed according to the manufacturer's protocol (Invitrogen), with slight modifications as described previously (13Balonova L. Hernychova L. Mann B.F. Link M. Bilkova Z. Novotny M.V. Stulik J. Multimethodological approach to identification of glycoproteins from the proteome of Francisella tularensis, an intracellular microorganism.J. Proteome Res. 2010; 9: 1995-2005Crossref PubMed Scopus (38) Google Scholar). The stained gels were visualized by illumination at 342 nm using a UV radiometer. After detection and excizing the glycoproteins, the gels were stained with a colloidal blue stain to detect all proteins as a control. The protein spots detected on the two-dimensional gels were excised and subjected to an in-gel tryptic digestion according to the procedure described recently (13Balonova L. Hernychova L. Mann B.F. Link M. Bilkova Z. Novotny M.V. Stulik J. Multimethodological approach to identification of glycoproteins from the proteome of Francisella tularensis, an intracellular microorganism.J. Proteome Res. 2010; 9: 1995-2005Crossref PubMed Scopus (38) Google Scholar). The digestion was stopped by acidifying the samples with TFA, bringing the pH to 2–3. Mass spectra acquired from the in-gel digestion of the proteins following Pro-Q Emerald staining were recorded in the positive reflectron mode on a 4800 MALDI-TOF/TOF mass spectrometer (AB Sciex, Foster City, CA) equipped with an Nd:YAG laser (355 nm) and operated in its delayed extraction mode. Internal calibration of mass spectra was conducted utilizing the tryptic autolytic peptides. Data acquisition and evaluation were performed as recently described (13Balonova L. Hernychova L. Mann B.F. Link M. Bilkova Z. Novotny M.V. Stulik J. Multimethodological approach to identification of glycoproteins from the proteome of Francisella tularensis, an intracellular microorganism.J. Proteome Res. 2010; 9: 1995-2005Crossref PubMed Scopus (38) Google Scholar). Acquired data were evaluated using GPS ExplorerTM software v.3.6 (AB Sciex), which integrates the Mascot search algorithm against F. tularensis OSU18 genome database (NC_008369.1, 1,555 sequence entries). Trypsin was selected as the proteolytic enzyme, and one missed cleavage was allowed. Carbamidomethylation of cysteine residues was set as a fixed modification, and oxidation of methionine was set as a variable modification. The mass tolerance values of the precursor and fragment ions were 100 ppm and 0.25 Da, respectively. The proteins were considered identified with confidence when the GPS protein score confidence interval (%) was equal to 100% and a minimum of two peptide sequences per protein were identified. PilA (FTH_0384) possesses the only tryptic peptide AQLGSDLSALGGAK with a theoretical mass of 1286.6830, which is measurable within the m/z range 800–4,000. Thus, the mass of 1287.6903 m/z [M+H]+ was added to the inclusion list of masses selected for fragmentation. A 1.0-μl aliquot of the in-gel digests obtained from the proteins DsbA and FTH_0069 was analyzed by C18 reversed phase liquid chromatography using either a Nanoflow 1100 HPLC (Agilent Technologies) coupled on-line to an LTQ-FT-ICR mass spectrometer (Thermo Finnigan) or an Ultimate 3000 (Dionex, Sunnyvale, CA) interfaced to an LTQ-Orbitrap (Thermo). The samples were desalted and preconcentrated on a C18 PepMap300 0.3 × 5-mm μ-precolumn containing 5-μm mean particle size silica, with a pore size of 300 Å (Dionex). After loading and washing the peptides for 10 min with mobile phase A (97/3/0.1, water/ACN/formic acid, v/v/v), the trapping column was switched in-line with the analytical column. The separation of the peptides was conducted at a flow rate of 250 nl/min with a column packed in-house (75-μm inner diameter × 150 mm) with Magic C18AQ (3-μm mean particle size with pores of 200 Å) from Michrom Bioresources, Inc. (Auburn, CA). A linear gradient from 3 to 55% phase B (99.9/0.1, ACN/formic acid, v/v) over a period of 45 min was followed by a steeper ramp from 55 to 80% phase B over 10 min. The column eluent was electrosprayed into the mass spectrometer using a 1.8 kV spray voltage. MS spectra were acquired in the mass range from 80 to 2,000 m/z at a low orifice potential, with fragmentation of the five most intense peaks in the linear ion trap. Collisional activation was performed using helium gas at normalized collision energy 35%. Diagnostic glycan oxonium ions were generated by an in-source CID at the orifice potential set at 100 V. The acquisition of the data was controlled using the Xcalibur software v2.0.7 (Thermo Finnigan). The acquired RAW data were processed using Turbo RAW2MGF converter v1.0.7 (Indiana University). The processing parameters were as follows: minimum ion count in MS/MS was at 5, absolute total ion intensity threshold was set to 100, smoothing was not applied, the percentage peak height at which centroids were calculated was 100%, and charge states were calculated. Peak lists were subjected to MASCOT (v2.2) searching against the F. tularensis subsp. holarctica (OSU18) protein sequences database (NC_008369.1, 1,555 sequence entries) using the following searching criteria: trypsin was used as the protease, allowing for one missed cleavage, 2+ and 3+ charge state ions; carbamidomethylation of cysteine residues was set as a fixed modification, and oxidation of methionine was selected as a variable modification. The mass tolerance of the precursor and fragment ions was set to 5 ppm and 0.8 Da, respectively. The obtained data were further filtered through ProteinParser v2.1 (24Mann B. Madera M. Sheng Q. Tang H. Mechref Y. Novotny M.V. ProteinQuant Suite: A bundle of automated software tools for label-free quantitative proteomics.Rapid. Commun. Mass Spectrom. 2008; 22: 3823-3834Cros

Abstract The host–pathogen interaction represents a complex and dynamic biological system. The outcome of this interaction is dependent on the microbial pathogen properties to establish infection and the ability of the host to control infection. Although bacterial pathogens have evolved a variety of strategies to subvert host defense functions, several general mechanisms have been shown to be shared among these pathogens. As a result, host effectors that are critical for pathogen entry, survival and replication inside the host cells have become a new paradigm for antimicrobial targeting. This review focuses on the potential utility of a proteomics approach in defining the host–pathogen interaction from the host's perspective.

Galectin-7 (Gal-7) is negatively regulated in cervical cancer, and appears to be a link between the apoptotic response triggered by cancer and the anti-tumoral activity of the immune system. Our understanding of how cervical cancer cells and their molecular networks adapt in response to the expression of Gal-7 remains limited.Meta-analysis of Gal-7 expression was conducted in three cervical cancer cohort studies and TCGA. In silico prediction and bisulfite sequencing were performed to inquire epigenetic alterations. To study the effect of Gal-7 on cervical cancer, we ectopically re-expressed it in the HeLa and SiHa cervical cancer cell lines, and analyzed their transcriptome and SILAC-based proteome. We also examined the tumor and microenvironment host cell transcriptomes after xenotransplantation into immunocompromised mice. Differences between samples were assessed with the Kruskall-Wallis, Dunn's Multiple Comparison and T tests. Kaplan-Meier and log-rank tests were used to determine overall survival.Gal-7 was constantly downregulated in our meta-analysis (p < 0.0001). Tumors with combined high Gal-7 and low galectin-1 expression (p = 0.0001) presented significantly better prognoses (p = 0.005). In silico and bisulfite sequencing assays showed de novo methylation in the Gal-7 promoter and first intron. Cells re-expressing Gal-7 showed a high apoptosis ratio (p < 0.05) and their xenografts displayed strong growth retardation (p < 0.001). Multiple gene modules and transcriptional regulators were modulated in response to Gal-7 reconstitution, both in cervical cancer cells and their microenvironments (FDR < 0.05 %). Most of these genes and modules were associated with tissue morphogenesis, metabolism, transport, chemokine activity, and immune response. These functional modules could exert the same effects in vitro and in vivo, even despite different compositions between HeLa and SiHa samples.Gal-7 re-expression affects the regulation of molecular networks in cervical cancer that are involved in diverse cancer hallmarks, such as metabolism, growth control, invasion and evasion of apoptosis. The effect of Gal-7 extends to the microenvironment, where networks involved in its configuration and in immune surveillance are particularly affected.

Although the role of high-risk human papillomaviruses (hrHPVs) as etiological agents in cancer development has been intensively studied during the last decades, there is still the necessity of understanding the impact of the HPV E6 and E7 oncogenes on host cells, ultimately leading to malignant transformation. Here, we used newly established immortalized human keratinocytes with a well-defined HPV16 E6E7 expression cassette to get a more complete and less biased overview of global changes induced by HPV16 by employing transcriptome sequencing (RNA-Seq) and stable isotope labeling by amino acids in cell culture (SILAC). This is the first study combining transcriptome and proteome data to characterize the impact of HPV oncogenes in human keratinocytes in comparison with their virus-negative counterparts. To enhance the informative value and accuracy of the RNA-Seq data, four different bioinformatic workflows were used. We identified potential novel upstream regulators (e.g., CNOT7, SPDEF, MITF, and PAX5) controlling distinct clusters of genes within the HPV-host cell network as well as distinct factors (e.g., CPPED1, LCP1, and TAGLN) with essential functions in cancer. Validated results in this study were compared to data sets from The Cancer Genome Atlas (TCGA), demonstrating that several identified factors were also differentially expressed in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and HPV-positive head and neck squamous cell carcinomas (HNSCs). This highly integrative approach allows the identification of novel HPV-induced cellular changes that are also reflected in cancer patients, providing a promising omics data set for future studies in both basic and translational research.IMPORTANCE Human papillomavirus (HPV)-associated cancers still remain a big health problem, especially in developing countries, despite the availability of prophylactic vaccines. Although HPV oncogenes have been intensively investigated for decades, a study applying recent advances in RNA-Seq and quantitative proteomic approaches to a precancerous model system with well-defined HPV oncogene expression alongside HPV-negative parental cells has been missing until now. Here, combined omics analyses reveal global changes caused by the viral oncogenes in a less biased way and allow the identification of novel factors and key cellular networks potentially promoting malignant transformation. In addition, this system also provides a basis for mechanistic research on novel key factors regulated by HPV oncogenes, especially those that are confirmed in vivo in cervical cancer as well as in head and neck cancer patient samples from TCGA data sets.

