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.

Department of Molecular Pathology and Biology Faculty of Military Health Sciences University of Defense Hradec Kralove Czechia

Faculty of Science Jan Evangelista Purkyně University Ústí nad Labem Czechia

Institute of Microbiology of the Czech Academy of Sciences Prague Czechia

Zobrazit více v PubMed

Apicella M. A., Post D. M. B., Fowler A. C., Jones B. D., Rasmussen J. A., Hunt J. R., et al. (2010). Identification, characterization and immunogenicity of an O-antigen capsular polysaccharide of Francisella tularensis. PLoS One 5:e11060. 10.1371/journal.pone.0011060 PubMed DOI PMC

Bandara A. B., Champion A. E., Wang X., Berg G., Apicella M. A., McLendon M. K., 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

Baumgarten T., Sperling S., Seifert J., Bergen M., von Steiniger F., Wick L. Y., et al. (2012). Membrane vesicle formation as a multiple-stress response mechanism enhances Pseudomonas putida DOT-T1E cell surface hydrophobicity and biofilm formation. Appl. Environ. Microbiol. 78 6217–6224. 10.1128/AEM.01525-12 PubMed DOI PMC

Benada O., Pokorný V. (1990). Modification of the Polaron sputter-coater unit for glow-discharge activation of carbon support films. J. Electron Microsc. Tech. 16 235–239. 10.1002/jemt.1060160304 PubMed DOI

Bhatnagar N. B., Elkins K. L., Fortier A. H. (1995). Heat stress alters the virulence of a rifampin-resistant mutant of Francisella tularensis LVS. Infect. Immun. 63 154–159. PubMed PMC

Bonnington K. E., Kuehn M. J. (2014). Protein selection and export via outer membrane vesicles. Biochim. Biophys. Acta Mol. Cell Res. 1843 1612–1619. 10.1016/j.bbamcr.2013.12.011 PubMed DOI PMC

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., Meyer L., Sun K., Lavander M., Sjöstedt A. (2012). Unique substrates secreted by the Type VI secretion system of Francisella tularensis during intramacrophage infection. PLoS One 7:e50473. 10.1371/journal.pone.0050473 PubMed DOI PMC

Brudal E., Lampe E. O., Reubsaet L., Roos N., Hegna I. K., Thrane I. M., et al. (2015). Vaccination with outer membrane vesicles from Francisella noatunensis reduces development of francisellosis in a zebrafish model. Fish Shellfish Immunol. 42 50–57. 10.1016/j.fsi.2014.10.025 PubMed DOI

Celli J., Zahrt T. C. (2013). Mechanisms of Francisella tularensis intracellular pathogenesis. Cold Spring Harb. Perspect. Med. 3:a010314. 10.1101/cshperspect.a010314 PubMed DOI PMC

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

Champion A. E., Bandara A. B., Mohapatra N., Fulton K. M., Twine S. M., Inzana T. J. (2018). Further characterization of the capsule-like complex (clc) produced by Francisella tularensis Subspecies tularensis: protective efficacy and similarity to outer membrane vesicles. Front. Cell. Infect. Microbiol. 8:182. 10.3389/fcimb.2018.00182 PubMed DOI 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

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

Clemens D. L., Ge P., Lee B.-Y., Horwitz M. A., Zhou Z. H. (2015). Atomic structure and mutagenesis of T6SS reveals interlaced array essential to function. Cell 160 940–951. 10.1016/j.cell.2015.02.005 PubMed DOI PMC

Clemens D. L., Horwitz M. A. (2007). Uptake and intracellular fate of Francisella tularensis in human macrophages. Ann. N. Y. Acad. Sci. 1105 160–186. 10.1196/annals.1409.001 PubMed DOI

Clemens D. L., Lee B.-Y., Horwitz M. A. (2004). Virulent and avirulent strains of Francisella tularensis prevent acidification and maturation of their phagosomes and escape into the cytoplasm in human macrophages. Infect. Immun. 72 3204–3217. 10.1128/IAI.72.6.3204-3217.2004 PubMed DOI PMC

