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.

Department of Biology of the Cell Nucleus Institute of Molecular Genetics ASCR v v i Prague Czechia

Department of Molecular Pathology Faculty of Military Health Science University of Defence Hradec Kralove Czechia

Division BIOCEV Laboratory of Epigenetics of the Cell Nucleus Institute of Molecular Genetics ASCR v v i Vestec Czechia

Microscopy Centre LM and EM Institute of Molecular Genetics ASCR v v i Prague Czechia

Zobrazit více v PubMed

Asare R., Abu Kwaik Y. (2010). Molecular complexity orchestrates modulation of phagosome biogenesis and escape to the cytosol of macrophages by Francisella tularensis. Environ. Microbiol. 12, 2559–2586. 10.1111/j.1462-2920.2010.02229.x PubMed DOI PMC

Bandara A. B., Champion A. E., Wang X., Berg G., Apicella M. A., McLendon M., et al. . (2011). Isolation and mutagenesis of a capsule-like complex (CLC) from Francisella tularensis, and contribution of the CLC to F. tularensis virulence in mice. PloS ONE 6:e19003. 10.1371/journal.pone.0019003 PubMed DOI PMC

Barel M., Hovanessian A. G., Meibom K., Briand J.-P., Dupuis M., Charbit A. (2008). A novel receptor - ligand pathway for entry of Francisella tularensis in monocyte-like THP-1 cells: interaction between surface nucleolin and bacterial elongation factor Tu. BMC Microbiol. 8:145. 10.1186/1471-2180-8-145 PubMed DOI PMC

Bocian-Ostrzycka K. M., Grzeszczuk M. J., Dziewit L., Jagusztyn-Krynicka E. K. (2015). Diversity of the Epsilonproteobacteria Dsb (disulfide bond) systems. Front. Microbiol. 6:570. 10.3389/fmicb.2015.00570 PubMed DOI PMC

Brissac T., Ziveri J., Ramond E., Tros F., Kock S., Dupuis M., et al. . (2015). Gluconeogenesis, an essential metabolic pathway for pathogenic Francisella. Mol. Microbiol. 98, 518–534. 10.1111/mmi.13139 PubMed DOI

Bröms J. E., Meyer L., Sjöstedt A. (2016). A mutagenesis-based approach identifies amino acids in the N-terminal part of Francisella tularensis IglE that critically control Type VI system-mediated secretion. Virulence 8, 821–847. 10.1080/21505594.2016.1258507 PubMed DOI PMC

Bröms J. E., Sjöstedt A., Lavander M. (2010). The role of the Francisella Tularensis pathogenicity island in type VI secretion, intracellular survival, and modulation of host cell signaling. Front. Microbiol. 1:136. 10.3389/fmicb.2010.00136 PubMed DOI PMC

Brotcke A., Monack D. M. (2008). Identification of fevR, a novel regulator of virulence gene expression in Francisella novicida. Infect. Immun. 76, 3473–3480. 10.1128/IAI.00430-08 PubMed DOI PMC

Brotcke A., Weiss D. S., Kim C. C., Chain P., Malfatti S., Garcia E., et al. . (2006). Identification of MglA-regulated genes reveals novel virulence factors in Francisella tularensis. Infect. Immun. 74, 6642–6655. 10.1128/IAI.01250-06 PubMed DOI PMC

Celli J. (2008). Intracellular localization of Brucella abortus and Francisella tularensis in primary murine macrophages. Methods Mol. Biol. 431, 133–145. 10.1007/978-1-60327-032-8_11 PubMed DOI

Chamberlain R. E. (1965). Evaluation of live tularemia vaccine prepared in a chemically defined medium. Appl. Microbiol. 13, 232–235. PubMed PMC

Chandler J. C., Sutherland M. D., Harton M. R., Molins C. R., Anderson R. V., Heaslip D. G., et al. . (2015). Francisella tularensis LVS surface and membrane proteins as targets of effective post-exposure immunization for tularemia. J. Proteome Res. 14, 664–675. 10.1021/pr500628k PubMed DOI PMC

