A Tale of Two Pathways: Secretin Assembly in Vibrio cholerae

Abstract

The Type 2 Secretion System (T2SS) is responsible for the transport of toxins and enzymes across the outer membrane of many Gram-negative bacteria. A crucial component of the T2SS is a large pore, composed of a multimer of EpsD, named the secretin. This pore inserts in the outer membrane with the assistance of a pilotin (EpsS) or assembly factors (EpsAB), both of which are present within the genome of Vibrio cholerae. The goal of this study was to determine whether or not both assembly mechanisms operate on the same secretin assembly in V. cholerae. Protease deficient mutants generated from an insertion transposon library in V. cholerae epsAB were analyzed. The transposon was found to disrupt the operon encoding VC1702 and epsS. Mutant strains of V. cholerae were constructed or obtained that are deficient in epsA, epsB, epsC, and epsS. Double mutants were constructed that were deficient in epsA and epsS or epsB and epsS. These mutants were tested for assembly of the secretin and secretion of lipase, protease, and cholera toxin. The epsA and epsB mutants have slightly reduced levels of secretion and secretin assembly, while the levels in the pilotin mutant are drastically reduced. The double mutants had little to no assembly, and secretion was reduced to the levels of the control mutant epsC. In an attempt to restore function epsAB was over-expressed in all strains. It successfully complemented the epsA and epsB mutants, and restored levels of secretion to epsS levels in the double mutant, epsAS. In a similar manner to epsAB complementation, epsS was over-expressed. It was found to require the preceding gene VC1702 to complement. The operon, encoding both epsS and VC1702, could complement both epsA and epsS mutations and over-expression increased secretin assembly and secretion to levels greater than wild-type levels. Lastly, a phylogenomic analysis demonstrates that the EpsAB protein complex is found in most orders of the gamma proteobacteria and is ancestral. The pilotins appear to be a late acquisition as they are only found in the family Enterobacteriales.