Clemens D. L., Lee B.-Y., Horwitz M. A. (2009). Francisella tularensis phagosomal escape does not require acidification of the phagosome. Infect. Immun. 77 1757–1773. 10.1128/IAI.01485-8 PubMed DOI PMC

Cooke A. C., Nello A. V., Ernst R. K., Schertzer J. W. (2019). Analysis of Pseudomonas aeruginosa biofilm membrane vesicles supports multiple mechanisms of biogenesis. PLoS One 14:e0212275. 10.1371/journal.pone.0212275 PubMed DOI PMC

Cox J., Mann M. (2008). MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26 1367–1372. 10.1038/nbt.1511 PubMed DOI

Dennis D. T., Inglesby T. V., Henderson D. A., Bartlett J. G., Ascher M. S., Eitzen E., et al. (2001). Tularemia as a biological weapon: medical and public health management. JAMA 285 2763–2773. 10.1001/jama.285.21.2763 PubMed DOI

Ellis T. N., Kuehn M. J. (2010). Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiol. Mol. Biol. Rev. 74 81–94. 10.1128/MMBR.00031-9 PubMed DOI PMC

Emiola A., Andrews S. S., Heller C., George J. (2016). Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli. Proc. Natl. Acad. Sci. 113 3108–3113. 10.1073/pnas.1521168113 PubMed DOI PMC

Forestal C. A., Gil H., Monfett M., Noah C. E., Platz G. J., Thanassi D. G., et al. (2008). A conserved and immunodominant lipoprotein of Francisella tularensis is proinflammatory but not essential for virulence. Microb. Pathog. 44 512–523. 10.1016/j.micpath.2008.01.003 PubMed DOI PMC

Gil H., Benach J. L., Thanassi D. G. (2004). Presence of pili on the surface of Francisella tularensis. Infect. Immun. 72 3042–3047. 10.1128/IAI.72.5.3042-3047.2004 PubMed DOI PMC

Harris J. R., Gebauer W., Markl J. (1995). Keyhole limpet haemocyanin: negative staining in the presence of trehalose. Micron 26 25–33. 10.1016/0968-4328(94)00049-V PubMed DOI

Haurat M. F., Aduse-Opoku J., Rangarajan M., Dorobantu L., Gray M. R., Curtis M. A., et al. (2011). Selective sorting of cargo proteins into bacterial membrane vesicles. J. Biol. Chem. 286 1269–1276. 10.1074/jbc.M110.185744 PubMed DOI PMC

Haurat M. F., Elhenawy W., Feldman M. F. (2015). Prokaryotic membrane vesicles: new insights on biogenesis and biological roles. Biol. Chem. 396 95–109. 10.1515/hsz-2014-0183 PubMed DOI

Hazlett K. R. O., Caldon S. D., McArthur D. G., Cirillo K. A., Kirimanjeswara G. S., Magguilli M. L., et al. (2008). Adaptation of Francisella tularensis to the mammalian environment is governed by cues which can be mimicked in vitro. Infect. Immun. 76 4479–4488. 10.1128/IAI.00610-8 PubMed DOI PMC

Hazlett K. R. O., Cirillo K. A. (2009). Environmental adaptation of Francisella tularensis. Microbes Infect. 11 828–834. 10.1016/j.micinf.2009.06.001 PubMed DOI PMC

Holland K. M., Rosa S. J., Kristjansdottir K., Wolfgeher D., Franz B. J., Zarrella T. M., et al. (2017). Differential growth of Francisella tularensis, which alters expression of virulence factors, dominant antigens, and surface-carbohydrate synthases, governs the apparent virulence of Ft SchuS4 to immunized animals. Front. Microbiol. 8:1158. 10.3389/fmicb.2017.01158 PubMed DOI PMC