The expression of calcium-binding protein S100A6 was investigated in normal colon tissue, in colon adenomas and in colorectal carcinomas. Using an immunoblotting approach we detected four S100A6 variants with Mwt of 10 kDa and pI of 5.05 (isoform I), 5.15 (isoform II), 5.23 (isoform III) and 5.32 (isoform IV) that were differentially expressed in the analysed samples. The quantitative examination of S100A6 variant expression in 25 pairs of colorectal carcinoma and matched control mucosa proved a statistically significant increased abundance of S100A6 isoforms I (P=0.004) and III (P=0.025) in malignant tissue, and conversely, an increased level of S100A6 isoform IV in healthy tissue (P=0.022). The expression of isoforms I and III and the loss of isoform IV were also observed in colon cancer cell lines. In addition, the immunohistochemical study of 16 primary colorectal carcinomas revealed both in the non-paired Student t-test and in the Mann–Whitney test the statistically significant accumulation of S100A6 protein (P<0.001) in the invasive margin of the tumour. The immunohistochemical analysis of S100A6 protein in polyps differing in clinical severity gave a strong staining that was maximal in dysplastic lesions. Thus, our results indicate a possible, statistically significant correlation (non-paired Student t-test P=0.036) between S100A6 expression and colon carcinoma progression.

Francisella tularensis is a Gram-negative, facultative intracellular bacterium causing disease in many mammalian species. The low infectious dose of F. tularensis and the ease of air-borne transmission are the main features responsible for the classification of this bacterium as a potential biological weapon. The live attenuated strain of F. tularensis live vaccine strain (LVS) is currently only effective vaccine against tularemia, however, this type of vaccine has not been approved for human use. In the presented study, sub-immunoproteome analysis was performed to search for new immunogenic proteins of Francisella tularensis LVS grown under different conditions. By this approach 35 immunoreactive antigens were identified, 19 of them showed to be novel immunogens. In conclusion, sub-immunoproteome analysis resulted in successful identification of novel immunoreactive proteins.

Pathogenic bacteria have developed various mechanisms to evade host immune defense systems. Invasion of pathogenic bacteria requires interaction of the pathogen with host receptors, followed by activation of signal transduction pathways and rearrangement of the cytoskeleton to facilitate bacterial entry. Numerous bacteria exploit specialized plasma membrane microdomains, commonly called membrane rafts, which are rich in cholesterol, sphingolipids and a special set of signaling molecules which allow entry to host cells and establishment of a protected niche within the host. This review focuses on the current understanding of the raft hypothesis and the means by which pathogenic bacteria subvert membrane microdomains to promote infection.

Francisella tularensis is a highly virulent intracellular pathogen responsible for tularemia. This bacterium is capable of infecting many mammalian species and various cell types, but little is known about the mechanisms of survival and interactions with host cells. We examined the number of infected host cells, cytotoxicity and the role of apoptosis or necrosis in infection-induced cell death. Our results demonstrate that F. tularensis LVS induces apoptosis of infected macrophages within 10 h. At later time points we were also able to detect a dramatic increase in the proportion of necrotic macrophages. We investigated the signalling pathways involved in infection-induced cell death by analysing three mitogen-activated protein kinase (MAPK) pathways that are known to be activated by LPS stimulation; p42/p44 MAPK (Erk1/2), transcription factor c-Jun and p38 MAPK. We identified post-translational activation of both p42 MAPK and p44 MAPK by phosphorylation at threonine and tyrosine residues after infection. Furthermore, treatment of infected cells with MEK1/2 inhibitors abrogated phosphorylation of p42/p44 MAPK and inhibited macrophage apoptosis and necrosis after infection. In contrast, phosphorylation and kinase activity of p38 MAPK was significantly lower in F. tularensis-infected cells, and inhibition of p38 MAPK activity induced apoptosis in uninfected cells. When we monitored JNK-dependent phosphorylation of the transcription factor c-Jun, we did not observe any reactivity with either SAPK/JNK or phospho-SAPK/JNK antibodies at any time point. In conclusion, we demonstrate that F. tularensis LVS infection induces macrophage apoptosis. This process requires activation of the p42/p44 MAPK pathway and is associated with reduced p38 MAPK activity, indicating that infection-induced cell death can be caused by perturbation of these two signalling pathways.

Immunity to Francisella tularensis is largely mediated by T lymphocytes but an important role of B lymphocytes in early stage of infection was previously uncovered. We wanted to find out if F. tularensis is able to infect B cells and/or influence them by direct contact. To investigate this possibility we infected B cell lines from mouse (A20) or humans (Ramos RA-1), or primary mouse spleen cells, with F. tularensis LVS and F. tularensis FSC200 in vitro. In all cases, we detected bacteria on the cell surface and inside the B cells using transmission electron microscopy. More than 20% cells were infected by microbes after 24 h. The number of bacteria, determined by CFU, increased about 1 log during 24 h. Infection with live bacteria led to apoptosis of Ramos cells and mouse CD19+ spleen cells. Approximately 30% of cells were apoptotic after 24 h and 70% after 48 h, independently of the F. tularensis strain, while only 10% of non-infected cell were apoptotic at either time point. Apoptosis was confirmed by Western blot using anti-PARP antibodies. Thus, this study demonstrates unique phenomenon – namely, the ability of the intracellular pathogen F. tularensis to invade and induce apoptosis in B cells.

Abstract The facultative intracellular bacterium Francisella tularensis is the causal agent of the serious infectious disease tularemia. Despite the dynamic progress, which has been made in last few years, important questions regarding Francisella pathogenicity still remain to be answered. Generally, secreted proteins play an important role in pathogenicity of intracellular microbes. In this study, we investigated the protein composition of the culture filtrate proteins of highly virulent F. tularensis subsp. tularensis , strain SCHU S4 and attenuated F. tularensis subsp. holarctica , live vaccine strain using a comparative proteomic analysis. The majority of proteins identified in this study have been implicated in virulence mechanisms of other pathogens, and several have been categorized as having moonlighting properties; those that have more than one unrelated function. This profiling study of secreted proteins resulted in the unique detection of acid phosphatase (precursor) A (AcpA), β‐lactamase, and hypothetical protein FTT0484 in the highly virulent strain SCHU S4 secretome. The release of AcpA may be of importance for F. tularensis subsp. tularensis virulence due to the recently described AcpA role in the F. tularensis escape from phagosomes.

The nucleoid-associated HU proteins are small abundant DNA-binding proteins in bacterial cell which play an important role in the initiation of DNA replication, cell division, SOS response, control of gene expression and recombination. HU proteins bind to double stranded DNA non-specifically, but they exhibit high affinity to abnormal DNA structures as four-way junctions, gaps or nicks, which are generated during DNA damage. In many pathogens HU proteins regulate expression of genes involved in metabolism and virulence. Here, we show that the Francisella tularensis subsp. holarctica gene locus FTS_0886 codes for functional HU protein which is essential for full Francisella virulence and its resistance to oxidative stress. Further, our results demonstrate that the recombinant FtHU protein binds to double stranded DNA and protects it against free hydroxyl radicals generated via Fenton's reaction. Eventually, using an iTRAQ approach we identified proteins levels of which are affected by the deletion of hupB, among them for example Francisella pathogenicity island (FPI) proteins. The pleiotropic role of HU protein classifies it as a potential target for the development of therapeutics against tularemia.

Release of outer membrane vesicles (OMV) is an important phenomenon in Gram-negative bacteria playing multiple roles in their lifestyle, including in relation to virulence and host-pathogen interaction. Francisella tularensis, unlike other bacteria, releases unusually shaped, tubular OMV. We present a proteomic comparison of OMV and membrane fractions from two F. tularensis strains: moderately virulent subsp. holarctica strain FSC200 and highly virulent subsp. tularensis strain SchuS4. Proteomic comparison studies routinely evaluate samples from the same proteome, but sometimes we must compare samples from closely related organisms. This raises quantification issues. We propose a novel approach to cross-species proteomic comparison based on an intersection protein database from the individual single-species databases. This is less prone to quantification errors arising from differences in the sequences. Consecutively comparing subproteomes of OMV and membranes of the two strains allows distinguishing differences in relative protein amounts caused by global expression changes from those caused by preferential protein packing to OMV or membranes. Among the proteins most differently packed into OMV between the two strains, we detected proteins involved in biosynthesis and metabolism of bacterial envelope components like O-antigen, lipid A, phospholipids, and fatty acids, as well as some major structural outer membrane proteins. The data are available via ProteomeXchange with identifier PXD022406.

Francisella tularensis is known to release unusually shaped tubular outer membrane vesicles (OMV) containing a number of previously identified virulence factors and immunomodulatory proteins. In this study, we present that OMV isolated from the F. tularensis subsp. holarctica strain FSC200 enter readily into primary bone marrow-derived macrophages (BMDM) and seem to reside in structures resembling late endosomes in the later intervals. The isolated OMV enter BMDM generally via macropinocytosis and clathrin-dependent endocytosis, with a minor role played by lipid raft-dependent endocytosis. OMVs proved to be non-toxic and had no negative impact on the viability of BMDM. Unlike the parent bacterium itself, isolated OMV induced massive and dose-dependent proinflammatory responses in BMDM. Using transmission electron microscopy, we also evaluated OMV release from the bacterial surface during several stages of the interaction of Francisella with BMDM. During adherence and the early phase of the uptake of bacteria, we observed numerous tubular OMV-like protrusions bulging from the bacteria in close proximity to the macrophage plasma membrane. This suggests a possible role of OMV in the entry of bacteria into host cells. On the contrary, the OMV release from the bacterial surface during its cytosolic phase was negligible. We propose that OMV play some role in the extracellular phase of the interaction of Francisella with the host and that they are involved in the entry mechanism of the bacteria into macrophages.

