Docking and Assembly of the Type II Secretion Complex of Vibrio cholerae

Abstract

Secretion of cholera toxin and other virulence factors from Vibrio cholerae is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (eps) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC-and GFP-EpsM-expressing strains disperse upon deletion of epsD, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.The type II secretion (T2S) pathway is widely used by pathogenic gram-negative bacteria for delivery of virulence factors into the extracellular milieu (11,17,46). Proteins destined for release through this pathway are first translocated across the cytoplasmic membrane via the Sec (24, 42) or Tat (59) machinery. Following folding and assembly in the periplasm, the proteins are transported across the outer membrane via the T2S machinery, a complex composed of 12 to 16 different gene products, depending on the species. In Vibrio cholerae, the elements of the T2S apparatus are encoded by the extracellular protein secretion (eps) genes, epsC through epsN and pilD (vcpD) (18,31,39,49,50). Together these proteins coordinate the outer membrane translocation of the major virulence factor, cholera toxin, as well as chitinase, lipase, hemagglutinin/ protease, and other proteases (12,27,49). Our studies are focused on better understanding how the T2S complex assembles in the cell envelope of V. cholerae to begin to elucidate the mechanism by which extracellular secretion is accomplished.

DNA-uptake machinery of naturally competent Vibrio cholerae

“…3). Interestingly, a recent study on the type II secretion system of V. cholerae also showed discrete fluorescent foci along the cell periphery for different components of this macromolecular complex (37). This finding is in contrast to the polar localization pattern described earlier (22), which most likely resulted from an overexpression artifact (37).…”

“…Indeed, Lybarger et al concluded that "chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins" (ref. 37, p. 3149), and such conditions were used throughout this study.…”

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

“…To overproduce protein encoded by the VC1200 loci, plasmid pVesB was used (50). The plasmids were introduced into the wild-type and various isogenic mutants of V. cholerae N16961 (as indicated in the text) by triparental conjugation (29).…”

“…CT A and B subunits were separated under denaturating and reducing conditions as indicated. Immunoblots were primarily blocked in 3% BSA in TBS buffer (50 mM Tris, 200 mM NaCl, pH 8), followed by incubation with polyclonal anti-CT antibodies (1:5000; Sigma) using as a positive control purified cholera toxin (Sigma) and finally developed (50). Immunoblots were developed using typhoon Trio (Amersham Biosciences).…”

On the path to uncover the bacterial type II secretion system

“…Moreover, while results obtained in K. oxytoca and V. cholerae [42][43][44] with GFP-fused Gsp proteins indicate a circumferential distribution of the machinery into foci, the P. aeruginosa Xcp secreton was proposed to be polar. This was shown by adding a Lumino tag onto XcpR or XcpS or by the visualization of protease secretion with an intramolecularly quenched casein conjugate [45].…”