Proteomic Characterization and Functional Analysis of Outer Membrane Vesicles of Francisella novicida Suggests Possible Role in Virulence and Use as a Vaccine

Abstract

We have isolated and characterized outer membrane vesicles (OMVs) from Francisella. Transport of effector molecules through secretion systems is a major mechanism by which Francisella tularensis alters the extracellular proteome and interacts with the host during infection. Outer membrane vesicles produced by Francisella were examined using TEM and AFM and found to be 43-125 nm in size, representing another potential mechanism for altering the extracellular environment. A proteomic analysis (LC-MS/MS) of OMVs from F. novicida and F. philomiragia identified 416 (F. novicida) and 238 (F. philomiragia) different proteins, demonstrating that OMVs are an important contributor to the extracellular proteome. Many of the identified OMV proteins have a demonstrated role in Francisella pathogenesis. Biochemical assays demonstrated that Francisella OMVs possess acid phosphatase and hemolytic activities that may affect host cells during infection, and are cytotoxic toward murine macrophages in cell culture. OMVs have been previously used as a human vaccine against Neisseria meningitidis . We hypothesized that Francisella OMVs could be useful as a novel Francisella vaccine. Vaccinated BALB/C mice challenged with up to 50 LD50 of Francisella showed statistically significant protection when compared to control mice. In the context of these new findings, we discuss the relevance of OMVs in Francisella pathogenesis as well as their potential use as a vaccine.

Identification of a Novel Francisella tularensis Factor Required for Intramacrophage Survival and Subversion of Innate Immune Response

“…Our hypothesis is that Francisella sheds OmpA-like protein during its residence in the mildly acidic environment of the phagosomes and during its rapid replication in the macrophage cytosol. The support for our hypothesis comes from a recent report demonstrating that Francisella produces outer membrane vesicles that may serve as a "vesicle-mediated secretion" system to deliver bacterial proteins in the extracellular milieu (68). Outer membrane vesicles are spherical fragments of bacterial outer membrane that are produced continuously without the concomitant bacterial lysis by several Gram-negative bacteria to mediate inflammatory response and virulence in vivo (49,69).…”

“…Outer membrane vesicles are spherical fragments of bacterial outer membrane that are produced continuously without the concomitant bacterial lysis by several Gram-negative bacteria to mediate inflammatory response and virulence in vivo (49,69). Interestingly, proteome characterization of outer membrane vesicles from F. novicida revealed the presence of FTN_0346, an ortholog of FTT0831c/FTL_0325 proteins of F. tularensis LVS and SchuS4, respectively (68). Based on these observations, we speculate that outer membrane vesicles containing surface-exposed OmpA-like protein shed abundantly by Francisella in the macrophage cytosol may interact with cytosolic NF-B-signaling components to mediate its suppressive effect.…”

Predatory activity of Myxococcus xanthus outer-membrane vesicles and properties of their hydrolase cargo

“…For instance, phosphatases (hydrolases that liberate inorganic phosphate from organophosphate molecules) are frequently found in OMVs (e.g. Bomberger et al, 2009;Horstman & Kuehn, 2000;Pierson et al, 2011). OMVs can lyse next to Gram-positive cells, releasing their contents next to a target cell.…”

Discovery of Salmonella Virulence Factors Translocated via Outer Membrane Vesicles to Murine Macrophages

“…OMV not only disseminate toxins and enzymes into host cells to protect bacterial survival but also serve as bacterium-like decoys to neutralize the host immune responses and facilitate Gram-negative pathogens' immune evasion (10,71). OMV delivery of antigens has been a large area of investigation, as OMV could improve vaccines by delivery to the major histocompatibility complex (MHC) class I pathway while simultaneously providing pathogen-associated molecular patterns as adjuvants (2,4,62,73). Based on the accumulative observations of OMV in a variety of microorganisms, multiple biological functions of OMV, including virulence attributes and its biogenesis, including production mechanism and cargo selectivity have been reviewed recently (1,21,46).…”