Chong A., Child R., Wehrly T. D., Rockx-Brouwer D., Qin A., Mann B. J., et al. . (2013). Structure-function analysis of DipA, a Francisella tularensis virulence factor required for intracellular replication. PLoS ONE 8:e67965. 10.1371/journal.pone.0067965 PubMed DOI PMC

Chung M.-C., Dean S., Marakasova E. S., Nwabueze A. O., van Hoek M. L. (2014). Chitinases are negative regulators of Francisella novicida biofilms. PLoS ONE 9:e93119. 10.1371/journal.pone.0093119 PubMed DOI PMC

Dankova V., Balonova L., Link M., Straskova A., Sheshko V., Stulik J. (2016). Inactivation of Francisella tularensis gene encoding putative ABC transporter has a pleiotropic effect upon production of various glycoconjugates. J. Proteome Res. 15, 510–524. 10.1021/acs.jproteome.5b00864 PubMed DOI

Dieppedale J., Gesbert G., Ramond E., Chhuon C., Dubail I., Dupuis M., et al. . (2013). Possible links between stress defense and the tricarboxylic acid (TCA) cycle in Francisella pathogenesis. Mol. Cell. Proteomics 12, 2278–2292. 10.1074/mcp.M112.024794 PubMed DOI PMC

Dieppedale J., Sobral D., Dupuis M., Dubail I., Klimentova J., Stulik J., et al. . (2011). Identification of a putative chaperone involved in stress resistance and virulence in Francisella tularensis. Infect. Immun. 79, 1428–1439. 10.1128/IAI.01012-10 PubMed DOI PMC

Egea L., Aguilera L., Giménez R., Sorolla M. A., Aguilar J., Badía J., et al. . (2007). Role of secreted glyceraldehyde-3-phosphate dehydrogenase in the infection mechanism of enterohemorrhagic and enteropathogenic Escherichia coli: interaction of the extracellular enzyme with human plasminogen and fibrinogen. Int. J. Biochem. Cell Biol. 39, 1190–1203. 10.1016/j.biocel.2007.03.008 PubMed DOI

Giménez R., Aguilera L., Ferreira E., Aguilar J., Baldoma L., Badia J. (2014). Glyceraldehyde-3-phosphate dehydrogenase as a moonlighting protien in bacteria, in Recent Advances in Pharmaceutical Sciences IV, eds Munoz-Torrero D., Vázquez-Carrera M., Estelrich J. (Kerala: Research Signpost; ), 165–180.

Guina T., Radulovic D., Bahrami A. J., Bolton D. L., Rohmer L., Jones-Isaac K. A., et al. . (2007). MglA regulates Francisella tularensis subsp. novicida (Francisella novicida) response to starvation and oxidative stress. J. Bacteriol. 189, 6580–6586. 10.1128/JB.00809-07 PubMed DOI PMC

Havlasová J., Hernychová L., Brychta M., Hubálek M., Lenco J., Larsson P., et al. . (2005). Proteomic analysis of anti-Francisella tularensis LVS antibody response in murine model of tularemia. Proteomics 5, 2090–2103. 10.1002/pmic.200401123 PubMed DOI

Henderson B., Martin A. (2011). Bacterial virulence in the moonlight: multitasking bacterial moonlighting proteins are virulence determinants in infectious disease. Infect. Immun. 79, 3476–3491. 10.1128/IAI.00179-11 PubMed DOI PMC

Heras B., Shouldice S. R., Totsika M., Scanlon M. J., Schembri M. A., Martin J. L. (2009). DSB proteins and bacterial pathogenicity. Nat. Rev. Microbiol. 7, 215–225. 10.1038/nrmicro2087 PubMed DOI

Inaba K. (2009). Disulfide bond formation system in Escherichia coli. J. Biochem. 146, 591–597. 10.1093/jb/mvp102 PubMed DOI

Jin H., Agarwal S., Agarwal S., Pancholi V. (2011). Surface export of GAPDH/SDH, a glycolytic enzyme, is essential for Streptococcus pyogenes virulence. Mbio 2, e00068-11. 10.1128/mBio.00068-11 PubMed DOI PMC