Horzempa J., Carlson P. E., O’Dee D. M., Shanks R. M., Nau G. J. (2008). Global transcriptional response to mammalian temperature provides new insight into Francisella tularensis pathogenesis. BMC Microbiol. 8:172. 10.1186/1471-2180-8-172 PubMed DOI PMC

Janovská S., Pávková I., Hubálek M., Lenčo J., Macela A., Stulík J. (2007). Identification of immunoreactive antigens in membrane proteins enriched fraction from Francisella tularensis LVS. Immunol. Lett. 108 151–159. 10.1016/j.imlet.2006.12.004 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

Jones C. L., Napier B. A., Sampson T. R., Llewellyn A. C., Schroeder M. R., Weiss D. S. (2012). Subversion of host recognition and defense systems by Francisella spp. Microbiol. Mol. Biol. Rev. 76 383–404. 10.1128/MMBR.05027-11 PubMed DOI PMC

Kaparakis-Liaskos M., Ferrero R. L. (2015). Immune modulation by bacterial outer membrane vesicles. Nat. Rev. Immunol. 15 375–387. 10.1038/nri3837 PubMed DOI

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

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

Krey J. F., Wilmarth P. A., Shin J.-B., Klimek J., Sherman N. E., Jeffery E. D., et al. (2014). Accurate label-free protein quantitation with high- and low-resolution mass spectrometers. J. Proteome Res. 13 1034–1044. 10.1021/pr401017h PubMed DOI PMC

Kulp A., Kuehn M. J. (2010). Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu. Rev. Microbiol. 64 163–184. 10.1146/annurev.micro.091208.073413 PubMed DOI PMC

Larsson P., Oyston P. C. F., Chain P., Chu M. C., Duffield M., Fuxelius H.-H., et al. (2005). The complete genome sequence of Francisella tularensis, the causative agent of tularemia. Nat. Genet. 37 153–159. 10.1038/ng1499 PubMed DOI

Lenco J., Hubálek M., Larsson P., Fucíková A., Brychta M., Macela A., et al. (2007). Proteomics analysis of the Francisella tularensis LVS response to iron restriction: induction of the F. tularensis pathogenicity island proteins IglABC. FEMS Microbiol. Lett. 269 11–21. 10.1111/j.1574-6968.2006.00595.x PubMed DOI

Lenco J., Link M., Tambor V., Zaková J., Cerveny L., Stulik A. J. (2009). iTRAQ quantitative analysis of Francisella tularensis ssp. holarctica live vaccine strain and Francisella tularensis ssp. tularensis SCHU S4 response to different temperatures and stationary phases of growth. Proteomics 9 2875–2882. 10.1002/pmic.200700820 PubMed DOI

Lenco J., Pavkova I., Hubalek M., Stulik J. (2005). Insights into the oxidative stress response in Francisella tularensis LVS and its mutant DeltaiglC1+2 by proteomics analysis. FEMS Microbiol. Lett. 246 47–54. 10.1016/j.femsle.2005.03.040 PubMed DOI

Li Y., Powell D. A., Shaffer S. A., Rasko D. A., Pelletier M. R., Leszyk J. D., et al. (2012). LPS remodeling is an evolved survival strategy for bacteria. Proc. Natl. Acad. Sci. U.S.A. 109 8716–8721. 10.1073/pnas.1202908109 PubMed DOI PMC

Lindgren H., Honn M., Salomonsson E., Kuoppa K., Forsberg Å, Sjöstedt A. (2011). Iron content differs between Francisella tularensis subspecies tularensis and subspecies holarctica strains and correlates to their susceptibility to H2O2-induced killing. Infect. Immun. 79 1218–1224. 10.1128/IAI.01116-0 PubMed DOI PMC

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.01717-6 PubMed DOI PMC

MacDonald I. A., Kuehn M. J. (2013). Stress-induced outer membrane vesicle production by Pseudomonas aeruginosa. J. Bacteriol. 195 2971–2981. 10.1128/JB.02267-12 PubMed DOI PMC