We have started the construction of a two-dimensional database of the proteome of Francisella tularensis, a bacterium that is responsible for the highly pathogenic disease tularemia. The genome of this intracellular pathogen is not completely sequenced yet and, currently, information about only 66 proteins is available from NCBI database. We have analyzed the F. tularensis live vaccine strain by two-dimensional gel electrophoresis with immobilized pH 3-10 gradient in the first dimension and 9-16% gradient or tricine SDS-PAGE in the second dimension. In both cases about 2000 spots were detected. Furthermore, we compared the protein pattern of the nonvirulent F. tularensis live vaccine strain with protein profiles of two wild type clinical isolates and more than 50 differentially expressed proteins were counted. The separated proteins are going to be identified by peptide mass fingerprinting. However, due to the lack of complete genome sequence data only eight proteins were unambiguously identified. Among them, acid phosphatase and the most basic isoform of a hypothetical 23 kDa protein are characteristic only for virulent strains.

Francisella tularensis is a facultative intracellular pathogen. Its capacity to induce disease depends on the ability to invade and multiply within a wide range of eukaryotic cells, such as professional phagocytes. The comparative disinterest in tularemia in the past relative to other human bacterial pathogens is reflected in the paucity of information concerning the mechanisms of pathogenesis. Only a few genes and gene products associated with Francisella virulence are known to date. The aim of this study was to find and identify proteins of F. tularensis live vaccine strain induced in the presence of hydrogen peroxide, and to investigate the role of the IglC protein in the regulation of genes expressed upon peroxide stress. The [35S]-radiolabelled protein patterns were examined for both the wild live vaccine strain and its ΔiglC1 + 2 mutant defective in synthesis of the IglC protein that was found to be strongly up-regulated during intracellular growth in murine macrophages in vitro and upon exposure to hydrogen peroxide. Globally, we found 21 protein spots whose levels were significantly altered in the presence of hydrogen peroxide in both the wild-type and mutant strains.

The facultative intracellular pathogen Francisella tularensis is the causative agent of the serious infectious disease tularemia. Despite intensive research, the virulence factors and pathogenetic mechanisms remain largely unknown. To identify novel putative virulence factors, we carried out a comparative proteome analysis of fractions enriched for membrane-associated proteins isolated from the highly virulent subspecies tularensis strain SCHU S4 and three representatives of subspecies holarctica of different virulence including the live vaccine strain. We identified six proteins uniquely expressed and four proteins expressed at significantly higher levels by SCHU S4 compared to the ssp. holarctica strains. Four other protein spots represented mass and charge variants and seven spots were charge variants of proteins occurring in the ssp. holarctica strains. The genes encoding proteins of particular interest were examined by sequencing in order to confirm and explain the findings of the proteome analysis. Our studies suggest that the subspecies tularensis-specific proteins represent novel potential virulence factors.

Francisella tularensis is a facultative intracellular, gram-negative bacterium that induces apoptosis in macrophages and B cells. Here we show apoptotic pathways that are activated in the Ramos human B cell line in the course of F. tularensis infection. Live bacteria F. tularensis FSC200 activate caspases 8, 9 and 3, as well as Bid; release cytochrome c and apoptosis-inducing factor from mitochondria; and induce depolarization of mitochondrial membrane potential in the Ramos cell line, thus leading these cells to apoptosis. Unlike live bacteria, killed F. tularensis FSC200 bacteria activated only caspase 3, and did not cause apoptosis of Ramos cells as measured by annexin V. Killed bacteria also caused accumulation of anti-apoptotic protein BclxL in mitochondrial membranes. Thus, live F. tularensis activates both caspase pathways (receptor-mediated and intrinsic) as well as caspase-independent mitochondrial death.

Investigation of protein-protein interactions has arisen as a comprehensive approach for understanding bacterial physiology as well as pathogenesis. From this point of view, bacterial membrane as a place of contact with outer milieu seems to be crucial compartment and therefore, investigation of lipophilic proteins and their interactions is inevitable. Unfortunately, several methods developed for the analysis of protein interaction suffer from their labour intensiveness and underrepresent integral membrane proteins. Therefore, blue native polyacrylamide gel electrophoresis (BN-PAGE) with its simplicity and suitability for lipophilic entities has been widely employed in microbiological research. For investigation of membrane proteins interactions BN-PAGE became a method of choice. The efficacy in this area was proven by the elucidation not only of the stoichiometry, but also dynamic changes of several complexes involved in energetic metabolism, secretion and transport systems, localized both in inner as well as in outer membrane. Moreover, BN-PAGE was also successfully applied on peripheral membrane and cytoplasmic proteins and enabled complex analysis of interactomes of several microorganisms. This review shows BN-PAGE as a potent tool in microbiological fundamental research ranging from Archaea, through Gram-positive and Gram-negative bacteria to Chlamydia.

Francisella tularensis is a highly infectious facultative intracellular bacterium and aetiological agent of tularaemia. The conserved hypothetical lipoprotein with homology to thiol/disulphide oxidoreductase proteins (FtDsbA) is an essential virulence factor in F. tularensis. Its protein sequence has two different domains: the DsbA_Com1_like domain (DSBA), with the highly conserved catalytically active site CXXC and cis-proline residue; and the domain amino-terminal to FKBP-type peptidyl-prolyl isomerases (FKBP_N). To establish the role of both domains in tularaemia infection models, site-directed and deletion mutagenesis affecting the active site (AXXA), the cis-proline (P286T) and the FKBP_N domain (ΔFKBP_N) were performed. The generated mutations led to high attenuation with the ability to induce full or partial host protective immunity. Recombinant protein analysis revealed that the active site CXXC as well as the cis-proline residue and the FKBP_N domain are necessary for correct thiol/disulphide oxidoreductase activity. By contrast, only the DSBA domain (and not the FKBP_N domain) seems to be responsible for the in vitro chaperone activity of the FtDsbA protein.

The field of microbial proteomics has currently experienced a boom in the discovery of glycosylated proteins of various pathogenic bacteria as potential mediators of host-pathogen interactions. The presence of glycoproteins has recently been discovered in a Gram-negative pathogenic bacterium Francisella tularensis, utilizing glycoprotein detection and isolation techniques in combination with mass spectrometry. The isolation of glycoproteins is a prerequisite for their subsequent mass-spectrometric identification. Current glycoprotein isolation/enrichment methods comprise lectin affinity chromatography, aminophenylboronic acid and hydrazide-based enrichment. The use of magnetic microspheres containing functional groups is nowadays among state-of-art separation methodologies owing to an ease of manipulation, a speed of separation, and a minimum of non-specific protein adsorption. In the present study, novel magnetic hydrazide-modified poly(2-hydroxyethyl methacrylate) (PHEMA) microspheres were developed using a multi-step swelling and polymerization method with subsequent precipitation of magnetic iron oxides within the pores of the particles. The microspheres had a regular shape, size of 4 μm and contained 0.18 mmol hydrazide groups per g; the magnetic microspheres were employed for specific enrichment of Francisella tularensis glycoproteins. Effectiveness of the newly prepared magnetic microspheres for glycoprotein enrichment was proved by comparison with commercial hydrazide-functionalized microparticles.

Francisella tularensis, the causative agent of tularemia, is a highly infectious intracellular pathogen with no licensed vaccine available today. The recent search for genome sequences involved in F. tularensis virulence mechanisms led to the identification of the 30-kb region defined as a Francisella pathogenicity island (FPI). In our previous iTRAQ study we described the concerted upregulation of some FPI proteins in different F. tularensis strains cultivated under stress conditions. Among them we identified the IglH protein whose role in Francisella virulence has not been characterized yet. In this work we deleted the iglH gene in a European clinical isolate of F. tularensis subsp. holarctica FSC200. We showed that the iglH gene is necessary for intracellular growth and escape of F. tularensis from phagosomes. We also showed that the iglH mutant is avirulent in a mouse model of infection and persists in the organs for about three weeks after infection. Importantly, mice vaccinated by infection with the iglH mutant were protected against subcutaneous challenge with the fully virulent parental FSC200 strain. This is the first report of a defined subsp. holarctica FPI deletion strain that provides protective immunity against subsequent subcutaneous challenge with a virulent isolate of F. tularensis subsp. holarctica.

The impact of Lps gene on the course of immune response against subcutaneous infection of mice with Francisella tularensis live vaccine strain was studied. Production and specificity of antibodies, cytotoxic responses of macrophages and NK-cells, spontaneous production ex vivo of cytokines IL-1α, IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α in spleen cell cultures in C3H/HeJ (Lpsd) mice in comparison with C3H/HeN (Lpsn) mice were tested. The value of LD50 was significantly different in the two strains of mice (8.0 × 103 cfu for C3H/HeJ versus 4.61 × 105 cfu for C3H/HeN mice after subcutaneous inoculation). The production of NO2 is also impaired in C3H/HeJ mice in the early intervals after infection. Thus, the defective Lps gene of C3H/HeJ mice influences both the level of innate resistance of mice to F. tularensis live vaccine strain infection and the process of induction and regulation of immune response against this intracellular bacterial pathogen.

Abstract Proteome analysis of Gram‐negative facultative intracellular pathogen Francisella tularensis ( F. tularensis ) live vaccine strain has been performed only on whole‐cell extracts so far. This is the first study dealing with the analysis of the membrane subproteome of this microorganism. A fraction enriched in membrane proteins obtained by carbonate extraction was separated using two‐dimensional electrophoresis and all visualized spots were identified by mass spectrometry. The reference map is the basis for further comparative analyses of virulent and non‐virulent F. tularensis strains.

