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 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 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 PubMed DOI PMC

Benada O., Pokorný V. (1990). Modification of the Polaron sputter-coater unit for glow-discharge activation of carbon support films. PubMed DOI

Bhatnagar N. B., Elkins K. L., Fortier A. H. (1995). Heat stress alters the virulence of a rifampin-resistant mutant of PubMed PMC

Bonnington K. E., Kuehn M. J. (2014). Protein selection and export via outer membrane vesicles. PubMed DOI PMC

Bröms J. E., Meyer L., Sjöstedt A. (2016). A mutagenesis-based approach identifies amino acids in the N-terminal part of 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 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 PubMed DOI

Celli J., Zahrt T. C. (2013). Mechanisms of PubMed DOI PMC

Chamberlain R. E. (1965). Evaluation of live tularemia vaccine prepared in a chemically defined medium. 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 PubMed DOI PMC

Chandler J. C., Sutherland M. D., Harton M. R., Molins C. R., Anderson R. V., Heaslip D. G., et al. (2015). PubMed DOI PMC

Chung M.-C., Dean S., Marakasova E. S., Nwabueze A. O., van Hoek M. L. (2014). Chitinases are negative regulators of 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. PubMed DOI PMC

Clemens D. L., Horwitz M. A. (2007). Uptake and intracellular fate of PubMed DOI

Clemens D. L., Lee B.-Y., Horwitz M. A. (2004). Virulent and avirulent strains of PubMed DOI PMC

Clemens D. L., Lee B.-Y., Horwitz M. A. (2009). PubMed DOI PMC

Cooke A. C., Nello A. V., Ernst R. K., Schertzer J. W. (2019). Analysis of 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. 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. PubMed DOI

Ellis T. N., Kuehn M. J. (2010). Virulence and immunomodulatory roles of bacterial outer membrane vesicles. 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 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 PubMed DOI PMC

Gil H., Benach J. L., Thanassi D. G. (2004). Presence of pili on the surface of PubMed DOI PMC

Harris J. R., Gebauer W., Markl J. (1995). Keyhole limpet haemocyanin: negative staining in the presence of trehalose. 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. PubMed DOI PMC

Haurat M. F., Elhenawy W., Feldman M. F. (2015). Prokaryotic membrane vesicles: new insights on biogenesis and biological roles. 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 PubMed DOI PMC

Hazlett K. R. O., Cirillo K. A. (2009). Environmental adaptation of 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 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 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 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. 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 PubMed DOI PMC

Kaparakis-Liaskos M., Ferrero R. L. (2015). Immune modulation by bacterial outer membrane vesicles. PubMed DOI

Kilmury S. L. N., Twine S. M. (2011). The 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 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. PubMed DOI PMC

Kulp A., Kuehn M. J. (2010). Biological functions and biogenesis of secreted bacterial outer membrane vesicles. 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 PubMed DOI

Lenco J., Hubálek M., Larsson P., Fucíková A., Brychta M., Macela A., et al. (2007). Proteomics analysis of the PubMed DOI

Lenco J., Link M., Tambor V., Zaková J., Cerveny L., Stulik A. J. (2009). iTRAQ quantitative analysis of PubMed DOI

Lenco J., Pavkova I., Hubalek M., Stulik J. (2005). Insights into the oxidative stress response in 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. PubMed DOI PMC

Lindgren H., Honn M., Salomonsson E., Kuoppa K., Forsberg Å, Sjöstedt A. (2011). Iron content differs between PubMed DOI PMC

Lindgren H., Shen H., Zingmark C., Golovliov I., Conlan W., Sjöstedt A. (2007). Resistance of PubMed DOI PMC

MacDonald I. A., Kuehn M. J. (2013). Stress-induced outer membrane vesicle production by PubMed DOI PMC

Malinverni J. C., Silhavy T. J. (2011). Assembly of outer membrane β-barrel proteins: the Bam complex. PubMed PMC

Manning A. J., Kuehn M. J. (2011). Contribution of bacterial outer membrane vesicles to innate bacterial defense. PubMed DOI PMC

Margolis J. J., El-Etr S., Joubert L.-M., Moore E., Robison R., Rasley A., et al. (2010). Contributions of 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. PubMed DOI PMC

McCaig W. D., Koller A., Thanassi D. G. (2013). Production of outer membrane vesicles and outer membrane tubes by PubMed DOI PMC

Melillo A. A., Bakshi C. S., Melendez J. A. (2010). PubMed DOI PMC

Mohapatra N. P., Soni S., Rajaram M. V. S., Strandberg K. L., Gunn J. S. (2013). Type A PubMed DOI PMC

Olsen I., Amano A. (2015). Outer membrane vesicles – offensive weapons or good Samaritans? PubMed DOI PMC

Oyston P. C. F. (2008). 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 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. 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 PubMed DOI

Powell D. A., Li Y., Ernst R. K. (2012). Turning up 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 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 PubMed DOI PMC

Rohmer L., Fong C., Abmayr S., Wasnick M., Larson Freeman T. J., Radey M., et al. (2007). Comparison of PubMed DOI PMC

Rowe H. M., Huntley J. F. (2015). From the outside-in: the 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 PubMed DOI

Shaffer S. A., Harvey M. D., Goodlett D. R., Ernst R. K. (2007). Structural heterogeneity and environmentally regulated remodeling of PubMed DOI PMC

Siebert C., Lindgren H., Ferré S., Villers C., Boisset S., Perard J., et al. (2019). PubMed DOI PMC

Sjöstedt A., Sandström G., Tärnvik A. (1992). Humoral and cell-mediated immunity in mice to a 17-kilodalton lipoprotein of PubMed PMC

Spidlova P., Stulik J. (2017). PubMed DOI PMC

Straskova A., Spidlova P., Mou S., Worsham P., Putzova D., Pavkova I., et al. (2015). PubMed DOI PMC

Thakran S., Li H., Lavine C. L., Miller M. A., Bina J. E., Bina X. R., et al. (2008). Identification of PubMed DOI

Traub A., Mager J., Grossowicz N. (1955). Studies on the nutrition of 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, 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. PubMed DOI

van Hoek M. L. (2013). Biofilms an advancement in our understanding of 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 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 PubMed DOI PMC

Zarrella T. M., Singh A., Bitsaktsis C., Rahman T., Sahay B., Feustel P. J., et al. (2011). Host-adaptation of PubMed DOI PMC

Zellner B., Huntley J. F. (2019). Ticks and tularemia: do we know what we don’t know? 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