The Ser/Thr kinase activity of the Yersinia protein kinase A (YpkA) is necessary for full virulence in the mouse, mollifying phagocytes, and disrupting the eukaryotic cytoskeleton

David J. Wiley, Roland Nordfeldth, Jason Rosenzweig, Christopher J. DaFonseca, Richard Gustin, Hans Wolf-Watz, Kurt Schesser

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

The Yersinia protein kinase A (YpkA) is injected into host cells by the yersinial type three secretion system (TTSS). YpkA is widely believed to function within the host cell based on the fact that its kinase domain is clearly homologous to eukaryotic Ser/Thr kinases and that its enzymatic activity, when assayed in vitro, is dependent on eukaryotic-derived host factors. Whether this activity is required for virulence has not been addressed. Here, we report that a Yersinia pseudotuberculosis strain expressing a kinase-inactive YpkAD270A variant is greatly attenuated in the mouse model of infection compared to the isogenic wild-type strain. The ypkAD270A mutant strain was likewise attenuated in a cell culture infection assay indicating that the kinase activity of YpkA enhances the viability of host cell-associated bacteria. To begin to understand what cellular activities are targeted, we expressed YpkA and its variants in two different yeast model systems. In agreement with previous studies, we found that when rapidly induced and expressed at high levels in Saccharomyces cerevisiae, YpkA-mediated toxicity occurred extremely swiftly. Under these conditions toxicity was dependent on the structurally distinct GTPase-binding domain of YpkA and was entirely independent of its kinase activity. Therefore, to probe for kinase-dependent effects we expressed YpkA and its kinase-inactive variant at comparatively moderate levels in the fission yeast Schizosaccharomyces pombe. S. pombe is particularly well suited for actin cytoskeletal studies due to its easily quantifiable, well defined pattern of actin localization. S. pombe transformed with a wild-type YpkA-encoding plasmid displayed a pronounced actin mislocalization phenotype, the severity of which was directly proportional to the level of YpkA expressed in the cell. In cells expressing the kinase-inactive YpkA variant, the degree of actin mislocalization was reduced, but not entirely abrogated, suggesting that YpkA affects the eukaryotic cytoskeleton through kinase-dependent and kinase-independent mechanisms. Collectively, our yeast-derived results show how critical expression levels and exposure periods are for assaying virulence factor activities in heterologous model systems. More generally, our finding that the 'eukaryotic-like' kinase domain of YpkA is important for virulence illustrates how a bacterium can utilize a host-like factor or activity in order to enhance its survival following host cell contact.

Original languageEnglish
Pages (from-to)234-243
Number of pages10
JournalMicrobial Pathogenesis
Volume40
Issue number5
DOIs
StatePublished - May 1 2006

Fingerprint

Yersinia
Phagocytes
Cyclic AMP-Dependent Protein Kinases
Cytoskeleton
Virulence
Phosphotransferases
Schizosaccharomyces
Actins
Yeasts
Yersinia pseudotuberculosis
Saccharomyces cerevisiae Proteins
Bacteria
GTP Phosphohydrolases
Virulence Factors
Infection
Cell Survival
Plasmids

Keywords

  • Fission yeast
  • Kinase
  • Pathogenesis
  • Yersinia
  • YpkA

ASJC Scopus subject areas

  • Microbiology
  • Infectious Diseases

Cite this

The Ser/Thr kinase activity of the Yersinia protein kinase A (YpkA) is necessary for full virulence in the mouse, mollifying phagocytes, and disrupting the eukaryotic cytoskeleton. / Wiley, David J.; Nordfeldth, Roland; Rosenzweig, Jason; DaFonseca, Christopher J.; Gustin, Richard; Wolf-Watz, Hans; Schesser, Kurt.

In: Microbial Pathogenesis, Vol. 40, No. 5, 01.05.2006, p. 234-243.

Research output: Contribution to journalArticle

Wiley, David J. ; Nordfeldth, Roland ; Rosenzweig, Jason ; DaFonseca, Christopher J. ; Gustin, Richard ; Wolf-Watz, Hans ; Schesser, Kurt. / The Ser/Thr kinase activity of the Yersinia protein kinase A (YpkA) is necessary for full virulence in the mouse, mollifying phagocytes, and disrupting the eukaryotic cytoskeleton. In: Microbial Pathogenesis. 2006 ; Vol. 40, No. 5. pp. 234-243.
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