Jin H., Song Y. P., Boel G., Kochar J., Pancholi V. (2005). Group A streptococcal surface GAPDH, SDH, recognizes uPAR/CD87 as its receptor on the human pharyngeal cell and mediates bacterial adherence to host cells. J. Mol. Biol. 350, 27–41. 10.1016/j.jmb.2005.04.063 PubMed DOI

Johansson A., Berglund L., Eriksson U., Göransson I., Wollin R., Forsman M., et al. . (2000). Comparative analysis of PCR versus culture for diagnosis of ulceroglandular tularemia. J. Clin. Microbiol. 38, 22–26. PubMed PMC

Kadzhaev K., Zingmark C., Golovliov I., Bolanowski M., Shen H., Conlan W., et al. . (2009). Identification of genes contributing to the virulence of Francisella tularensis SCHU S4 in a mouse intradermal infection model. PLoS ONE 4:e5463. 10.1371/journal.pone.0005463 PubMed DOI PMC

Kilmury S. L. N., Twine S. M. (2011). The Francisella tularensis proteome and its recognition by antibodies. Front. Microbiol. 1:143. 10.3389/fmicb.2010.00143 PubMed DOI PMC

Kim T.-H., Sebastian S., Pinkham J. T., Ross R. A., Blalock L. T., Kasper D. L. (2010). Characterization of the O-antigen polymerase (Wzy) of Francisella tularensis. J. Biol. Chem. 285, 27839–27849. 10.1074/jbc.M110.143859 PubMed DOI PMC

Konecna K., Hernychova L., Reichelova M., Lenco J., Klimentova J., Stulik J., et al. . (2010). Comparative proteomic profiling of culture filtrate proteins of less and highly virulent Francisella tularensis strains. Proteomics 10, 4501–4511. 10.1002/pmic.201000248 PubMed DOI

Lindgren H., Shen H., Zingmark C., Golovliov I., Conlan W., Sjöstedt A. (2007). Resistance of Francisella tularensis strains against reactive nitrogen and oxygen species with special reference to the role of KatG. Infect. Immun. 75, 1303–1309. 10.1128/IAI.01717-06 PubMed DOI PMC

Lo K. Y.-S., Chua M. D., Abdulla S., Law H. T., Guttman J. A. (2013). Examination of in vitro epithelial cell lines as models for Francisella tularensis non-phagocytic infections. J. Microbiol. Methods 93, 153–160. 10.1016/j.mimet.2013.03.004 PubMed DOI

Masuda T., Tomita M., Ishihama Y. (2008). Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J. Proteome Res. 7, 731–740. 10.1021/pr700658q PubMed DOI

McLendon M. K., Schilling B., Hunt J. R., Apicella M. A., Gibson B. W. (2007). Identification of LpxL, a late acyltransferase of Francisella tularensis. Infect. Immun. 75, 5518–5531. 10.1128/IAI.01288-06 PubMed DOI PMC

Meibom K. L., Charbit A. (2010). Francisella tularensis metabolism and its relation to virulence. Front. Microbiol. 1:140. 10.3389/fmicb.2010.00140 PubMed DOI PMC

Milton D. L., O'Toole R., Horstedt P., Wolf-Watz H. (1996). Flagellin A is essential for the virulence of Vibrio anguillarum. J. Bacteriol. 178, 1310–1319. PubMed PMC

Pancholi V., Fischetti V. A. (1992). A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate-dehydrogenase with multiple binding activity. J. Exp. Med. 176, 415–426. 10.1084/jem.176.2.415 PubMed DOI PMC

Pávková I., Brychta M., Strašková A., Schmidt M., Macela A., Stulík J. (2013). Comparative proteome profiling of host-pathogen interactions: insights into the adaptation mechanisms of Francisella tularensis in the host cell environment. Appl. Microbiol. Biotechnol. 97, 10103–10115. 10.1007/s00253-013-5321-z PubMed DOI

Pávková I., Hubálek M., Zechovská J., Lenco J., Stulík J. (2005). Francisella tularensis live vaccine strain: proteomic analysis of membrane proteins enriched fraction. Proteomics 5, 2460–2467. 10.1002/pmic.200401213 PubMed DOI