Immunoproteomic analysis was applied to study the immunoreactivity of serum samples collected at different time points from a laboratory assistant accidentally infected with highly virulent strain of Francisella tularensis subsp. tularensis. Immunoblotting showed that the spectrum of F. tularensis antigens recognized specifically by immune sera remained with the exception for 1 antigen stable for up to 16 years after infection. Using immunoproteomics approach 10 immunoreactive antigens were successfully identified. Several new immunogenic F. tularensis proteins were described for the first time.

Abstract Proteomics has been shown to significantly contribute to the investigation of the pathogenicity of the extremely infectious bacteria Francisella tularensis . In this study, the authors employed iTRAQ quantitative proteomic analysis in order to monitor alterations in proteomes of F. tularensis ssp. holarctica live vaccine strain and F. tularensis ssp. tularensis SCHU S4 associated with the cultivation at different temperatures or in the stationary phase. Correlated production of the identified proteins studied by the exploratory statistical analysis revealed novel candidates for virulence factors that were regulated in a similar manner to the genes encoded in the Francisella Pathogenicity Island. Moreover, the assessment of the adaptation of live vaccine strain and SCHU S4 strain to the examined stimuli uncovered differences in their physiological responses to the stationary phase of growth.

The study of membrane proteins and membrane protein complexes (MPC) provides crucial information in the field of bacterial physiology and pathogenesis research. The method of blue native polyacrylamide gel electrophoresis and its combination with SDS-PAGE (BN/SDS-PAGE) were here employed to study the membrane complexome of an intracellular bacterium Francisella tularensis, the causative agent of a severe disease tularemia. In the presented study we describe the subunit composition and stoichiometry of several MPC involved in various cell functions (oxidative phosphorylation, membrane transport, cell division, membrane or periplasmic proteins folding, iron storage, phospholipid and cell envelope biosynthesis). Moreover, some undocumented or hypothetical MPC with possible connection to virulence factors were also proposed and some newly detected subunits were assigned to known complexes. The BN/SDS-PAGE combined with mass spectrometry appeared to be a strong tool in the investigation of membrane proteins and complexes and thus successfully complements the traditional electrophoresis approaches.

Dendritic cells (DCs) infected by <i>Francisella tularensis</i> are poorly activated and do not undergo classical maturation process. Although reasons of such unresponsiveness are not fully understood, their impact on the priming of immunity is well appreciated. Previous attempts to explain the behavior of <i>Francisella</i>-infected DCs were hypothesis-driven and focused on events at later stages of infection. Here, we took an alternative unbiased approach by applying methods of global phosphoproteomics to analyze the dynamics of cell signaling in primary DCs during the first hour of infection by <i>Francisella tularensis</i>. Presented results show that the early response of DCs to <i>Francisella</i> occurs in phases and that ERK and p38 signaling modules induced at the later stage are differentially regulated by virulent and attenuated Δ<i>dsbA</i> strain. These findings imply that the temporal orchestration of host proinflammatory pathways represents the integral part of <i>Francisella</i> life-cycle inside hijacked DCs.

The initiation of T-cell signaling is critically dependent on the function of the member of Src family tyrosine kinases (SFKs), Lck. Upon TCR triggering, Lck kinase activity induces the nucleation of signal-transducing hubs that regulate the formation of complex signaling network and cytoskeletal rearrangement. In addition, the delivery of Lck function requires rapid and targeted membrane redistribution, but the mechanism underpinning this process is largely unknown. To gain insight into this process, we considered previously described proteins that could assist in this process via their capacity to interact with kinases and regulate their intracellular translocations. An adaptor protein, Receptor for Activated C Kinase 1 (RACK1), was chosen as a viable option and its capacity to bind Lck and aid the process of activation-induced redistribution of Lck was assessed. Our microscopic observation showed that T-cell activation induces a rapid, concomitant and transient co-redistribution of Lck and RACK1 into the forming immunological synapse. Consistent with this observation, the formation of transient RACK1-Lck complexes were detectable in primary CD4+ T-cells with their maximum levels peaking 10 seconds after TCR-CD4 co-aggregation. Moreover, RACK1 preferentially binds to a pool of kinase active pY394Lck which co-purifies with high molecular weight cellular fractions. The formation of RACK1-Lck complexes depends on functional SH2 and SH3 domains of Lck and includes several other signaling and cytoskeletal elements that transiently bind the complex. Notably, the F-actin-crosslinking protein, α-actinin-1, binds to RACK1 only in the presence of kinase active Lck suggesting that the formation of RACK1-pY394Lck-α-actinin-1 complex serves as a signal module coupling actin cytoskeleton bundling with productive TCR/CD4 triggering. In addition, the treatment of CD4+ T-cells with nocodazole, which disrupts the microtubular network, also blocked the formation of RACK1-Lck complexes. Importantly, activation-induced Lck redistribution was diminished in primary CD4+ T-cells by an adenoviral-mediated knock-down of RACK1. These results demonstrate that in T cells, RACK1 as an essential component of the multiprotein complex which upon TCR engagement links the binding of kinase active Lck to elements of the cytoskeletal network and affects the subcellular redistribution of Lck.

Francisella tularensis is a highly infectious Gram-negative coccobacillus which causes the disease tularemia. The potential for its misuse as a biological weapon has led disease control and prevention centers to classify this bacterium as a category A agent. Bacterial outer membrane vesicles (OMVs) are spherical particles 20-250 nm in size produced by all Gram-negative bacteria and constitute one of the major secretory pathways. Bacteria use them in interacting with both other bacterial cells and eukaryotic (host) cells. OMVs of Francisella contain number of its so far described virulence factors and immunomodulatory proteins. Their role in host-pathogen interactions can therefore be presumed, and the possibility exists also for their potential use in a subunit vaccine. Moreover, Francisella microbes produce both usual spherical and unusual tubular OMVs. Because OMVs emerge from the outermost surface of the bacterial cell, we focused on the secretion of OMVs in several mutant Francisella strains with disrupted surface structures (namely the O-antigen). O-antigen in Francisella is not only the structural component of LPS but also forms another important virulence factor: the O-antigen polysaccharide capsule. Mutant strain phenotypes were evaluated by growth curves, vesiculation rates, their sensitivity to the complement contained in serum, and proliferation inside murine bone marrow macrophages. Morphologies of both OMVs and the bacteria were visualized by electron microscopy. The O-antigen mutant strains were considerably attenuated in serum resistance and intracellular proliferation. All the strains showed lower ability to form the tubular OMVs. Some strains formed tubular protrusions from their outer membrane but their stability was weak. Some hypervesiculating strains were revealed that will serve as source of OMVs for further studies of their protective potential. Our results suggest the presence of LPS and the O-antigen capsule on the surface of Francisella to be critical not only for its virulence but also for the exceptional tubular shape of its OMVs.

Francisella tularensis secretes tubular outer membrane vesicles (OMVs) that contain a number of immunoreactive proteins as well as virulence factors. We have reported previously that isolated Francisella OMVs enter macrophages, cumulate inside, and induce a strong pro-inflammatory response. In the current article, we present that OMVs treatment of macrophages also enhances phagocytosis of the bacteria and suppresses their intracellular replication. On the other hand, the subsequent infection with Francisella is able to revert to some extent the strong pro-inflammatory effect induced by OMVs in macrophages. Being derived from the bacterial surface, isolated OMVs may be considered a "non-viable mixture of Francisella antigens" and as such, they present a promising protective material. Immunization of mice with OMVs isolated from a virulent F. tularensis subsp. holarctica strain FSC200 prolonged the survival time but did not fully protect against the infection with a lethal dose of the parent strain. However, the sera of the immunized animals revealed unambiguous cytokine and antibody responses and proved to recognize a set of well-known Francisella immunoreactive proteins. For these reasons, Francisella OMVs present an interesting material for future protective studies.

The role of antibodies in the course of Francisella tularensis (F. tularensis) infection is still a subject of debate. The understanding of the poorly described role of humoral immunity is more than important for the effort to develop effective prophylactic procedure against the infection with Francisella virulent strains. We utilized the model of gamma-irradiated mice for the studies of the protective role of anti-F. tularensis antibodies in order to partially eliminate cellular responses. The model of gamma-irradiated mice can also demonstrate the responses of immunocompromised host to intracellular bacterial infection. The gamma-irradiation by doses greater than 3 Gy completely impairs the resistance to infection and causes a disbalance of cytokine production in mice. In this study, we demonstrate that passive transfer of immune sera protected irradiated mice against subsequent infection with strains of F. tularensis subsp. holarctica. Naïve mice of BALB/c or C3H/CBi strains were subjected to passive transfer of sera obtained from immunized mice with live vaccine strain (LVS) F. tularensis LVS, F. tularensis subsp. holarctica strain 15, heat-killed F. tularensis LVS, or heat-killed strain 15 two hours before infection with lethal doses of LVS or strain 15. The passive transfer of sera obtained from immunized mice conferred full protection of naïve unirradiated as well as sublethally irradiated mice against low lethal doses of infection with F. tularensis LVS or strain 15, in all variants of the experiments. In addition, the passively protected mice that survived the primary infection with F. tularensis LVS were protected also against further secondary challenge with a highly virulent strain of F. tularensis subsp. tularensis SchuS4. Moreover, the first evidence of combination of successful passive transfer of immunity by specific antisera and subsequent active immunization of immunocompromised animals is demonstrated. In summary, we demonstrate that B cell-mediated effector responses together with the induction of T cell-mediated immunity both play an important role in naïve and also in immunocompromised mice and this fact it would be appropriate to take into the account in the design of new vaccines.