Malinverni J. C., Silhavy T. J. (2011). Assembly of outer membrane β-barrel proteins: the Bam complex. EcoSal Plus 4 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231818/ PubMed PMC

Manning A. J., Kuehn M. J. (2011). Contribution of bacterial outer membrane vesicles to innate bacterial defense. BMC Microbiol. 11:258. 10.1186/1471-2180-11-258 PubMed DOI PMC

Margolis J. J., El-Etr S., Joubert L.-M., Moore E., Robison R., Rasley A., et al. (2010). Contributions of Francisella tularensis subsp. novicida chitinases and sec secretion system to biofilm formation on chitin. Appl. Environ. Microbiol. 76 596–608. 10.1128/AEM.02037-9 PubMed DOI PMC

McBroom A. J., Kuehn M. J. (2007). Release of outer membrane vesicles by gram-negative bacteria is a novel envelope stress response. Mol. Microbiol. 63 545–558. 10.1111/j.1365-2958.2006.05522.x PubMed DOI PMC

McCaig W. D., Koller A., Thanassi D. G. (2013). Production of outer membrane vesicles and outer membrane tubes by Francisella novicida. J. Bacteriol. 195 1120–1132. 10.1128/JB.02007-12 PubMed DOI PMC

Melillo A. A., Bakshi C. S., Melendez J. A. (2010). Francisella tularensis antioxidants harness reactive oxygen species to restrict macrophage signaling and cytokine production. J. Biol. Chem. 285:27553. 10.1074/jbc.M110.144394 PubMed DOI PMC

Mohapatra N. P., Soni S., Rajaram M. V. S., Strandberg K. L., Gunn J. S. (2013). Type A Francisella tularensis acid phosphatases contribute to pathogenesis. PLoS One 8:56834. 10.1371/journal.pone.0056834 PubMed DOI PMC

Olsen I., Amano A. (2015). Outer membrane vesicles – offensive weapons or good Samaritans? J. Oral Microbiol. 7:27468. 10.3402/jom.v7.27468 PubMed DOI PMC

Oyston P. C. F. (2008). Francisella tularensis: unravelling the secrets of an intracellular pathogen. J. Med. Microbiol. 57 921–930. 10.1099/jmm.0.2008/000653-0 PubMed DOI

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

Perez-Riverol Y., Csordas A., Bai J., Bernal-Llinares M., Hewapathirana S., Kundu D. J., et al. (2019). The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 47 D442–D450. 10.1093/nar/gky1106 PubMed DOI PMC

Pierson T., Matrakas D., Taylor Y. U., Manyam G., Morozov V. N., Zhou W., et al. (2011). Proteomic characterization and functional analysis of outer membrane vesicles of Francisella novicida suggests possible role in virulence and use as a vaccine. J. Proteome Res. 10 954–967. 10.1021/pr1009756 PubMed DOI

Powell D. A., Li Y., Ernst R. K. (2012). Turning up Francisella pathogenesis. Virulence 3 594–595. 10.4161/viru.22496 PubMed DOI PMC

Prior J. L., Prior R. G., Hitchen P. G., Diaper H., Griffin K. F., Morris H. R., et al. (2003). Characterization of the O antigen gene cluster and structural analysis of the O antigen of Francisella tularensis subsp. tularensis. J. Med. Microbiol. 52 845–851. 10.1099/jmm.0.05184-0 PubMed DOI

Raynaud C., Meibom K. L., Lety M.-A., Dubail I., Candela T., Frapy E., et al. (2007). Role of the wbt locus of Francisella tularensis in lipopolysaccharide O-antigen biogenesis and pathogenicity. Infect. Immun. 75 536–541. 10.1128/IAI.01429-6 PubMed DOI PMC

Rohmer L., Fong C., Abmayr S., Wasnick M., Larson Freeman T. J., Radey M., et al. (2007). Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains. Genome Biol. 8:R102. 10.1186/gb-2007-8-6-r102 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

