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. IMPORTANCESecretins 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.

The Phage Shock Protein Response

“…Klebsiella oxytoca PulD secretin multimers inserted into the E. coli IM were visualized by microscopy, and this event was correlated with increased PspA synthesis (34). Furthermore, in some cases, pilotin proteins facilitate secretin insertion into the OM (57), but those secretins become more potent Psp inducers when their pilotin is absent (e.g., 17,37,79). Other important observations have also implicated the IM as the site of Psp induction.…”

Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system

“…We therefore screened for additional interactors of SctG by co-immunoprecipitation. Notably, while the pilotin mostly fractionates with the OM in membrane preparations [18,25,59], many of the proteins found to interact with the labeled pilotin in the co-immunoprecipitation assay are integral IM proteins or IM-associated proteins. The used SctG-sfGFP fusion is expressed from its native genetic location, supports native secretion (Fig.…”

“…A prime candidate to establish the site of assembly for new injectisomes is the pilotin, a lipoprotein anchored in the inner leaflet of the outer membrane (OM). Pilotins are required for the proper integration and oligomerization of the secretin protein, which forms a large channel in the OM in the T3SS of animal pathogens, but also type II secretion systems (T2SS) and type IV pili (T4P) [15][16][17][18][19][20][21][22][23][24][25][26][27]. Since the secretin ring is one of the nucleation structures of the injectisome [6,28] and anchors the T3SS at its final position in the peptidoglycan mesh [29] (Fig.…”

The YsrS Paralog DygS Has the Capacity To Activate Expression of the Yersinia enterocolitica Ysa Type III Secretion System

“…The ϳ30-kb ysa locus contains a long operon with genes encoding the secretion apparatus, translocon proteins, and one effector protein. Adjacent to this operon, located on the opposite strand, are a few open reading frames encoding the recently identified YsaP pilotin protein (36) and other proteins that are likely part of the secretion apparatus. The ysrRST genes are just downstream of this region, and it was their proximity to the ysa genes that led us to investigate their role in ysa gene expression (22).…”