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.

Department of Biology of the Cell Nucleus Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czechia

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

Electron Microscopy Core Facility Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czechia

Laboratory of Immunobiology Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czechia

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Acevedo R., Fernández S., Zayas C., Acosta A., Sarmiento M. E., Ferro V. A., et al. (2014). Bacterial outer membrane vesicles and vaccine applications. Front. Immunol. 5:121. 10.3389/fimmu.2014.00121 PubMed DOI PMC

Athman J. J., Wang Y., McDonald D. J., Boom W. H., Harding C. V., Wearsch P. A. (2015). Bacterial membrane vesicles mediate the release of Mycobacterium tuberculosis lipoglycans and lipoproteins from infected macrophages. J. Immunol. 195 1044–1053. 10.4049/jimmunol.1402894 PubMed DOI PMC

Avila-Calderón E. D., Araiza-Villanueva M. G., Cancino-Diaz J. C., López-Villegas E. O., Sriranganathan N., Boyle S. M., et al. (2014). Roles of bacterial membrane vesicles. Arch. Microbiol. 197 1–10. 10.1007/s00203-014-1042-7 PubMed DOI

Balonova L., Mann B. F., Cerveny L., Alley W. R., Chovancova E., Forslund A.-L., et al. (2012). Characterization of protein glycosylation in Francisella tularensis subsp. holarctica. Mol. Cell. Proteomics 11:M111.015016. 10.1074/mcp.M111.015016 PubMed DOI PMC

Bauler T. J., Chase J. C., Wehrly T. D., Bosio C. M. (2014). Virulent Francisella tularensis destabilize host mRNA to rapidly suppress inflammation. J. Innate Immun. 6 793–805. 10.1159/000363243 PubMed DOI PMC

Behrouzi A., Vaziri F., Riazi Rad F., Amanzadeh A., Fateh A., Moshiri A., et al. (2018). Comparative study of pathogenic and non-pathogenic Escherichia coli outer membrane vesicles and prediction of host-interactions with TLR signaling pathways. BMC Res. Notes 11:539. 10.1186/s13104-018-3648-3 PubMed DOI PMC

Bielaszewska M., Rüter C., Bauwens A., Greune L., Jarosch K.-A., Steil D., et al. (2017). Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: intracellular delivery, trafficking and mechanisms of cell injury. PLoS Pathog. 13:e1006159. 10.1371/journal.ppat.1006159 PubMed DOI PMC

Bitto N. J., Kaparakis-Liaskos M. (2017). The therapeutic benefit of bacterial membrane vesicles. Int. J. Mol. Sci. 18:1287. 10.3390/ijms18061287 PubMed DOI PMC

Cai W., Kesavan D. K., Wan J., Abdelaziz M. H., Su Z., Xu H. (2018). Bacterial outer membrane vesicles, a potential vaccine candidate in interactions with host cells based. Diagn. Pathol. 13:95. 10.1186/s13000-018-0768-y PubMed DOI PMC

Celli J. (2008). “Intracellular localization of Brucella abortus and Francisella tularensis in primary murine macrophages,” in Bacterial Pathogenesis: Methods and Protocols Methods in Molecular BiologyTM , eds DeLeo F. R., Otto M. (Totowa, NJ: Humana Press; ), 133–145. 10.1007/978-1-60327-032-8_11 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

Chmiela M., Walczak N., Rudnicka K. (2018). Helicobacter pylori outer membrane vesicles involvement in the infection development and Helicobacter pylori-related diseases. J. Biomed. Sci. 25:78. 10.1186/s12929-018-0480-y 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. (2005). Francisella tularensis enters macrophages via a novel process involving pseudopod loops. Infect. Immun. 73 5892–5902. 10.1128/IAI.73.9.5892-5902.2005 PubMed DOI PMC

Clemens D. L., Lee B.-Y., Horwitz M. A. (2012). O-antigen-deficient Francisella tularensis live vaccine strain mutants are ingested via an aberrant form of looping phagocytosis and show altered kinetics of intracellular trafficking in human macrophages. Infect. Immun. 80 952–967. 10.1128/IAI.05221-11 PubMed DOI PMC