This article refers to:A mutagenesis-based approach identifies amino acids in the N-terminal part of Francisella tularensis IglE that critically control Type VI system-mediated secretion

The DsbA homolog of Francisella tularensis was previously demonstrated to be required for intracellular replication and animal death. Disruption of the dsbA gene leads to a pleiotropic phenotype that could indirectly affect a number of different cellular pathways. To reveal the broad effects of DsbA, we compared fractions enriched in membrane proteins of the wild-type FSC200 strain with the dsbA deletion strain using a SILAC-based quantitative proteomic analysis. This analysis enabled identification of 63 proteins with significantly altered amounts in the dsbA mutant strain compared to the wild-type strain. These proteins comprise a quite heterogeneous group including hypothetical proteins, proteins associated with membrane structures, and potential secreted proteins. Many of them are known to be associated with F. tularensis virulence. Several proteins were selected for further studies focused on their potential role in tularemia's pathogenesis. Of them, only the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme of glycolytic pathway, was found to be important for full virulence manifestations both in vivo and in vitro. We next created a viable mutant strain with deleted gapA gene and analyzed its phenotype. The gapA mutant is characterized by reduced virulence in mice, defective replication inside macrophages, and its ability to induce a protective immune response against systemic challenge with parental wild-type strain. We also demonstrate the multiple localization sites of this protein: In addition to within the cytosol, it was found on the cell surface, outside the cells, and in the culture medium. Recombinant GapA was successfully obtained, and it was shown that it binds host extracellular serum proteins like plasminogen, fibrinogen, and fibronectin.

Francisella tularensis subspecies tularensis is a highly virulent intracellular bacterial pathogen, causing the disease tularemia. However, a safe and effective vaccine for routine application against F. tularensis has not yet been developed. We have recently constructed the deletion mutants for the DsbA homolog protein (ΔdsbA/FSC200) and a hypothetical protein IglH (ΔiglH/FSC200) in the type B F. tularensis subsp. holarctica FSC200 strain, which exerted different protection capacity against parental virulent strain. In this study, we further investigated the immunological correlates for these different levels of protection provided by ΔdsbA/FSC200 and ΔiglH/FSC200 mutants. Our results show that ΔdsbA/FSC200 mutant, but not ΔiglH/FSC200 mutant, induces an early innate inflammatory response leading to strong Th1-like antibody response. Furthermore, vaccination with ΔdsbA/FSC200 mutant, but not with ΔiglH/FSC200, elicited protection against the subsequent challenge with type A SCHU S4 strain in mice. An immunoproteomic approach was used to map a spectrum of antigens targeted by Th1-like specific antibodies, and more than 80 bacterial antigens, including novel ones, were identified. Comparison of tularemic antigens recognized by the ΔdsbA/FSC200 post-vaccination and the SCHU S4 post-challenge sera then revealed the existence of 22 novel SCHU S4 specific antibody clones.

Due to their cardiac origin, H9c2 cells rank among the most popular cell lines in current cardiovascular research, yet molecular phenotype remains elusive. Hence, in this study we used proteomic approach to describe molecular phenotype of H9c2 cells in their undifferentiated (i.e., most frequently used) state, and its functional response to cardiotoxic drug doxorubicin. Of 1671 proteins identified by iTRAQ IEF/LC–MSMS analysis, only 12 proteins were characteristic for striated muscle cells and none was cardiac phenotype-specific. Targeted LC-SRM and western blot analyses confirmed that undifferentiated H9c2 cells are phenotypically considerably different to both primary neonatal cardiomyocytes and adult myocardium. These cells lack proteins essential for formation of striated muscle myofibrils or they express only minor amounts thereof. They also fail to express many proteins important for metabolism of muscle cells. The challenge with clinically relevant concentrations of doxorubicin did not induce a proteomic signature that has been previously noted in primary cardiomyocytes or adult hearts. Instead, several alterations previously described in other cells of mesodermal origin, such as fibroblasts, were observed (e.g., severe down-regulation of collagen synthesis pathway). In conclusion, the molecular phenotype of H9c2 cells resembles very immature myogenic cells with skeletal muscle commitment upon differentiation and thus, translatability of findings obtained in these cells deserves caution.

A bstract : The whole cell lysate of Coxiella burnetii strain RSA 493 was separated by two‐dimensional electrophoresis and more than 500 protein spots were found on silver‐stained reference map. Spots from the gels were subjected to identification based on peptide mass fingerprinting (PMF). In order to identify additional proteins, tandem mass spectrometry (MS/MS) using electrospray and matrix‐assisted laser desorption/ionization techniques was applied. The three independent approaches resulted in the identification of 197 open reading frames (ORFs). Fifty‐two proteins were identified by PMF and at least with one of the MS/MS methods, 37 proteins with both MS/MS instruments, and 19 proteins with all three techniques applied. All predicted C. burnetii ORFs were compared with the Clusters of Orthologous Groups database. The data related to identified proteins were stored and indexed in a file that can be read and searched using Microsoft Access.

Purpose: To determine whether radiation‐induced changes in protein abundance can be correlated with their differential gene expression in a murine fibroblast L929 cell line.Materials and methods: L929 cells were irradiated with 6 Gy. Cell lysates were collected at different points in time (20 min, 12, 24, 36, 48 and 72 h). The extracted proteins were separated by two‐dimensional gel electrophoresis and quantified using computerized image analysis. Proteins exhibiting a differential expression equal to or more than twofold were identified by mass spectrometry following trypsin digestion. From these, 10 proteins characterized by large changes of radiation‐induced abundance were selected in order to measure their corresponding gene expression using RTQ‐PCR (real‐time quantitative polymerase chain reaction).Results: Up to 15‐fold changes in the abundance of these 10 proteins were associated with no detectable changes more than twofold on the gene expression level. However, one gene (VEGF‐D) showed a significant (p=0.005) up‐regulation (1.8‐fold).Conclusions: Deducing protein abundance from mRNA expression levels and vice versa appears to be of limited use. Furthermore, examination of transcriptional and translational changes provides different but complementary information.

Dendritic cells (DCs) serve as the primers of adaptive immunity, which is indispensable for the control of the majority of infections. Interestingly, some pathogenic intracellular bacteria can subvert DC function and gain the advantage of an ineffective host immune reaction. This scenario appears to be the case particularly with so-called stealth pathogens, which are the causative agents of several under-diagnosed chronic diseases. However, there is no consensus how less explored stealth bacteria like Coxiella, Brucella and Francisella cross-talk with DCs. Therefore, the aim of this review was to explore the issue and to summarize the current knowledge regarding the interaction of above mentioned pathogens with DCs as crucial hosts from an infection strategy view. Evidence indicates that infected DCs are not sufficiently activated, do not undergo maturation and do not produce expected proinflammatory cytokines. In some cases, the infected DCs even display immunosuppressive behaviour that may be directly linked to the induction of tolerogenicity favouring pathogen survival and persistence.

The success of pathogens depends on their ability to circumvent immune defences. Francisella tularensis is one of the most infectious bacteria known. The remarkable virulence of Francisella is believed to be due to its capacity to evade or subvert the immune system, but how remains obscure. Here, we show that Francisella triggers but concomitantly inhibits the Toll-like receptor, RIG-I-like receptor, and cytoplasmic DNA pathways. Francisella subverts these pathways at least in part by inhibiting K63-linked polyubiquitination and assembly of TRAF6 and TRAF3 complexes that control the transcriptional responses of pattern recognition receptors. We show that this mode of inhibition requires a functional type VI secretion system and/or the presence of live bacteria in the cytoplasm. The ability of Francisella to enter the cytosol while simultaneously inhibiting multiple pattern recognition receptor pathways may account for the notable capacity of this bacterium to invade and proliferate in the host without evoking a self-limiting innate immune response.

In-depth proteome discovery analysis represents new strategy in an effort to identify novel reliable specific protein markers for hypertrophic cardiomyopathy and other life threatening cardiovascular diseases. To systematically identify novel protein biomarkers of cardiovascular diseases with high mortality we employed an isobaric tag for relative and absolute quantitation (iTRAQ) proteome technology to make comparative analysis of plasma samples obtained from patients suffering from non-obstructive hypertrophic cardiomyopathy, stable dilated cardiomyopathy, aortic valve stenosis, chronic stable coronary artery disease and stable arterial hypertension. We found 128 plasma proteins whose abundances were uniquely regulated among the analyzed cardiovascular pathologies. 49 of them have not been described yet. Additionally, application of statistical exploratory analyses of the measured protein profiles indicated the relationship in pathophysiology of the examined cardiovascular pathologies.

D-alanyl-D-alanine carboxypeptidase, product of dacD gene in Francisella, belongs to penicillin binding proteins (PBPs) and is involved in remodeling of newly synthetized peptidoglycan. In E. coli, PBPs are synthetized in various growth phases and they are able to substitute each other to a certain extent. The DacD protein was found to be accumulated in fraction enriched in membrane proteins from severely attenuated dsbA deletion mutant strain. It has been presumed that the DsbA is not a virulence factor by itself but that its substrates, whose correct folding and topology are dependent on the DsbA oxidoreductase and/or isomerase activities, are the primary virulence factors. Here we demonstrate that Francisella DacD is required for intracellular replication and virulence in mice. The dacD insertion mutant strain showed higher sensitivity to acidic pH, high temperature and high osmolarity when compared to the wild-type. Eventually, transmission electron microscopy revealed differences in mutant bacteria in both the size and defects in outer membrane underlying its SDS and serum sensitivity. Taken together these results suggest DacD plays an important role in Francisella pathogenicity.