Pavkova I., Reichelova M., Larsson P., Hubalek M., Vackova J., Forsberg A., et al. . (2006). Comparative proteome analysis of fractions enriched for membrane-associated proteins from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica strains. J. Proteome Res. 5, 3125–3134. 10.1021/pr0601887 PubMed DOI

Pérez N., Johnson R., Sen B., Ramakrishnan G. (2016). Two parallel pathways for ferric and ferrous iron acquisition support growth and virulence of the intracellular pathogen Francisella tularensis Schu S4. Microbiologyopen 5, 453–468. 10.1002/mbo3.342 PubMed DOI PMC

Qin A., Scott D. W., Rabideau M. M., Moore E. A., Mann B. J. (2011). Requirement of the CXXC motif of novel Francisella infectivity potentiator protein B FipB, and FipA in virulence of F. tularensis subsp. tularensis. PLoS ONE 6:e24611. 10.1371/journal.pone.0024611 PubMed DOI PMC

Qin A., Zhang Y., Clark M. E., Moore E. A., Rabideau M. M., Moreau G. B., et al. . (2016). Components of the type six secretion system are substrates of Francisella tularensis Schu S4 DsbA-like FipB protein. Virulence 7, 882–894. 10.1080/21505594.2016.1168550 PubMed DOI PMC

Qin A., Zhang Y., Clark M. E., Rabideau M. M., Millan Barea L. R., Mann B. J. (2014). FipB, an essential virulence factor of Francisella tularensis subsp. tularensis, has dual roles in disulfide bond formation. J. Bacteriol. 196, 3571–3581. 10.1128/JB.01359-13 PubMed DOI PMC

Raghunathan A., Shin S., Daefler S. (2010). Systems approach to investigating host-pathogen interactions in infections with the biothreat agent Francisella. Constraints-based model of Francisella tularensis. BMC Syst. Biol. 4:118. 10.1186/1752-0509-4-118 PubMed DOI PMC

Ramakrishnan G., Meeker A., Dragulev B. (2008). fslE Is necessary for siderophore-mediated iron acquisition in Francisella tularensis Schu S4. J. Bacteriol. 190, 5353–5361. 10.1128/JB.00181-08 PubMed DOI PMC

Ren G., Champion M. M., Huntley J. F. (2014). Identification of disulfide bond isomerase substrates reveals bacterial virulence factors. Mol. Microbiol. 94, 926–944. 10.1111/mmi.12808 PubMed DOI PMC

Robertson G. T., Child R., Ingle C., Celli J., Norgard M. V. (2013). IglE is an outer membrane-associated lipoprotein essential for intracellular survival and murine virulence of type A Francisella tularensis. Infect. Immun. 81, 4026–4040. 10.1128/IAI.00595-13 PubMed DOI PMC

Rodriguez S. A., Davis G., Klose K. E. (2009). Targeted gene disruption in Francisella tularensis by group II introns. Methods 49, 270–274. 10.1016/j.ymeth.2009.04.011 PubMed DOI PMC

Rowe H. M., Huntley J. F. (2015). From the outside-in: the Francisella tularensis envelope and virulence. Front. Cell. Infect. Microbiol. 5:94. 10.3389/fcimb.2015.00094 PubMed DOI PMC

Salomonsson E. N., Forslund A.-L., Forsberg A. (2011). Type IV Pili in Francisella - a virulence trait in an intracellular pathogen. Front. Microbiol. 2:29. 10.3389/fmicb.2011.00029 PubMed DOI PMC

Sambrook J., Russel D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Schmidt M., Klimentova J., Rehulka P., Straskova A., Spidlova P., Szotakova B., et al. . (2013). Francisella tularensis subsp. holarctica DsbA homologue: a thioredoxin-like protein with chaperone function. Microbiol. Read. Engl. 159, 2364–2374. 10.1099/mic.0.070516-0 PubMed DOI