Craven R. R., Hall J. D., Fuller J. R., Taft-Benz S., Kawula T. H. (2008). Francisella tularensis invasion of lung epithelial cells. Infect. Immun. 76 2833–2842. 10.1128/IAI.00043-08 PubMed DOI PMC

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.00031-09 PubMed DOI PMC

Elmi A., Nasher F., Jagatia H., Gundogdu O., Bajaj-Elliott M., Wren B., et al. (2016). Campylobacter jejuni outer membrane vesicle-associated proteolytic activity promotes bacterial invasion by mediating cleavage of intestinal epithelial cell E-cadherin and occludin. Cell. Microbiol. 18 561–572. 10.1111/cmi.12534 PubMed DOI

Fabrik I., Link M., Putzova D., Plzakova L., Lubovska Z., Philimonenko V., et al. (2018). The early dendritic cell signaling induced by virulent Francisella tularensis strain occurs in phases and involves the activation of Extracellular Signal-Regulated Kinases (ERKs) and p38 in the later stage. Mol. Cell. Proteomics 17 81–94. 10.1074/mcp.RA117.000160 PubMed DOI PMC

Fernandez-Moreira E., Helbig J. H., Swanson M. S. (2006). Membrane vesicles shed by Legionella pneumophila inhibit fusion of phagosomes with lysosomes. Infect. Immun. 74 3285–3295. 10.1128/IAI.01382-05 PubMed DOI PMC

Galkina S. I., Romanova J. M., Bragina E. E., Tiganova I. G., Stadnichuk V. I., Alekseeva N. V., et al. (2011). Membrane tubules attach Salmonella typhimurium to eukaryotic cells and bacteria. FEMS Immunol. Med. Microbiol. 61 114–124. 10.1111/j.1574-695X.2010.00754.x PubMed DOI

Gao L., van der Veen S. (2020). Role of outer membrane vesicles in bacterial physiology and host cell interactions. Infect. Microbes Dis. 2 3–9. 10.1097/IM9.0000000000000017 DOI

Gerritzen M. J. H., Salverda M. L. M., Martens D. E., Wijffels R. H., Stork M. (2019). Spontaneously released Neisseria meningitidis outer membrane vesicles as vaccine platform: production and purification. Vaccine 37 6978–6986. 10.1016/j.vaccine.2019.01.076 PubMed DOI

Gill S., Catchpole R., Forterre P. (2018). Extracellular membrane vesicles in the three domains of life and beyond. FEMS Microbiol. Rev. 43 273–303. 10.1093/femsre/fuy042 PubMed DOI PMC

Gillette D. D., Curry H. M., Cremer T., Ravneberg D., Fatehchand K., Shah P. A., et al. (2014). Virulent Type A Francisella tularensis actively suppresses cytokine responses in human monocytes. Front. Cell. Infect. Microbiol. 4:45. 10.3389/fcimb.2014.00045 PubMed DOI PMC

Guidi R., Levi L., Rouf S. F., Puiac S., Rhen M., Frisan T. (2013). Salmonella enterica delivers its genotoxin through outer membrane vesicles secreted from infected cells. Cell. Microbiol. 15 2034–2050. 10.1111/cmi.12172 PubMed DOI

Hall J. D., Craven R. R., Fuller J. R., Pickles R. J., Kawula T. H. (2007). Francisella tularensis replicates within alveolar Type II epithelial cells in vitro and in vivo following inhalation. Infect. Immun. 75 1034–1039. 10.1128/IAI.01254-06 PubMed DOI PMC

Holland K. M., Rosa S. J., Hazlett K. R. O. (2016). Francisella tularensis–immune cell activator, suppressor, or stealthy evader: the evolving view from the petri dish. J. Bioterror. Biodef. 7:144. 10.4172/2157-2526.1000144 PubMed DOI PMC

Hu R., Lin H., Li J., Zhao Y., Wang M., Sun X., et al. (2020). Probiotic Escherichia coli Nissle 1917-derived outer membrane vesicles enhance immunomodulation and antimicrobial activity in RAW264.7 macrophages. BMC Microbiol. 20:268. 10.1186/s12866-020-01953-x PubMed DOI PMC

