Proteolytic Cleavage of the FlhB Homologue YscU of Yersinia pseudotuberculosis Is Essential for Bacterial Survival but Not for Type III Secretion

Abstract

The pathogenic species of the genus Yersinia (Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis) cause infections of highly varied severity in humans. Y. pestis causes plague and is transmitted by flea bites or infectious aerosols, whereas Y. enterocolitica and Y. pseudotuberculosis are enteric pathogens that cause gastroenteritis after the ingestion of contaminated food or water (for reviews, see references 9 and 35). Still, the virulence mechanisms of the different species show a lot of similarities. One such similarity is the ability to inhibit phagocytosis, which enables the pathogens to replicate in lymphoid tissues. This is conferred by an ca. 70-kb plasmid that is required for virulence in all three species. The plasmid encodes a type III secretion system (TTSS) that delivers antihost proteins or virulence effectors called Yops (Yersinia outer proteins) into the cytosol of eukaryotic cells (12, 13). Yop secretion is normally triggered by eukaryotic cell contact (36, 37), but it can also be induced in vitro by growing the bacteria in calcium-depleted medium at 37°C (13).TTSSs are found in several gram-negative animal and plant pathogens (20, 39). The overall mechanism of secretion appears to be conserved in the different systems. Typically, 20 to 25 proteins are required to assemble a functional secretion system. Nine of these proteins are conserved not only in the TTSSs of different pathogens but also in the bacterial flagellar export apparatus (for reviews, see references 1, 20, and 25). For several animal pathogens, the type III secretion organelle, also referred to as the secreton, has been isolated and analyzed (5,6,21,22,40,44). The basal body of this structure possesses two sets of rings resembling the flagellar basal body. The components common to the virulence associated and the flagellar TTSS are believed either to associate with the cytoplasmic face of the basal body-like structure or to form a pore in the inner membrane ring (15,42). A common feature of secretons isolated so far is a needle-like structure that protrudes from the ring structure located in the outer membrane. This needle is required for secretion, suggesting that the combination of the basal portion and needle extension (needle complex) constitutes an intact secretion organelle (5,6,21,22,40,44). In Yersinia spp. the needle-like structure is comprised of the YscF protein and localizes to the bacterial cell surface prior to eukaryotic cell contact (18; P. Edqvist, J. Olsson, M. Lavander, L. Sundberg, Å. Forsberg, H. Wolf-Watz, and S. Lloyd, unpublished data).The proteins forming the actual secretion apparatus are believed to somehow identify the type III secretion substrates to enable their secretion through the basal body-like structure. One key protein in the export of flagellar components is FlhB, a membrane protein with a large cytoplasmic C-terminal domain (29,30). YscU, the corresponding protein of the TTSS of Yersinia spp. has also been shown to localize to the cytoplasmic membrane (3). The flagellum is a tripar...

YscP and YscU Regulate Substrate Specificity of theYersiniaType III Secretion System

“…In any event, this suppression suggests that YscU, together with YscP, has a role in regulating the secretion of Yop effectors as well as YscF in the Yersinia TTSS. Like FlhB (30), the C-terminal cytoplasmic part of YscU is organized into two domains, as it was demonstrated that YscU was specifically proteolyzed in vivo (25). Although there is no evidence that the proteolysis of YscU per se is important for the function of the Yersinia TTSS, it clearly reflects the conformation of the YscU cytoplasmic domains.…”

Organization and coordinated assembly of the type III secretion export apparatus

“…In addition to the structural components of the inner and outer rings of the NC (PrgH, PrgK, and InvG) (6) and the needle and inner rod proteins (PrgI and PrgJ) (17,18), we detected the export apparatus components InvA (19), SpaP, and SpaS (20) (Table S1). To validate the interaction of the export apparatus components with the NC base, we immunoprecipitated the complex from a strain carrying a carboxy terminal FLAG-epitope tagged allele of SpaS (SpaS N258A ), which carries a mutation in its catalytic, autoprocessing site, thus preventing the cleavage of its carboxyl terminus (21,22). Proteins affinity purified with an anti-FLAG antibody were separated by BN-PAGE, and the band corresponding to the NC was analyzed by LC-MS/MS.…”

FlhB Regulates Ordered Export of Flagellar Components via Autocleavage Mechanism

“…In Yersinia pseudotuberculosis, mutations in the cytoplasmic domain of the inner membrane protein YscU (FlhB homologue) suppress the yscP (fliK homologue) polyneedle phenotype (39). In addition, it has been shown that specific cleavage occurs at the equivalent site (Asn-263/Pro-264) of YscU, and mutations at this site have complex effects on late substrate secretion and growth (40).…”