Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases

Abstract

The type II secretion (T2S) system is responsible for extracellular secretion of a broad range of proteins, including toxins and degradative enzymes that play important roles in the pathogenesis and life cycle of many Gram-negative bacteria. In Vibrio cholerae, the etiological agent of cholera, the T2S machinery transports cholera toxin, which induces profuse watery diarrhea, a hallmark of this life-threatening disease. Besides cholera toxin, four other proteins have been shown to be transported by the T2S machinery, including hemagglutinin protease, chitinase, GbpA, and lipase. Here, for the first time, we have applied proteomic approaches, including isotope tagging for relative and absolute quantification coupled with multidimensional liquid chromatography and tandem mass spectrometry, to perform an unbiased and comprehensive analysis of proteins secreted by the T2S apparatus of the V. cholerae El Tor strain N16961 under standard laboratory growth conditions. This analysis identified 16 new putative T2S substrates, including sialidase, several proteins participating in chitin utilization, two aminopeptidases, TagA-related protein, cytolysin, RbmC, three hypothetical proteins encoded by VCA0583, VCA0738, and VC2298, and three serine proteases VesA, VesB, and VesC. Focusing on the initial characterization of VesA, VesB, and VesC, we have confirmed enzymatic activities and T2S-dependent transport for each of these proteases. In addition, analysis of single, double, and triple protease knock-out strains indicated that VesA is the primary protease responsible for processing the A subunit of cholera toxin during in vitro growth of the V. cholerae strain N16961.Gram-negative bacteria have evolved at least six secretion pathways devoted to the transport of proteins through the cell envelope into either the extracellular environment or directly into host cells (1, 2). The type II secretion (T2S) 2 system was first discovered in Klebsiella oxytoca and has been shown to be widely distributed among ␥-proteobacteria (3-6). Depending on the bacterial species, the T2S complex consists of 12-16 different constituents that form a multiprotein apparatus spanning the entire cell envelope (7,8). The conserved components of the T2S machinery include the cytoplasmic ATPase (T2S E), the inner membrane platform (T2S C, F, L, and M), a pilus-like structure (T2S G-K), a protein responsible for the processing of pseudopilins (T2S O), and the secretion pore (T2S D) embedded in the outer membrane (9). The exoprotein precursors are synthesized with N-terminal signal peptides that direct them into the periplasmic space via either the Sec or Tat transport systems (10, 11). After obtaining tertiary conformation, the exoproteins enter the T2S machinery and are subsequently translocated into the extracellular milieu (12, 13). Many key steps in the secretion process are still not well understood, including how the exoproteins are recognized by the T2S system, and a specific secretion signal common to known substrates has not yet been identified....

Type II Secretion-Dependent Degradative and Cytotoxic Activities Mediated by Stenotrophomonas maltophilia Serine Proteases StmPr1 and StmPr2

“…and human (i.e., Legionella pneumophila, Pseudomonas aeruginosa, Vibrio spp., and enterohemorrhagic Escherichia coli) pathogens (22). Among these pathogens, serine proteases have been identified as T2S substrates in Xanthomonas campestris and Vibrio cholerae (41)(42)(43). BLASTP analysis revealed that StmPr1 produced by strain K279a is most homologous with an annotated serine protease of Pseudomonas geniculata (69% identity), followed by annotated serine proteases of Xanthomonas sacchari (62% identity) and X. campestris (57% identity).…”

Regulation of bacterial virulence gene expression by cell envelope stress responses

“…V. cholerae employs a T2SS to export at least 19 proteins, including its major virulence factor cholera toxin that is responsible for the profuse watery diarrhea that defines the disease. 74,75 The epsC-N operon and the pilD gene are required for production of the V. cholerae T2SS. 73,76 Aside from its export role, some studies have also implicated this T2SS in maintaining cell envelope integrity.…”

Nanotechnology: the word is new but the concept is old. An overview of the science and technology in food and food products at the nanoscale level

“…V. cholera enters the body through an oral route of infection with contaminated water or food, and colonises the small intestine where it produces and secretes cholera toxin (Sikora, Zielke, Lawrence, Andrews, & Sandkvist, 2011). Its type II secretion system is responsible for extracellular secretion of a broad range of proteins including cholera toxin, hemagglutinin/protease, two aminopeptidases, and three serine proteases VesA, VesB, and VesC among others (Sikora et al, 2011). V. cholera secretes its own protease(s) that is/are responsible for cleaving (and thus activation) of cholera toxin (Booth, Boesmanfinkelstein, & Finkelstein, 1984).…”

“…V. cholera secretes its own protease(s) that is/are responsible for cleaving (and thus activation) of cholera toxin (Booth, Boesmanfinkelstein, & Finkelstein, 1984). VesA, VesB, and VesC were just recently confirmed to have enzymatic activities, and knock-out strains indicated that VesA is the primary protease responsible for processing the A subunit of cholera toxin during in vitro growth of a V. cholerae strain (N16961) (Sikora et al, 2011), presumably indicating that VesA has a high degree of specificity. We suggest that nanocarriers containing crosslinking peptides having cleavage sites specific to either hemagglutinin/protease or VesA could be intelligent and effective strategies for the prevention and halting of this devastating disease, respectively.…”