Chapter 6 : Toxins and Type II Secretion Systems

Assembly and secretion of CT. The individual A and B subunits of CT are transported as precursor proteins across the cytoplasmic membrane via a Sec-like mechanism. The signal peptides are removed, and the subunits fold and assemble with the assistance of disulfide isomerase DsbA into the AB5 complex in the periplasmic compartment. The assembled toxin is then translocated across the outer membrane via the type II secretion apparatus that is encoded by the eps genes (C to N) and vcpD (O).

Model of pilus-mediated secretion by the type II apparatus. Assembled CT (AB5) is targeted to the secretion apparatus via specific recognition of B5 in a process that may involve components C and/or D (I). Upon binding, conformational changes in the type II apparatus may result in accommodation of the AB5 complex and lead to polymerization of component G and the other pilin-like proteins into a piston-like structure that extends from the E-F-L-M platform in the cytoplasmic membrane (II). Through the active process of pilin subunit polymerization, AB5 is then pushed through the channel to the extracellular environment in a process that is thought to be regulated and/or energized by the E component (III). Proteins A, B, N, and S are not shown in this model since they are not present in all organisms that encode and assemble a type II pathway and these proteins may not be required for secretion in every case.

Polar localization of GFP-EpsL and GFP-EpsM protein fusions. Fluorescent microscopy, with the aid of an FITC filter, was used to determine the location within the cell of different GFP constructs. (A) GFP in V. cholerae epsL mutant. (B) GFP-EpsL in V. cholerae epsL mutant. (C) GFP-EpsM in V. cholerae epsM mutant. (D) GFP-EpsM in E. coli MC1061. (E) GFP-EpsL in E. coli MC1061. (F) GFP-EpsL and EpsM in E. coli MC1061.

Polar Eps-dependent protease secretion in single cells. Cells of wildtype V. cholerae TRH7000 (A) and epsM mutant PU3 (B) that expressed IPTGinducible HA/protease from plasmid (pHAP) were embedded in agarose supplemented with M9 salts, amino acids, IPTG, and intramolecularly quenched casein. Casein became highly fluorescent when cleaved by secreted HA/protease. The location of HA/protease secreted from single cells via the Eps apparatus was determined by fluorescence microscopy with the use of a TRITC filter after overnight incubation at 37°C. Next to panels (A) and (B) are the corresponding phase-contrast images.