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.

• MeSH
• bakteriální proteiny * chemie   genetika   metabolismus   MeSH
• buněčné linie MeSH
• chromatografie kapalinová metody   MeSH
• faktory virulence chemie   genetika   metabolismus   MeSH
• Francisella tularensis * genetika   metabolismus   patogenita   MeSH
• hmotnostní spektrometrie metody   MeSH
• isoelektrická fokusace MeSH
• lidé MeSH
• makrofágy cytologie   metabolismus   MeSH
• membránové proteiny * chemie   genetika   metabolismus   MeSH
• míra přežití MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• peptidy chemie   genetika   metabolismus   MeSH
• proteindisulfidisomerasy * chemie   genetika   metabolismus   MeSH
• proteom analýza   MeSH
• proteomika metody   MeSH
• tularemie metabolismus   mortalita   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• faktory virulence MeSH
• membránové proteiny * MeSH
• peptidy MeSH
• proteindisulfidisomerasy * MeSH
• proteom MeSH

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.

• Klíčová slova
• DsbA, Francisella tularensis, SILAC, glyceraldehyde-3-phosphate dehydrogenase, moonlighting,
• MeSH
• delece genu * MeSH
• faktory virulence analýza   MeSH
• Francisella tularensis enzymologie   imunologie   patogenita   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy nedostatek   metabolismus   MeSH
• krevní proteiny metabolismus   MeSH
• mikrobiální viabilita MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• proteindisulfidisomerasy nedostatek   MeSH
• proteom analýza   MeSH
• salmonelová infekce u zvířat mikrobiologie   patologie   MeSH
• vazba proteinů MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• faktory virulence MeSH
• glyceraldehyd-3-fosfátdehydrogenasy MeSH
• krevní proteiny MeSH
• proteindisulfidisomerasy MeSH
• proteom MeSH

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.

• MeSH
• faktory virulence genetika   metabolismus   MeSH
• Francisella tularensis enzymologie   genetika   MeSH
• molekulární chaperony genetika   metabolismus   MeSH
• mutační analýza DNA MeSH
• mutageneze cílená MeSH
• mutantní proteiny genetika   metabolismus   MeSH
• proteindisulfidisomerasy genetika   metabolismus   MeSH
• sekvenční delece MeSH
• thioredoxiny genetika   metabolismus   MeSH
• virulence MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• faktory virulence MeSH
• molekulární chaperony MeSH
• mutantní proteiny MeSH
• proteindisulfidisomerasy MeSH
• thioredoxiny MeSH

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.

• Klíčová slova
• chaperone, copper ions, oligomerization, protein disulphide isomerase, virulence factor,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• faktory virulence genetika   metabolismus   MeSH
• Francisella tularensis genetika   růst a vývoj   patogenita   MeSH
• lipoproteiny genetika   metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• mutantní proteiny genetika   metabolismus   MeSH
• myši inbrední BALB C MeSH
• sekvenční delece MeSH
• tularemie mikrobiologie   patologie   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• faktory virulence MeSH
• lipoproteiny MeSH
• membránové proteiny MeSH
• mutantní proteiny MeSH

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.

• Klíčová slova
• DacD, Francisella, carboxypeptidase, membrane defects, penicillin binding proteins, phagosomal escape, virulence,
• MeSH
• antibakteriální látky farmakologie   MeSH
• buněčná stěna metabolismus   MeSH
• Francisella tularensis účinky léků   růst a vývoj   patogenita   MeSH
• karboxypeptidasa štěpící D-Ala-D-Ala vazby serinového typu genetika   metabolismus   MeSH
• kultivované buňky MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• peptidoglykan biosyntéza   MeSH
• proteindisulfidisomerasy genetika   MeSH
• proteiny vázající penicilin genetika   metabolismus   MeSH
• transmisní elektronová mikroskopie MeSH
• tularemie mikrobiologie   patologie   MeSH
• virulence genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• antibakteriální látky MeSH
• karboxypeptidasa štěpící D-Ala-D-Ala vazby serinového typu MeSH
• peptidoglykan MeSH
• proteindisulfidisomerasy MeSH
• proteiny vázající penicilin MeSH

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.