Jan A. T. (2017). Outer Membrane Vesicles (OMVs) of gram-negative bacteria: a perspective update. Front. Microbiol. 8:1053. 10.3389/fmicb.2017.01053 PubMed DOI PMC

Jin J. S., Kwon S.-O., Moon D. C., Gurung M., Lee J. H., Kim S. I., et al. (2011). Acinetobacter baumannii secretes cytotoxic outer membrane protein a via outer membrane vesicles. PLoS One 6:e17027. 10.1371/journal.pone.0017027 PubMed DOI PMC

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 E. J., Booth C., Fonseca S., Parker A., Cross K., Miquel-Clopés A., et al. (2020). The uptake, trafficking, and biodistribution of bacteroides thetaiotaomicron generated outer membrane vesicles. Front. Microbiol. 11:57. 10.3389/fmicb.2020.00057 PubMed DOI PMC

Jung A. L., Stoiber C., Herkt C. E., Schulz C., Bertrams W., Schmeck B. (2016). Legionella pneumophila-derived outer membrane vesicles promote bacterial replication in macrophages. PLoS Pathog. 12:e1005592. 10.1371/journal.ppat.1005592 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

Kingry L. C., Petersen J. M. (2014). Comparative review of Francisella tularensis and Francisella novicida. Front. Cell. Infect. Microbiol. 4:35. 10.3389/fcimb.2014.00035 PubMed DOI PMC

Klimentova J., Pavkova I., Horcickova L., Bavlovic J., Kofronova O., Benada O., et al. (2019). Francisella tularensis subsp. holarctica releases differentially loaded outer membrane vesicles under various stress conditions. Front. Microbiol. 10:2304. 10.3389/fmicb.2019.02304 PubMed DOI PMC

Klimentova J., Rehulka P., Pavkova I., Kubelkova K., Bavlovic J., Stulik J. (2021). Cross-species proteomic comparison of outer membrane vesicles and membranes of Francisella tularensis subsp. tularensis versus subsp. holarctica. J. Proteome Res. 20 1716–1732. 10.1021/acs.jproteome.0c00917 PubMed DOI

Kunsmann L., Rüter C., Bauwens A., Greune L., Glüder M., Kemper B., et al. (2015). Virulence from vesicles: novel mechanisms of host cell injury by Escherichia coli O104:H4 outbreak strain. Sci. Rep. 5:13252. 10.1038/srep13252 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

MacDonald I. A., Kuehn M. J. (2012). Offense and defense: microbial membrane vesicles play both ways. Res. Microbiol. 163 607–618. 10.1016/j.resmic.2012.10.020 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

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

Metruccio M. M. E., Evans D. J., Gabriel M. M., Kadurugamuwa J. L., Fleiszig S. M. J. (2016). Pseudomonas aeruginosa outer membrane vesicles triggered by human mucosal fluid and lysozyme can prime host tissue surfaces for bacterial adhesion. Front. Microbiol. 7:871. 10.3389/fmicb.2016.00871 PubMed DOI PMC

Mondal A., Tapader R., Chatterjee N. S., Ghosh A., Sinha R., Koley H., et al. (2016). Cytotoxic and inflammatory responses induced by outer membrane vesicle-associated biologically active proteases from Vibrio cholerae. Infect. Immun. 84 1478–1490. 10.1128/IAI.01365-15 PubMed DOI PMC

O’Donoghue E. J., Krachler A. M. (2016). Mechanisms of outer membrane vesicle entry into host cells. Cell. Microbiol. 18 1508–1517. 10.1111/cmi.12655 PubMed DOI PMC

O’Donoghue E. J., Sirisaengtaksin N., Browning D. F., Bielska E., Hadis M., Fernandez-Trillo F., et al. (2017). Lipopolysaccharide structure impacts the entry kinetics of bacterial outer membrane vesicles into host cells. PLoS Pathog 13:e1006760. 10.1371/journal.ppat.1006760 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