Francisella tularensis, an intracellular pathogen causing the disease tularemia, utilizes surface glycoconjugates such as lipopolysaccharide, capsule, and capsule-like complex for its protection against inhospitable conditions of the environment. Francisella species also possess a functional glycosylation apparatus by which specific proteins are O-glycosidically modified. We here created a mutant with a nonfunctional FTS_1402 gene encoding for a putative glycan flippase and studied the consequences of its disruption. The mutant strain expressed diminished glycosylation similarly to, but to a lesser extent than, that of the oligosaccharyltransferase-deficient ΔpglA mutant. In contrast to ΔpglA, inactivation of FTS_1402 had a pleiotropic effect, leading to alteration in glycosylation and, importantly, to decrease in lipopolysaccharide, capsule, and/or capsule-like complex production, which were reflected by distinct phenotypes in host–pathogen associated properties and virulence potential of the two mutant strains. Disruption of FTS_1402 resulted in enhanced sensitivity to complement-mediated lysis and reduced virulence in mice that was independent of diminished glycosylation. Importantly, the mutant strain induced a protective immune response against systemic challenge with homologous wild-type FSC200 strain. Targeted disruption of genes shared by multiple metabolic pathways may be considered a novel strategy for constructing effective live, attenuated vaccines.

Subtractive two-dimensional gel electrophoresis (2-DE) has been used for the study of the protein patterns of the normal colonic mucosa and the specimens collected from patients diagnosed for inflammatory bowel disease (IBD), colonic polyps and colorectal cancer. We found a 13 kDa protein that was detected in five of seven adenomas and in 13 of 15 colorectal carcinomas while it was absent or only slightly expressed in normal colonic mucosa. Furthermore, this protein occured in all specimens collected from patients suffering from IBD and its quantity reflected the increased severity of inflammation. The combination of microsequencing and mass spectrometry led to the identification of the 13 kDa spot as calgranulin B. Our results indicate that the production of calgranulin B is unregulated in inflammatory, preneoplastic and neoplastic lesions of colonic mucosa.

Analyses of the protein expression profiles of irradiated cells may be beneficial for identification of new biomolecules of radiation-induced cell damage. Therefore, in this study we exploited the proteomic approach to identify proteins whose expression is significantly altered in gamma-irradiated human T-lymphocyte leukemia cells. MOLT-4 cells were irradiated with 7.5 Gy and the cell lysates were collected at different times after irradiation (2, 5 and 12 h). The proteins were separated by two-dimensional electrophoresis and quantified using an image evaluation system. Proteins exhibiting significant radiation-induced alterations in abundance were identified by peptide mass fingerprinting. We identified 14 proteins that were either up- or down-regulated. Cellular levels of four of the proteins (Rho GDP dissociation inhibitor 1 and 2, Ran binding protein 1, serine/threonine protein kinase PAK2) were further analyzed by two-dimensional immunoblotting to confirm the data obtained from proteome analysis. All identified proteins were classified according to their cellular function, including their participation in biochemical and signaling pathways. Taken together, our results suggest the feasibility of the proteome method for monitoring of cellular radiation responses.

To confirm the initial iTRAQ-based proteomic findings related to the plasma fibronectin level and hypertrophic cardiomyopathy using an antibody-based assay.The iTRAQ technique was used for the discovery proteomic analysis of the pooled plasma samples. The ELISA was used for verification of fibronectin plasma concentration in individual samples. Additional five related plasma proteins were assessed: matrix metalloproteinase 2, matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 1, tissue inhibitor of metalloproteinase 2 and brain natriuretic peptide.The plasma fibronectin level in patients with hypertrophic cardiomyopathy was significantly lower in comparison to the healthy subjects [215.5±47.3μg/ml, n=17 vs. 376.7±134.8μg/ml, n=17; p<0.0001].In this study we present and confirm our initial proteomic findings and we suggest fibronectin as a potential indicator of an extracellular matrix remodeling related to the hypertrophic cardiomyopathy.

Regulation of gene transcription is the initial step in the complex process that controls gene expression within bacteria. Transcriptional control involves the joint effort of RNA polymerases and numerous other regulatory factors. Whether global or local, positive or negative, regulators play an essential role in the bacterial cell. For instance, some regulators specifically modify the transcription of virulence genes, thereby being indispensable to pathogenic bacteria. Here, we provide a comprehensive overview of important transcription factors and DNA-binding proteins described for the virulent bacterium Francisella tularensis, the causative agent of tularemia. This is an unexplored research area, and the poorly described networks of transcription factors merit additional experimental studies to help elucidate the molecular mechanisms of pathogenesis in this bacterium, and how they contribute to disease.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known for its multifunctionality in several pathogenic bacteria. Our previously reported data suggest that the GAPDH homologue of Francisella tularensis, GapA, might also be involved in other processes beyond metabolism. In the present study, we explored GapA’s potential implication in pathogenic processes at the host cell level. Using immunoelectron microscopy, we demonstrated the localization of this bacterial protein inside infected macrophages and its peripheral distribution in bacterial cells increasing with infection time. A quantitative proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) combined with pull-down assay enabled the identification of several of GapA’s potential interacting partners within the host cell proteome. Two of these partners were further confirmed by alternative methods. We also investigated the impact of gapA deletion on the transcription of selected cytokine genes and the activation of the main signaling pathways. Our results show that ∆gapA-induced transcription of genes encoding several cytokines whose expressions were not affected in cells infected with a fully virulent wild-type strain. That might be caused, at least in part, by the detected differences in ERK/MAPK signaling activation. The experimental observations together demonstrate that the F. tularensis GAPDH homologue is directly implicated in multiple host cellular processes and, thereby, that it participates in several molecular mechanisms of pathogenesis.

Using two-dimensional electrophoresis we investigated the effect of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT; induction with 1 mM ALA for 4 h followed by blue light dose of 18 J/cm2) on the protein expression in HL60 leukemia cells. ALA-PDT resulted in extensive qualitative and quantitative changes in the protein pattern of HL60 cell lysates. Of more than 1350 protein spots recognized on the protein maps of ALA-induced cells, seven proteins were enhanced and 17 suppressed following irradiation. Three of these, calreticulin precursor, p58 microsomal protein (ERp57) and protein disulfide isomerase (p55) have been identified by matrix-assisted laser desorption and ionization-mass spectrometry and the pI/molecular weight parameters of the affected proteins were estimated by computer analysis. The findings suggest participation of endoplasmic reticulum Ca(2+)-binding chaperones and/or Ca2+ signaling in ALA-PDT mediated cytotoxicity.

The ovaries of the common wasp, Vespula germanica are polytrophic-meroistic and consist of 2-3 (workers) or 7 (queens) ovarioles. The ovarioles are differentiated into three regions: a terminal filament, a germarium, and a vitellarium. The germaria of both castes consist of two zones: an anterior zone of germ-cell cluster formation and a posterior one of germ-cell cluster differentiation. The vitellaria comprise 4-6 (workers) or 7-10 (queens) ovarian follicles (egg chambers). Each chamber consists of an oocyte and about 60 isodiametric nurse cells (trophocytes). The egg chambers have been arbitrarily classified into four developmental categories: early and late previtellogenic, vitellogenic, and choriogenic. The process of oogenesis in workers proceeds only up to the onset of the late previtellogenesis. Neither vitellogenic nor choriogenic egg chambers were observed in this caste. During early and late previtellogenesis the envelope of the oocyte nucleus proliferates and becomes highly folded. This process leads to the formation of characteristic organelles, termed accessory nuclei (AN). Although AN arise in the oocytes of both queens and workers, their number in the latter caste is always considerably lower. At the onset of the late previtellogenesis AN start to migrate towards the periphery of the oocyte where they reside till the end of oogenesis. The physiological state of the worker ovaries is discussed in the light of the presented results.

Protein concentration measurement in the urine can be problematic in the presence of Bence Jones protein. We have carried out an external quality control assessment with the participation of 79 clinical biochemistry laboratories from the Czech Republic and Slovakia.The laboratories received a reference urine sample obtained from a patient with multiple myeloma and lambda free light chain proteinuria and were asked to type the paraprotein using immunofixation and to measure total urinary protein using their established method, most commonly turbidimetry, pyrogallol red assay, and biuret assay.There was a very wide inter-laboratory variability in the protein concentration readouts with up to three-fold difference in some cases. High-resolution two-dimensional electrophoresis and linear mass spectrometry showed that a high proportion of the urinary paraprotein was composed of lambda light chain fragments with molecular weight of 12kDa.Our results highlight the challenges of reliable and reproducible measurement of urinary protein concentration in the presence of Bence Jones protein.

Francisella tularensis is a highly infectious intracellular pathogen that has evolved an efficient strategy to subvert host defense response to survive inside the host. The molecular mechanisms regulating these host–pathogen interactions and especially those that are initiated at the time of the bacterial entry via its attachment to the host plasma membrane likely predetermine the intracellular fate of pathogen. Here, we provide the evidence that infection of macrophages with F. tularensis leads to changes in protein composition of macrophage-derived lipid rafts, isolated as detergent-resistant membranes (DRMs). Using SILAC-based quantitative proteomic approach, we observed the accumulation of autophagic adaptor protein p62 at the early stages of microbe–host cell interaction. We confirmed the colocalization of the p62 with ubiquitinated and LC3-decorated intracellular F. tularensis microbes with its maximum at 1 h postinfection. Furthermore, the infection of p62-knockdown host cells led to the transient increase in the intracellular number of microbes up to 4 h after in vitro infection. Together, these data suggest that the activation of the autophagy pathway in F. tularensis infected macrophages, which impacts the early phase of microbial proliferation, is subsequently circumvented by ongoing infection.