• Klíčová slova
• FTS_1067 protein, Francisella tularensis, bacterial two-hybrid assay, immunopurification,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• faktory virulence genetika   metabolismus   MeSH
• Francisella tularensis genetika   metabolismus   patogenita   MeSH
• lipoproteiny genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• faktory virulence MeSH
• lipoproteiny MeSH

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.

• MeSH
• faktory virulence chemie   genetika   metabolismus   MeSH
• Francisella tularensis genetika   metabolismus   patogenita   MeSH
• glykosylace MeSH
• molekulární sekvence - údaje MeSH
• multigenová rodina MeSH
• mutace MeSH
• O-antigeny chemie   genetika   metabolismus   MeSH
• proteiny fimbrií chemie   genetika   metabolismus   MeSH
• sacharidové sekvence MeSH
• sekvence aminokyselin MeSH
• tandemová hmotnostní spektrometrie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• faktory virulence MeSH
• O-antigeny MeSH
• PilA protein, Francisella tularensis MeSH Prohlížeč
• proteiny fimbrií MeSH

Recently, it was shown that electrochemical methods can be used for analysis of poorly water-soluble proteins and for study of their structural changes and intermolecular (protein-ligand) interactions. In this study, we focused on complex electrochemical investigation of recombinant protein FTT1103, a disulfide oxidoreductase with structural similarity to well described DsbA proteins. This thioredoxin-like periplasmic lipoprotein plays an important role in virulence of bacteria Francisella tularensis. For electrochemical analyses, adsorptive transfer (ex situ) square-wave voltammetry with pyrolytic graphite electrode, and alternating-current voltammetry and constant-current chronopotentiometric stripping analysis with mercury electrodes, including silver solid amalgam electrode (AgSAE) were used. AgSAE was used in poorly water-soluble protein analysis for the first time. In addition to basic redox, electrocatalytic and adsorption/desorption characterization of FTT1103, electrochemical methods were also used for sensitive determination of the protein at nanomolar level and study of its interaction with surface of AgSA microparticles. Proposed electrochemical protocol and AgSA surface-inhibition approach presented here could be used in future for biochemical studies focused on proteins associated with membranes as well as on those with disulfide oxidoreductase activity.

• Klíčová slova
• Disulfide bond forming protein, Electrochemical sensing, Membrane proteins, Oxidoreductase, Surface inhibition,
• MeSH
• adsorpce MeSH
• elektrochemické techniky * MeSH
• elektrody MeSH
• molekulární modely MeSH
• povrchové vlastnosti MeSH
• prášky, zásypy, pudry chemie   MeSH
• proteindisulfidreduktasa (glutathion) analýza   antagonisté a inhibitory   metabolismus   MeSH
• rekombinantní proteiny analýza   metabolismus   MeSH
• rtuť chemie   MeSH
• stříbro chemie   farmakologie   MeSH
• uhlík chemie   MeSH
• velikost částic MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• prášky, zásypy, pudry MeSH
• proteindisulfidreduktasa (glutathion) MeSH
• rekombinantní proteiny MeSH
• rtuť MeSH
• stříbro MeSH
• uhlík MeSH

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.

• Klíčová slova
• antibody response, cytokines, immunoproteomics, protection, tularemia,
• MeSH
• atenuované vakcíny aplikace a dávkování   genetika   imunologie   MeSH
• bakteriální vakcíny aplikace a dávkování   genetika   imunologie   MeSH
• cytokiny metabolismus   MeSH
• faktory virulence nedostatek   MeSH
• Francisella tularensis klasifikace   enzymologie   imunologie   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední BALB C MeSH
• proteindisulfidisomerasy nedostatek   MeSH
• Th1 buňky imunologie   MeSH
• tularemie imunologie   prevence a kontrola   MeSH
• tvorba protilátek * MeSH
• zkřížená ochrana * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• atenuované vakcíny MeSH
• bakteriální vakcíny MeSH
• cytokiny MeSH
• faktory virulence MeSH
• proteindisulfidisomerasy MeSH