Sampath V., McCaig W. D., Thanassi D. G. (2017). Amino acid deprivation and central carbon metabolism regulate the production of outer membrane vesicles and tubes by Francisella. Mol. Microbiol. 107 523–541. 10.1111/mmi.13897 PubMed DOI

Shaffer S. A., Harvey M. D., Goodlett D. R., Ernst R. K. (2007). Structural heterogeneity and environmentally regulated remodeling of Francisella tularensis subspecies novicida lipid A characterized by tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 18 1080–1092. 10.1016/j.jasms.2007.03.008 PubMed DOI PMC

Siebert C., Lindgren H., Ferré S., Villers C., Boisset S., Perard J., et al. (2019). Francisella tularensis: FupA mutation contributes to fluoroquinolone resistance by increasing vesicle secretion and biofilm formation. Emerg. Microbes Infect. 8 808–822. 10.1080/22221751.2019.1615848 PubMed DOI PMC

Sjöstedt A., Sandström G., Tärnvik A. (1992). Humoral and cell-mediated immunity in mice to a 17-kilodalton lipoprotein of Francisella tularensis expressed by Salmonella typhimurium. Infect. Immun. 60 2855–2862. PubMed PMC

Spidlova P., Stulik J. (2017). Francisella tularensis type VI secretion system comes of age. Virulence 8 628–631. 10.1080/21505594.2016.1278336 PubMed DOI PMC

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

Thakran S., Li H., Lavine C. L., Miller M. A., Bina J. E., Bina X. R., et al. (2008). Identification of Francisella tularensis lipoproteins that stimulate the toll-like receptor (TLR) 2/TLR1 heterodimer. J. Biol. Chem. 283 3751–3760. 10.1074/jbc.M706854200 PubMed DOI

Traub A., Mager J., Grossowicz N. (1955). Studies on the nutrition of Pasteurella tularensis. J. Bacteriol. 70 60–69. PubMed PMC

Twine S. M., Mykytczuk N. C. S., Petit M. D., Shen H., Sjöstedt A., Wayne Conlan J., et al. (2006). In vivo proteomic analysis of the intracellular bacterial pathogen, Francisella tularensis, isolated from mouse spleen. Biochem. Biophys. Res. Commun. 345 1621–1633. 10.1016/j.bbrc.2006.05.070 PubMed DOI

Tyanova S., Temu T., Sinitcyn P., Carlson A., Hein M. Y., Geiger T., et al. (2016). The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13 731–740. 10.1038/nmeth.3901 PubMed DOI

van Hoek M. L. (2013). Biofilms an advancement in our understanding of Francisella species. Virulence 4 833–846. 10.4161/viru.27023 PubMed DOI PMC

Wallqvist A., Memišević V., Zavaljevski N., Pieper R., Rajagopala S. V., Kwon K., et al. (2015). Using host-pathogen protein interactions to identify and characterize Francisella tularensis virulence factors. BMC Genomics 16:1106. 10.1186/s12864-015-2351-1 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

Zarrella T. M., Singh A., Bitsaktsis C., Rahman T., Sahay B., Feustel P. J., et al. (2011). Host-adaptation of Francisella tularensis alters the bacterium’s surface-carbohydrates to hinder effectors of innate and adaptive immunity. PLoS One 6:e22335. 10.1371/journal.pone.0022335 PubMed DOI PMC

Zellner B., Huntley J. F. (2019). Ticks and tularemia: do we know what we don’t know? Front. Cell. Infect. Microbiol. 9:146. 10.3389/fcimb.2019.00146 PubMed DOI PMC

Priority order of neonatal colonization by a probiotic or pathogenic Escherichia coli strain dictates the host response to experimental colitis

Protective potential of outer membrane vesicles derived from a virulent strain of Francisella tularensis

Bacterial nucleoid-associated protein HU as an extracellular player in host-pathogen interaction

Francisella tularensis Outer Membrane Vesicles Participate in the Early Phase of Interaction With Macrophages

Control of Francisella tularensis Virulence at Gene Level: Network of Transcription Factors