The adenylate cyclase toxin (CyaA) of the whooping cough agent Bordetella pertussis subverts immune functions of host myeloid cells expressing the αMβ2 integrin (CD11b/CD18, CR3 or Mac-1). CyaA delivers into cytosol of cells an extremely catalytically active adenylyl cyclase enzyme, which disrupts the innate and adaptive immune functions of phagocytes through unregulated production of the key signaling molecule cAMP. We have used phosphoproteomics to analyze cAMP signaling of CyaA in murine bone marrow-derived dendritic cells. CyaA action resulted in alterations of phosphorylation state of a number of proteins that regulate actin cytoskeleton homeostasis, including Mena, Talin-1 and VASP. CyaA action repressed mTOR signaling through activation of mTORC1 inhibitors TSC2 and PRAS40 and altered phosphorylation of multiple chromatin remodelers, including the class II histone deacetylase HDAC5. CyaA toxin action further elicited inhibitory phosphorylation of SIK family kinases involved in modulation of immune response and provoked dephosphorylation of the transcriptional coactivator CRTC3, indicating that CyaA-promoted nuclear translocation of CRTC3 may account for CyaA-induced IL-10 production. These findings document the complexity of subversive physiological manipulation of myeloid phagocytes by the CyaA toxin, serving in immune evasion of the pertussis agent.

Natural resistance to Mycobacterium bovis bacillus Calmette-Guérin (BCG) is determined by the Bcg gene (Nramp1), which is exclusively expressed by mature macrophages. The Nramp1 gene is a dominant autosomal gene that has two allelic forms; r confers resistance and s confers susceptibility to infection with intracellular pathogen. Although the wide range of pleiotropic immunological effects of the Nramp1 gene has been described, the exact mechanism of its action remains elusive. In this study we searched for differentially expressed proteins that might provide clues in the studies on Nramp1 gene function. We performed two-dimensional gel electrophoresis of cellular proteins prepared from a B10R macrophage line derived from mice carrying the r allele of the Nramp1 gene, B10S macrophages carrying the s allele, and B10R-Rb macrophages transfected with Nramp1-ribozyme. The classification of protein patterns and selection of distinct proteins characteristic of r or s allele-carrying macrophages was performed using the principal component analysis. We found differential expression of four proteins with the following isoelectric point/molecular weight (pI/Mr) in B10R macrophages compared to B10S and B10R-Rb macrophages: 6.6/25, 7.0/22, 9.1/31.5, and 5.3/8.5. The protein 7.0/22 has been identified as Mn-superoxide dismutase and the best candidate for protein p6.6/25 seems to be Bcl-2 according to the immunoblot analysis. When the splenic macrophages carrying the r or s allele were analyzed, the changes in relative abundance for proteins 6.6/25 and p7.0/22 were satisfactorily reproduced. Overall, the two identified proteins are important in the regulation of intracellular redox balance and the regulation of apoptosis in macrophages, respectively. Our findings may suggest their possible biological role in the innate immunity against intracellular pathogens.

Although dendritic cells (DCs) control the priming of the adaptive immunity response, a comprehensive description of their behavior at the protein level is missing. The introduction of the quantitative proteomic technique of metabolic labeling (SILAC) into the field of DC research would therefore be highly beneficial. To achieve this, we applied SILAC labeling to primary bone marow-derived DCs (BMDCs). These cells combine both biological relevance and experimental feasibility, as their in vitro generation permits the use of 13C/15N-labeled amino acids. Interestingly, BMDCs appear to exhibit a very active arginine metabolism. Using standard cultivation conditions, ∼20% of all protein-incorporated proline was a byproduct of heavy arginine degradation. In addition, the dissipation of 15N from labeled arginine to the whole proteome was observed. The latter decreased the mass accuracy in MS and affected the natural isotopic distribution of peptides. SILAC-connected metabolic issues were shown to be enhanced by GM-CSF, which is used for the differentiation of DC progenitors. Modifications of the cultivation procedure suppressed the arginine-related effects, yielding cells with a proteome labeling efficiency of ≥90%. Importantly, BMDCs generated according to the new cultivation protocol preserved their resemblance to inflammatory DCs in vivo, as evidenced by their response to LPS treatment.

The posttranscriptional regulatory protein Hfq was shown to be an important determinant of the stress resistance and full virulence in the dangerous human pathogen Francisella tularensis. Transcriptomics brought rather limited clues to the precise contribution of Hfq in virulence. To reveal the molecular basis of the attenuation caused by hfq inactivation, we employed iTRAQ in the present study and compared proteomes of the parent and isogenic Δhfq strains. We show that Hfq modulates the level of 76 proteins. Most of them show decreased abundance in the ∆hfq mutant, thereby indicating that Hfq widely acts rather as a positive regulator of Francisella gene expression. Several key Francisella virulence factors including those encoded within the Francisella pathogenicity island were found among the downregulated proteins, which is in a good agreement with the attenuated phenotype of the Δhfq strain. To further validate the iTRAQ exploratory findings, we subsequently performed targeted LC-SRM analysis of selected proteins. This accurate quantification method corroborated the trends found in the iTRAQ data.

Background . Hypertrophic cardiomyopathy (HCM) is mostly autosomal dominant disease of the myocardium, which is characterized by myocardial hypertrophy. Vascular endothelial growth factor (VEGF) is involved in myocyte function, growth, and survival. The aim of study was to analyze the clinical significance of VEGF in structural and functional changes in patient with HCM. Methods . In a group of 21 patients with nonobstructive HCM, we assessed serum VEGF and analyzed its association with morphological and functional parameters. Compared to healthy controls, serum VEGF was increased: 199 (IQR: 120.4–260.8) ng/L versus 20 (IQR: 14.8–37.7) ng/L, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M1"><mml:mi>P</mml:mi><mml:mo>&lt;</mml:mo><mml:mn fontstyle="italic">0.001</mml:mn></mml:math>. VEGF levels were associated with left atrium diameter (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M2"><mml:mi>r</mml:mi><mml:mo>=</mml:mo><mml:mn fontstyle="italic">0.51</mml:mn></mml:math>, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M3"><mml:mi>P</mml:mi><mml:mo>=</mml:mo><mml:mn fontstyle="italic">0.01</mml:mn></mml:math>), left ventricle ejection fraction (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M4"><mml:mi>r</mml:mi><mml:mo>=</mml:mo><mml:mo>-</mml:mo><mml:mn fontstyle="italic">0.56</mml:mn></mml:math>, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M5"><mml:mi>P</mml:mi><mml:mo>=</mml:mo><mml:mn fontstyle="italic">0.01</mml:mn></mml:math>), fractional shortening (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M6"><mml:mi>r</mml:mi><mml:mo>=</mml:mo><mml:mo>-</mml:mo><mml:mn fontstyle="italic">0.54</mml:mn></mml:math>, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M7"><mml:mi>P</mml:mi><mml:mo>=</mml:mo><mml:mn fontstyle="italic">0.02</mml:mn></mml:math>), left ventricular mass (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M8"><mml:mi>r</mml:mi><mml:mo>=</mml:mo><mml:mn fontstyle="italic">0.61</mml:mn></mml:math>, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M9"><mml:mi>P</mml:mi><mml:mo>=</mml:mo><mml:mn fontstyle="italic">0.03</mml:mn></mml:math>), LV mass index (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M10"><mml:mi>r</mml:mi><mml:mo>=</mml:mo><mml:mn fontstyle="italic">0.46</mml:mn></mml:math>, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M11"><mml:mi>P</mml:mi><mml:mo>=</mml:mo><mml:mn fontstyle="italic">0.04</mml:mn></mml:math>), vena cava inferior diameter (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M12"><mml:mi>r</mml:mi><mml:mo>=</mml:mo><mml:mn fontstyle="italic">0.65</mml:mn></mml:math>, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M13"><mml:mi>P</mml:mi><mml:mo>=</mml:mo><mml:mn fontstyle="italic">0.01</mml:mn></mml:math>), and peak gradient of tricuspid regurgitation (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M14"><mml:mi>r</mml:mi><mml:mo>=</mml:mo><mml:mn fontstyle="italic">0.46</mml:mn></mml:math>, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M15"><mml:mi>P</mml:mi><mml:mo>=</mml:mo><mml:mn fontstyle="italic">0.03</mml:mn></mml:math>). Conclusions . Increased VEGF level is associated with structural and functional parameters in patients with HCM and serves as a potential tool for diagnostic process of these patients.

Tularemia, otherwise known as &#8220;rabbit fever&#8221;, is a zoonotic disease caused by a gram-negative intracellular bacterium - Francisella tularensis. The species is considered as a potential bioterrorism agent due to its high infectivity, the fact of being relatively easy to culture, the absence of human vaccine, and the potential for spreading through aerosol. In the Czech Republic, infection is usually caused by a tick bite, less frequently by a mosquito bite, direct contact with infected animals, or ingestion of contaminated water. The aim of this review is to provide a comprehensive view of tularemia, its diagnosis, clinical symptoms and treatment, along with the military perspective on a potential risk of F. tularensis to be misused as a biological weapon.