Shouldice S. R., Heras B., Walden P. M., Totsika M., Schembri M. A., Martin J. L. (2011). Structure and Function of DsbA, a key bacterial oxidative folding catalyst. Antioxid. Redox Signal. 14, 1729–1760. 10.1089/ars.2010.3344 PubMed DOI

Simon R., Priefer U., Puhler A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nat. Biotechnol. 1, 784–791. 10.1038/nbt1183-784 DOI

Smith R. P., Paxman J. J., Scanlon M. J., Heras B. (2016). Targeting bacterial Dsb proteins for the development of anti-virulence agents. Molecules 21:811. 10.3390/molecules21070811 PubMed DOI PMC

Strádalová V., Gaplovská-Kyselá K., Hozák P. (2008). Ultrastructural and nuclear antigen preservation after high-pressure freezing/freeze-substitution and low-temperature LR White embedding of HeLa cells. Histochem. Cell Biol. 130, 1047–1052. 10.1007/s00418-008-0504-x PubMed DOI

Straskova A., Pavkova I., Link M., Forslund A.-L., Kuoppa K., Noppa L., et al. . (2009). Proteome analysis of an attenuated Francisella tularensis dsbA mutant: identification of potential DsbA substrate proteins. J. Proteome Res. 8, 5336–5346. 10.1021/pr900570b PubMed DOI

Straskova A., Spidlova P., Mou S., Worsham P., Putzova D., Pavkova I., et al. . (2015). Francisella tularensis type B ΔdsbA mutant protects against type A strain and induces strong inflammatory cytokine and Th1-like antibody response in vivo. Pathog. Dis. 73:ftv058. 10.1093/femspd/ftv058 PubMed DOI PMC

Su J., Yang J., Zhao D., Kawula T. H., Banas J. A., Zhang J.-R. (2007). Genome-wide identification of Francisella tularensis virulence determinants. Infect. Immun. 75, 3089–3101. 10.1128/IAI.01865-06 PubMed DOI PMC

Thomas R. M., Twine S. M., Fulton K. M., Tessier L., Kilmury S. L. N., Ding W., et al. . (2011). Glycosylation of DsbA in Francisella tularensis subsp. tularensis. J. Bacteriol. 193, 5498–5509. 10.1128/JB.00438-11 PubMed DOI PMC

Tunio S. A., Oldfield N. J., Ala'Aldeen D. A., Wooldridge K. G., Turner D. P. (2010). The role of glyceraldehyde 3-phosphate dehydrogenase (GapA-1) in Neisseria meningitidis adherence to human cells. BMC Microbiol. 10:280. 10.1186/1471-2180-10-280 PubMed DOI PMC

Vizcaíno J. A., Csordas A., Del-Toro N., Dianes J. A., Griss J., Lavidas I., et al. (2016). 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44, 11033 10.1093/nar/gkw880 PubMed DOI PMC

Wehrly T. D., Chong A., Virtaneva K., Sturdevant D. E., Child R., Edwards J. A., et al. . (2009). Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophages. Cell. Microbiol. 11, 1128–1150. 10.1111/j.1462-5822.2009.01316.x PubMed DOI PMC

Weiss D. S., Brotcke A., Henry T., Margolis J. J., Chan K., Monack D. M. (2007). In vivo negative selection screen identifies genes required for Francisella virulence. Proc. Natl. Acad. Sci. U.S.A. 104, 6037–6042. 10.1073/pnas.0609675104 PubMed DOI PMC

Francisella tularensis Glyceraldehyde-3-Phosphate Dehydrogenase Is Relocalized during Intracellular Infection and Reveals Effect on Cytokine Gene Expression and Signaling

Identification of Bacterial Protein Interaction Partners Points to New Intracellular Functions of Francisella tularensis Glyceraldehyde-3-Phosphate Dehydrogenase

Diverse Localization and Protein Binding Abilities of Glyceraldehyde-3-Phosphate Dehydrogenase in Pathogenic Bacteria: The Key to its Multifunctionality?

Francisella tularensis D-Ala D-Ala Carboxypeptidase DacD Is Involved in Intracellular Replication and It Is Necessary for Bacterial Cell Wall Integrity