Ozanic M., Marecic V., Abu Kwaik Y., Santic M. (2015). The divergent intracellular lifestyle of Francisella tularensis in evolutionarily distinct host cells. PLoS Pathog. 11:e1005208. 10.1371/journal.ppat.1005208 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

Pollak C. N., Delpino M. V., Fossati C. A., Baldi P. C. (2012). Outer membrane vesicles from Brucella abortus promote bacterial internalization by human monocytes and modulate their innate immune response. PLoS One 7:e50214. 10.1371/journal.pone.0050214 PubMed DOI PMC

Prados-Rosales R., Baena A., Martinez L. R., Luque-Garcia J., Kalscheuer R., Veeraraghavan U., et al. (2011). Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice. J. Clin. Invest. 121 1471–1483. 10.1172/JCI44261 PubMed DOI PMC

Putzova D., Panda S., Härtlova A., Stulík J., Gekara N. O. (2017). Subversion of innate immune responses by Francisella involves the disruption of TRAF3 and TRAF6 signalling complexes. Cell. Microbiol. 19:e12769. 10.1111/cmi.12769 PubMed DOI

Rodal S. K., Skretting G., Garred Ø, Vilhardt F., van Deurs B., Sandvig K. (1999). Extraction of cholesterol with methyl-β-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. Mol. Biol. Cell 10 961–974. PubMed PMC

Sampath V., McCaig W. D., Thanassi D. G. (2018). 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

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., et al. (2012). Fiji–an open source platform for biological image analysis. Nat. Methods 9 676–682. 10.1038/nmeth.2019 PubMed DOI PMC

Schwechheimer C., Kuehn M. J. (2015). Outer-membrane vesicles from gram-negative bacteria: biogenesis and functions. Nat. Rev. Microbiol. 13 605–619. 10.1038/nrmicro3525 PubMed DOI PMC

Singh A., Rahman T., Malik M., Hickey A. J., Leifer C. A., Hazlett K. R. O., et al. (2013). Discordant results obtained with Francisella tularensis during In vitro and in vivo immunological studies are attributable to compromised bacterial structural integrity. PLoS One 8:e58513. 10.1371/journal.pone.0058513 PubMed DOI PMC

Telepnev M., Golovliov I., Grundström T., Tärnvik A., Sjöstedt A. (2003). Francisella tularensis inhibits toll-like receptor-mediated activation of intracellular signalling and secretion of TNF-α and IL-1 from murine macrophages. Cell. Microbiol. 5 41–51. 10.1046/j.1462-5822.2003.00251.x PubMed DOI

Turner L., Bitto N. J., Steer D. L., Lo C., D’Costa K., Ramm G., et al. (2018). Helicobacter pylori outer membrane vesicle size determines their mechanisms of host cell entry and protein content. Front. Immunol. 9:1466. 10.3389/fimmu.2018.01466 PubMed DOI PMC

Vanaja S. K., Russo A. J., Behl B., Banerjee I., Yankova M., Deshmukh S. D., et al. (2016). Bacterial outer membrane vesicles mediate cytosolic localization of LPS and caspase-11 activation. Cell 165 1106–1119. 10.1016/j.cell.2016.04.015 PubMed DOI PMC

Vdovikova S., Luhr M., Szalai P., Nygård Skalman L., Francis M. K., Lundmark R., et al. (2017). A novel role of listeria monocytogenes membrane vesicles in inhibition of autophagy and cell death. Front. Cell. Infect. Microbiol. 7:154. 10.3389/fcimb.2017.00154 PubMed DOI PMC

Yang Y., Hong Y., Cho E., Kim G. B., Kim I.-S. (2018). Extracellular vesicles as a platform for membrane-associated therapeutic protein delivery. J. Extracell. Vesicles 7:1440131. 10.1080/20013078.2018.1440131 PubMed DOI PMC

Yoon H., Ansong C., Adkins J. N., Heffron F. (2011). Discovery of Salmonella virulence factors translocated via outer membrane vesicles to murine macrophages. Infect. Immun. 79 2182–2192. 10.1128/IAI.01277-10 PubMed DOI PMC

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