Analysis of the function of the Yersinia protein kinase YpkA and its role in bacterial pathogenesis.

摘要

Pathogenic species of Yersinia utilize a type III secretion apparatus to translocate six effector proteins into the host cell. These proteins downregulate the immune response of the host, thereby insuring bacterial survival. One effector protein, YpkA, contains a eukaryotic serine/threonine protein kinase domain. The experiments described here analyze the role of YpkA in Yersinia pathogenesis and identify its target within the host cell.; Transient overexpression of YpkA in mammalian cells and in vitro kinase assays revealed that YpkA is expressed as an active kinase in mammalian cells. However, recombinant YpkA was completely inactive in vitro, suggesting that mammalian cells contain an activator of YpkA. Indeed, addition of eukaryotic cell extracts to recombinant YpkA resulted in the stimulation of YpkA kinase activity. Biochemical purification identified actin as an activator of YpkA. Addition of purified actin to YpkA resulted in a dose-dependent activation of YpkA, with maximal activity measured at stoichiometric ratios of YpkA and actin. Deletion of the C-terminal amphipathic helix in YpkA disrupted both YpkA kinase activity and the interaction between YpkA and actin. Transient overexpression of YpkA in HeLa cells resulted in a cytotoxic phenotype with a complete disruption of actin stress fibers. In vitro kinase assays revealed that both G- and F-actin are substrates for YpkA. Although, phosphorylation of actin does not inhibit in vitro polymerization, it may inhibit actin dynamics in vivo.; To identify novel YpkA substrates, a technique termed chemical genetics was employed. This technique identified a novel 36-kDa substrate for YpkA within mammalian extracts. This 36-kDa protein was identified as human protein FLJ20113 using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. FLJ20113 is not only an in vitro substrate for YpkA, but it also co-immunoprecipitates with YpkA and the actin cytoskeleton. FLJ20113 resembles the ovarian tumor cysteine protease superfamily, suggesting a role for a novel protease in the regulation of actin dynamics. Collectively, these studies reveal a mechanism of YpkA regulation and clarify its role in Yersinia pathogenesis.