Identification of YsaP, the Pilotin of the Yersinia enterocolitica Ysa Type III Secretion System.

Abstract

Secretins are multimeric outer membrane pore-forming proteins found in complex export systems in Gram-negative bacteria. All type III secretion systems (T3SSs) have a secretin, and one of these is the YsaC secretin of the chromosomally encoded Ysa T3SS of Yersinia enterocolitica. In some cases, pilotin proteins, which are outer membrane lipoproteins, are required for their cognate secretins to multimerize and/or localize to the outer membrane. However, if secretin multimers mislocalize to the inner membrane, this can trigger the protective phage shock protein (Psp) stress response. During a screen for mutations that suppress YsaC toxicity to a psp null strain, we isolated several independent mutations predicted to increase expression of the YE3559 gene within the Ysa pathogenicity island. YE3559, which we have named ysaP, is predicted to encode a small outer membrane lipoprotein, and this location was confirmed by membrane fractionation. Elevated ysaP expression increased the steady-state level of YsaC but made it less toxic to a psp null strain, and it also decreased YsaC-dependent induction of psp gene expression. Subsequent experiments showed that YsaP was not required for YsaC multimerization but was required for the multimers to localize to the outer membrane. Consistent with this, a ysaP null mutation compromised protein export by the Ysa T3SS. All these observations suggest that YsaP is the pilotin for the YsaC secretin. This is only the second pilotin to be characterized for Yersinia and one of only a small number of pilotins described for all bacteria. Secretins are essential for the virulence of many bacterial pathogens and also play roles in surface attachment, motility, and competence. This has generated considerable interest in understanding how secretins function. However, their fundamental differences from typical outer membrane proteins have raised various questions about secretins, including how they are assembled into outer membrane multimers. Pilotin proteins facilitate the assembly of some secretins, but only a small number of pilotins have been identified, slowing efforts to understand common and distinct features of secretin assembly. This study provides an important advance by identifying a novel member of the pilotin family and also demonstrating a method of pilotin discovery that could be broadly applied.