Proteomic analysis of Vibrio cholerae outer membrane vesicles

Abstract

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria provide an interesting research material for defining cell-envelope proteins without experimental cell disruption. OMVs are also promising immunogenic platforms and may play important roles in bacterial survival and pathogenesis. We used in-solution trypsin digestion coupled to mass spectrometry to identify 90 proteins present in OMVs of Vibrio cholerae when grown under conditions that activate the TCP pilus virulence regulatory protein (ToxT) virulence regulon. The ToxT expression profile and potential contribution to virulence of these proteins were assessed using ToxT and in vivo RNA-seq, Tn-seq, and cholera stool proteomic and other genome-wide data sets. Thirteen OMV-associated proteins appear to be essential for cell growth, and therefore may represent antibacterial drug targets. Another 12 nonessential OMV proteins, including DegP protease, were required for intestinal colonization in rabbits. Comparative proteomics of a degP mutant revealed the importance of DegP in the incorporation of nine proteins into OMVs, including ones involved in biofilm matrix formation and various substrates of the type II secretion system. Taken together, these results suggest that DegP plays an important role in determining the content of OMVs and also affects phenotypes such as intestinal colonization, proper function of the type II secretion system, and formation of biofilm matrix.

Keywords: CTX phage; HtrA family; biofilm formation; in-solution digestion.

Fig. 1.

TcpA was identified in OMVs produced by V. cholerae El Tor C6706. El Tor C6706 cells were grown in the presence (A) or absence (B) of sodium bicarbonate induction and imaged by transmission electron microscopy with an α-TcpA monoclonal antibody. The white arrow indicates TCP and flagella. (“3x” indicates 3× zoom into the selected region). (C) Amino acid sequence of TcpA highlighting the transmembrane domain (underlined) and peptides identified by tryptic digestion of OMVs (in red).

Fig. 2.

CTXΦ-K_m phage-infected assays. Susceptibility of V. cholerae O1 El Tor and classical strains to CTX-K_mΦ under ToxT-induced in vitro condition (LB with sodium bicarbonate) and normal laboratory condition (LB). El Tor strains C6706 wild type, Haiti H1 wild type, C6706 Δ tcpA, and classical strain O395 were tested. Values represent the averages of three independent observations. Significance was determined by t test: **P < 0.01. Mean with stand error of mean (SEM) is shown.

Fig. 3.

(A–C) Venn diagrams showing the cellular location and putative function for identified OMV proteins. The 90 proteins identified on the outer membrane vesicles of V. cholerae grouped into families according to (A) their predicted subcellular localization, (B) proportion of identified proteins to the whole V. cholerae theoretical proteome and (C) function. (D–I) Venn diagrams comparing OMV proteins with previous genomics and proteomics research of V. cholerae. (D) OMVs and ToxT-induced transcriptome (10). (E) OMVs and in vivo-overexpressed genes (48). (F) OMVs and ToxR regulon (49). (G) OMVs and proteins identified in patient stool samples (50). (H) OMVs and V. cholerae essential genes (51). (I) OMVs and important in vivo fitness genes (52).

Fig. 4.

Competition of V. cholerae strains using infant mouse and rabbit colonization models. (A) Ability of six different selected mutant strains to colonize the infant mouse intestine was compared with the parental strain. The dotted line indicates a competitive index of 1. Error bars represent SDs from at least four mice. The major virulence regulatory protein ToxT mutant was used as a negative control. Significance was determined by t test relative to colonization ratio of parental strains WT C6706 versus C6706 lacZ:Tn. ****P < 0.0001. Mean with SEM is shown. (B) Ability of degP mutant (EC956) strain to colonize the infant rabbit intestine was compared with the parental strain. The competitive index was determined for a phenotypically LacZ+ TnFGL3 insertion in lacZ (lacZ::Tn) when competed against its parental strain ΔlacZ WT strain (WT). The competitive index was determined for a phenotypically LacZ+ TnFGL3 insertion in degP mutant (EC956) when competed against its ΔlacZ parental strain (WT) either from the cecal fluid (Center) or distal small intestine (Right). ****P value < 0.0001 and **P < 0.01. Mean with SEM is shown.

Fig. 5.

Effect of degP mutation on biofilm formation in V. cholerae. The wild-type and degP mutant (EC956) strains were inoculated into culture tubes and allowed to grow without shaking at room temperature for 24 h. Data presented are averages of three replicates and error bars represent calculated SDs (OD₅₇₀ of the crystal violet-stained biofilm).