The induction, regulation and expression of protective immunity against Francisella tularensis LVS infection is dependent on the results of primary interaction between the cells of host's immunoregulatory system and the microbe. The early events, at least on the side of macrophages, are under the genetic control. To determine the impact of genes that might be involved in the control of resistance to Francisella tularensis LVS infection, we have used three different inbred strains of mice with increasing resistance to this infection in order C3H/HeJ (Lpsd), C3H/HeN (Lpsn"), and C57B1/10N (Lpsn"). The controlled production of IL-10, IFN-gamma, and TNF-alpha coupled with increased production of reactive oxygen metabolites during early phase of infection distinguished less susceptible C3H/HeN mice from their more susceptible cogenic C3H/HeJ counterparts. The enhancement of oxidative metabolism that appeared on day 5 after the infection of both C3H/HeN and C57B1/10N mice closely correlated with increasing resistance of these two strains of mice to Francisella tularensis LVS infection. These mice were also capable to reach the highest level of TNF-alpha on day 5 after the infection. At the same time interval, only C57B1/10N mice produced significantly enhanced level of nitric oxide. Overall, these parameters may suggest their possible biological role in early-phase resistance to Francisella tularensis LVS infection and their subsequent consequences for ultimate control of infection and its clearance.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is well known for its involvement in numerous non-metabolic processes inside mammalian cells. Alternative functions of prokaryotic GAPDH are mainly deduced from its extracellular localization ability to bind to selected host proteins. Data on its participation in intracellular bacterial processes are scarce as there has been to date only one study dealing with this issue. We previously have reported several points of evidence that the GAPDH homolog of Francisella tularensis GapA might also exert additional non-enzymatic functions. Following on from our earlier observations we decided to identify GapA's interacting partners within the bacterial proteome to explore its new roles at intracellular level. The quantitative proteomics approach based on stable isotope labeling of amino acids in cell culture (SILAC) in combination with affinity purification mass spectrometry enabled us to identify 18 proteins potentially interacting with GapA. Six of those interactions were further confirmed by alternative methods. Half of the identified proteins were involved in non-metabolic processes. Further analysis together with quantitative label-free comparative analysis of proteomes isolated from the wild-type strain strain with deleted gapA gene suggests that GapA is implicated in DNA repair processes. Absence of GapA promotes secretion of its most potent interaction partner the hypothetical protein with peptidase propeptide domain (PepSY) thereby indicating that it impacts on subcellular distribution of some proteins.

Francisella tularensis is a highly infectious bacterium that causes the potentially lethal disease tularemia. This extremely virulent bacterium is able to replicate in the cytosolic compartments of infected macrophages. To invade macrophages and to cope with their intracellular environment, Francisella requires multiple virulence factors, which are still being identified. Proteins containing tetratricopeptide repeat (TPR)-like domains seem to be promising targets to investigate, since these proteins have been reported to be directly involved in virulence-associated functions of bacterial pathogens. Here, we studied the role of the FTS_0201, FTS_0778, and FTS_1680 genes, which encode putative TPR-like proteins in Francisella tularensis subsp. holarctica FSC200. Mutants defective in protein expression were prepared by TargeTron insertion mutagenesis. We found that the locus FTS_1680 and its ortholog FTT_0166c in the highly virulent Francisella tularensis type A strain SchuS4 are required for proper intracellular replication, full virulence in mice, and heat stress tolerance. Additionally, the FTS_1680-encoded protein was identified as a membrane-associated protein required for full cytopathogenicity in macrophages. Our study thus identifies FTS_1680/FTT_0166c as a new virulence factor in Francisella tularensis.

Francisella tularensis the etiological agent of tularaemia is one of the most infectious human pathogen known. Our knowledge about its key virulence factors has increased recently but it still remains a lot to explore. One of the described essential virulence factors is membrane lipoprotein FTS_1067 (nomenclature of F. tularensis subsp. holarctica strain FSC200) with homology to the protein family of disulphide oxidoreductases DsbA. Lipoprotein consists of two different domains: the C-terminal DsbA_Com1-like domain (DSBA-like) and the N-terminal FKBP-type peptidyl-prolyl cis/trans isomerases (FKBP_N-like). To uncover the biological role of these domains, we created bacterial strain with deletion of the DSBA-like domain. This defect in gene coding for lipoprotein FTS_1067 led to high in vivo attenuation associated with the ability to induce host protective immunity. Analyses performed with the truncated recombinant protein showed that the absence of DSBA-like domain revealed the loss of thiol/disulphide oxidoreductase activity and, additionally, confirmed the role of the FKBP_N-like domain in the FTS_1067 oligomerization and chaperone-like function. Finally, we verified that only full-length form of FTS_1067 recombinant protein possesses the isomerase activity. Based on our results, we proposed that for the correct FTS_1067 protein function both domains are needed.

Francisella tularensis is a highly virulent intracellular pathogen with the capacity to infect a variety of hosts including humans. One of the most important proteins involved in F. tularensis virulence and pathogenesis is the protein DsbA. This protein is annotated as a lipoprotein with disulfide oxidoreductase/isomerase activity. Therefore, its interactions with different substrates, including probable virulence factors, to assist in their proper folding are anticipated. We aimed to use the immunopurification approach to find DsbA (gene locus FTS_1067) interacting partners in F. tularensis subsp. holarctica strain FSC200 and compare the identified substrates with proteins which were found in our previous comparative proteome analysis. As a result of our work two FTS_1067 substrates, D-alanyl-D-alanine carboxypeptidase family protein and HlyD family secretion protein, were identified. Bacterial two-hybrid systems were further used to test their relevance in confirming FTS_1067 protein interactions.

Intracellular pathogen F. tularensis is a causative agent of tularemia disease and belongs to the most hazardeous pathogen worldwide, categorized by the Center for Disease Control and Prevention, USA (CDC) as a category A agent.However, no safe and licensed vaccine for prevention a F. tularensis infection is available for vaccination.Tularemia is manifested by several forms depending on a route of infection and virulence of a F. tularensis strain.Essential to a development of the disease is the ability to infect, survive and proliferate inside the mononuclear phagocytes, such as macrophages or dendritic cells.Therefore, this review will discuss aspects of F. tularensis intracellular fate within host macrophages, modulate host signaling pathways to benefit Francisella infection and finally, summarize bacterial determinats involved in the process of phagosomal escape and intracellular replication.

The history of national tularemia research started in 1936 when the first outbreak was recognized in south-east Moravia.Since then in average about one hundred cases have been recorded annually.As tularemia was endemic in former Czechoslovakia, three research groups which concentrated on this disease were formed during decades.The first two groups have worked from sixties and were associated with Jiri Libich (Prague) and Darina Gurycova (Bratislava).The third group which concentrated on the research of natural foci started during late seventies in Valtice (Zdenek Hubalek).The experimental research was, and still is, mainly associated with military research, recently with the Proteomic Center (Faculty of Military Health Sciences, University of Defence) in Hradec Kralove.This center opens molecular approaches to the analysis of Francisella tularensis microbes on one side and the studies on mutual host-pathogen interaction on the other side.One of the significant aims of the research is searching for the new typing and diagnostic markers of Francisella tularensis for the military and medical practice.Thus, scientists from former Czechoslovakia and the Czech Republic contributed significantly to current knowledge on Francisella pathogenesis and their results were highly appraised by international scientific community.

Abstract Principal component analysis was applied to two‐dimensional (2‐D) gel electrophoresis patterns, obtained in various phases of infection. Untreated controls could be satisfactorily differentiated from patterns after infection on days 3 and 7 whereas day 10 of infection was grouped with the controls. Comparison of host cellular protein patterns could help to classify in vivo developing infection without requiring any so‐called immune marker functions. Immunoaffinity separation of infected cells treated with detergent, followed by 2‐D electrophoresis of negative as well as positive eluates, did not reveal radiolabeled bacterial protein antigens.

Sera containing IgD paraprotein present problems in identifying patients with monoclonal gammopathies because only a small or even no spike may be present on standard serum protein electrophoresis. We have detected heavy chains of IgD monoclonal protein by means of high resolution two-dimensional gel electrophoresis. Besides clearly identifying delta heavy chains in maps of serum proteins, we also found size and charge heterogeneity of monoclonal immunoglobulins. The results demonstrate the usefulness of two-dimensional gel electrophoresis in the analysis of selected cases of immunoglobulin malignancies.

Heavy chain disease with monoclonal incomplete μ-heavy chains (μ-HCD) is a rare disorder usually associated with an underlying lymphoproliferative malignancy. Laboratory diagnosis of patients with μ-HCD is usually challenging and the monoclonal protein is not detected by electrophoresis in up to 75% of μ-HCD cases. We describe a patient with multiple malignancies in whom we detected and characterized monoclonal μ-heavy chains using immunofixation electrophoresis, capillary zone electrophoresis with immunotyping, and high resolution two-dimensional electrophoresis. The high resolution 2D electrophoresis enabled us to determine the molecular weight of the μ-heavy chains. The abnormal protein concentration in the serum was unusually high, 38 g/l measured in our patient is the highest reported value in the literature so far.

Using two-dimensional electrophoresis we investigated the effect of 5-aminolevulinic acid (ALA)–based photodynamic therapy (PDT; induction with 1 mM ALA for 4 h followed by blue light dose of 18 J/cm2) on the protein expression in HL60 leukemia cells. ALA-PDT resulted in extensive qualitative and quantitative changes in the protein pattern of HL60 cell lysates. Of more than 1350 protein spots recognized on the protein maps of ALA-induced cells, seven proteins were enhanced and 17 suppressed following irradiation. Three of these, calreticulin presursor, p58 microsomal protein (ERp57) and protein disulfide isomerase (p55) have been identified by matrix-assisted laser desorption and ionization-mass spectrometry and the pI/molecular weight parameters of the affected proteins were estimated by computer analysis. The findings suggest participation of endoplasmic reticulum Ca2+-binding chaperones and/or Ca2+ signaling in ALA-PDT mediated cytotoxicity.

Francisella tularensis influences several host molecular/signaling pathways during infection. Ubiquitination and deubiquitination are among the most important regulatory mechanisms and respectively occur through attachment or removal of the ubiquitin molecule. The process is necessary not only to mark molecules for degradation, but also, for example, to the activation of signaling pathways leading to pro-inflammatory host response. Many intracellular pathogens, including Francisella tularensis , have evolved mechanisms of modifying such host immune responses to escape degradation. Here, we describe that F. tularensis interferes with the host’s ubiquitination system. We show increased total activity of deubiquitinating enzymes (DUBs) in human macrophages after infection, while confirm reduced enzymatic activities of two specific DUBs (USP10 and UCH-L5), and demonstrate increased activity of USP25. We further reveal the enrichment of these three enzymes in exosomes derived from F. tularensis -infected cells. The obtained results show the regulatory effect on ubiquitination mechanism in macrophages during F. tularensis infection.