Cytotoxic and Inflammatory Responses Induced by Outer Membrane Vesicle-Associated Biologically Active Proteases from Vibrio cholerae

Abstract

e Proteases in Vibrio cholerae have been shown to play a role in its pathogenesis. V. cholerae secretes Zn-dependent hemagglutinin protease (HAP) and calcium-dependent trypsin-like serine protease (VesC) by using the type II secretion system (TIISS). Our present studies demonstrated that these proteases are also secreted in association with outer membrane vesicles (OMVs) and transported to human intestinal epithelial cells in an active form. OMV-associated HAP induces dose-dependent apoptosis in Int407 cells and an enterotoxic response in the mouse ileal loop (MIL) assay, whereas OMV-associated VesC showed a hemorrhagic fluid response in the MIL assay, necrosis in Int407 cells, and an increased interleukin-8 (IL-8) response in T84 cells, which were significantly reduced in OMVs from VesC mutant strain. Our results also showed that serine protease VesC plays a role in intestinal colonization of V. cholerae strains in adult mice. In conclusion, our study shows that V. cholerae OMVs secrete biologically active proteases which may play a role in cytotoxic and inflammatory responses. Vibrio cholerae is the causative agent of the life-threatening disease cholera. Cholera epidemics in Haiti in 2010 provide evidence that it still remains an ongoing public health threat (1). Strains of the El Tor biotype O1 serogroup are responsible for seventh pandemic and recent cholera outbreaks (2). Cholera toxin (CT) and toxin-coregulated pilus (TCP) have been identified as the major virulence factors for V. cholerae pathogenesis. CT is responsible for profuse watery diarrhea, whereas TCP is essential for sustaining colonization of human small intestine. V. cholerae also secretes several proteases which may also play a role in its pathogenesis (3).The major protease secreted by V. cholerae strains is the 35-kDa hemagglutinin protease (HAP) (4). HAP has been reported to accelerate the bacterial detachment from cultured cells by digestion of V. cholerae adhesins (5). HAP modulates the enterotoxigenicity of cholera toxin by nicking the A subunit of CT (6). HAP also plays a role in processing hemolysin to its mature form by removal of a 15-kDa N-terminal peptide (7). As shown in the above-mentioned studies, HAP plays an indirect role in V. cholerae pathogenesis. The possibility of its direct role in pathogenesis has been shown by Ghosh et al. (8). They reported that purified HAP from a V. cholerae non-O1, non-O139 strain showed a hemorrhagic response in rabbit ileal loops (RILs) and an increase in intestinal short-circuit current in Ussing's chamber (8). Besides HAP, the other major well-characterized metalloprotease is a 97-kDa Vibrio cholerae protease, PrtV which has been shown to play a role in virulence in the Caenorhabditis elegans infection model (9). In an earlier study, we also reported the presence of a novel serine protease encoded by VC1649 (VesC) in a hapA prtV mutant Vibrio cholerae O1 strain and showed its role in the hemorrhagic response in the rabbit ileal loop model (10).Gram-negative bacteria, including V. cholerae, us...

Mechanisms of outer membrane vesicle entry into host cells

“…Induction of pro‐inflammatory cytokines following infection with OMVs was measured using ELISA, and cytotoxicity determined using flow cytometry. Both responses were reduced after treatment with mbcd to deplete cholesterol, but no effect was observed after dynamin inhibition (Mondal et al, ). The oral pathogen Porphyromonas gingivalis secretes OMVs containing virulence factors such as gingipains and fimbriae, and these OMVs were shown to enter HeLa and gingival epithelial cells in a Rac1/lipid raft dependent manner, and independent of caveolin, clathrin, and dynamin (Furuta et al, ).…”

Bacterial membrane vesicles: Biogenesis, immune regulation and pathogenesis

“…Interactions between OMVs and epithelial cells can modulate cellular mechanisms that control proliferation (Li et al, ), apoptosis (Kunsmann et al, ; Mondal et al, ), and ultimately immune responses. Cronobacter sakazakii , a causative agent of infant meningitis and enterocolitis, produces OMVs that are internalized by intestinal epithelial cells inducing cell proliferation, IL‐8 secretion and facilitating bacterial persistence (Alzahrani, Winter, Boocock, De Girolamo, & Forsythe, ).…”

Association of Vibrio cholerae 569B outer membrane vesicles with host cells occurs in a GM1‐independent manner

“…Cholesterol-rich lipid rafts have been commonly reported to be involved in the uptake of OMVs produced by a number of species, including enterotoxigenic Escherichia coli (ETEC), Haemophilus influenzae , Campylobacter jejuni , and Pseudomonas aeruginosa , among others (Bomberger et al, 2009; Elmi et al, 2012; Kaparakis et al, 2010; Kesty, Mason, Reedy, Miller, & Kuehn, 2004; Mondal et al, 2016; Sharpe, Kuehn, & Mason, 2011), while OMVs produced by other organisms, including Helicobacter pylori and enterohemorrhagic E. coli (EHEC), have been reported to enter cells in cholesterol-independent endocytic processes (Bielaszewska et al, 2017; Bielaszewska et al, 2013; Canas et al, 2016; Kunsmann et al, 2015; Parker, Chitcholtan, Hampton, & Keenan, 2010). …”