Virulence protein secretion in Gram-negative bacteria via the type V pathway

Type V secretion is among the multiple protein secretion pathways present in Gram-negative bacteria and include the autotransporter (AT) and two-partner secretion (TPS) systems. An AT polypeptide has three domains: a cleavable N-terminal signal sequence, an internal passenger domain, and a C-terminal translocator domain. In the TPS system, the passenger and translocator domains are encoded on two separate polypeptides. The translocator of either system forms a beta-barrel in the outer membrane, allowing the passenger to be transported to the cell surface. The biological functions of the secreted substrates are associated with the passenger domains, most of which are virulence determinants implicated in bacterial pathogenesis.;To study the secretion mechanism of ATs, Tsh from the serine protease autotransporters of the Enterobacteriaceae (SPATE) family was used as a model protein. We asked if conserved residues in the SPATE beta-barrel forming region played important roles in passenger secretion. Site-directed mutagenesis and cell fractionation revealed that more than half of the Tsh mutants showed different degrees and a variety of abnormal phenotypes, including processing errors, cell lysis, and accumulation of proteins in the periplasm. The diversity of these defects implies that conserved residues in the beta-barrel forming region carry out different functions in SPATE secretion. Based on the data, the secretion roles of several conserved residues were interpreted and their involvement in SPATE secretion was deduced.;To identify novel virulence proteins whose secretion might be relevant for the pathogenicity of Yersinia pestis, the plague-causing agent, the genome of Y. pestis KIM was screened by bioinformatics methods, which revealed the components of all the secretion systems known to date in Gram-negative bacteria, including 14 type V systems. RT-PCR analysis confirmed that 13 genes encoding the type V secretion substrates are expressed in this organism. Five AT genes were cloned into Escherichia coli K12 relying on native promoters, and the expression and localization of the proteins (Yaps) in the cloning host were established. Western analysis demonstrated protein expression and secretion of YapA in Y. pestis . Three Yaps also demonstrated cell adherence activities. The study provides evidence that Y. pestis type V systems are functional and virulent.