Yersinia pestis YopM: thrombin binding and overexpression

Abstract

In previous studies, Yersinia pestis YopM has been shown through mutational analysis to be necessary for virulence in mice and found to have homology with the thrombin-binding domain of the platelet receptor GPIba. In this study, YopM was purified and shown by dot blot and chemical cross-linking tests to bind to human ct-thrombin. No cross-linked product could be detected when human prothrombin was incubated with YopM. As a functional test of thrombin binding, it was shown that native but not boiled YopM inhibits thrombin-induced aggregation of human platelets. Control tests showed that YopM did not inactivate the platelets themselves, nor was its effect a nonspecific consequence of its very acidic isoelectric point. Microsequencing of YopM revealed an intact N terminus, indicating that functional YopM is not processed at the N terminus or secreted by a mechanism involving a cleavable signal sequence. Further characterization was made of an interesting effect on yopM expression that had been noticed in a previous study. A 1.5-kb HaeTI subclone overexpressed YopM in both Y. pestis and Escherichia coli compared with a larger clone containing the 5.3-kb HindHI-F fragment. To search for a possible regulator ofYopM expression, the Hindm-F fragment was sequenced, revealing several open reading frames and three large repeated sequences. Deletional analysis showed that these were not involved in regulation ofyopM. The data implicated a DNA structure 5' to yopM in moderating yopM expression.

Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells.

“…Four of these, YopH, YopE, YopM and YpkA, have been shown by site-directed mutagenesis to be indispensable for virulence Forsberg and Wolf-Watz, 1988;Leung et al, 1990;Galyov et al, 1993). YopM, which shares high homology with GpIbat, the a-chain of the platelet receptor of the von Willebrand factor, prevents platelet aggregation by interaction with thrombin (Reisner and Straley, 1992). YpkA exhibits a Ser/Thr phosphokinase activity that shows extensive homology with corresponding enzymes of eukaryotic origin (Galyov et al, 1993).…”

Isolation of a new insertion element of Yersinia intermedia closely related to remnants of mobile genetic elements present on Yersinia plasmids harboring the Yop virulon

“…Under the assumption that IS1635 elements may form a composite transposon, it is conceivable that the yopM gene was integrated into the Yersinia virulence plasmids as part of a related mobile genetic element. This was also discussed by Reisner and Straley [18], who discovered the R1, R2 and R3 sequences of the pCD1 plasmid of Y. pestis. However, they did not ¢nd an experimental support for this hypothesis, as plasmid DNA of Shigella £exneri, which harbored the yopM homolog ipaH, did not hybridize to an R2 probe.…”

“…The pCD1 plasmid of Y. pestis harbors three repetitive sequences (R1, R2, R3) with a length of 189 bases (R1), 274 bases (R2) and 275 bases (R3) described by Reisner and Straley [18]. To de¢ne the length of these IS1635-like remnants more precisely a detailed sequence comparison has been performed.…”

“…White rectangles indicate the region of homology to IS1635, gray rectangles inside the Y. pestis (Y.p.) elements show the repetitive sequences R1, R2 and R3 described in [18] dicate a speci¢c target sequence. In addition, no sequence duplication was found.…”

The species-spanning family of LPX-motif harbouring effector proteins

“…The figure was produced using Servier Medical Art Thus, recent findings stating that due to a conserved N-terminal Cys-Leu-Asp sequence motif Y. pestis-derived YopM functions as an E3 ubiquitin ligase targeting NLRP3 (NLR family pyrin domain containing 3) for Lys63-linked ubiquitinylation, which results in the induction of necrotic cell death , must clearly be validated for YopM proteins derived from different strains and other Yersinia species. In addition to extracellular host proteins including α-thrombin, which upon interaction with YopM is no longer able to induce platelet aggregation (Leung, Reisner, & Straley, 1990;Reisner & Straley, 1992), and α 1 -antitrypsin, which is specifically bound but whose antiprotease activity is not affected by YopM (Heusipp, Spekker, Brast, Fälker, & Schmidt, 2006) (Chung et al, 2016;Ratner et al, 2016). Additionally, YopM antagonises inflammasome formation either by direct interaction with caspase-1 or indirectly by targeting the regulatory protein IQGAP1 (IQ motif-containing GTPase-activating protein 1; Chung et al, 2014;LaRock & Cookson, 2012).…”