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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, DJ ; Nordfeldth, R ; Rosenzweig, J ; DaFonseca, CJ ; Gustin, R ; Wolf-Watz, H and Schesser, Kurt LU (2006) In Microbial Pathogenesis 40(5). p.234-243
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 YpkA(D270A) variant is greatly attenuated in the mouse model of infection compared to the isogenic wild-type strain. The YPkA(D270A) mutant strain was likewise attenuated in a cell culture infection assay indicating... (More)
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 YpkA(D270A) variant is greatly attenuated in the mouse model of infection compared to the isogenic wild-type strain. The YPkA(D270A) 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. (c) 2006 Elsevier Ltd. All rights reserved. (Less)
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author
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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Yersinia, pathogenesis, YpkA, fission yeast, kinase
in
Microbial Pathogenesis
volume
40
issue
5
pages
234 - 243
publisher
Academic Press
external identifiers
  • wos:000238060600006
  • scopus:33747780120
  • pmid:16626927
ISSN
1096-1208
DOI
10.1016/j.micpath.2006.02.001
language
English
LU publication?
yes
id
688b9000-2f1e-4994-96be-75a191f0e0da (old id 407374)
date added to LUP
2016-04-01 11:34:35
date last changed
2021-10-06 02:25:56
@article{688b9000-2f1e-4994-96be-75a191f0e0da,
  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 YpkA(D270A) variant is greatly attenuated in the mouse model of infection compared to the isogenic wild-type strain. The YPkA(D270A) 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. (c) 2006 Elsevier Ltd. All rights reserved.},
  author       = {Wiley, DJ and Nordfeldth, R and Rosenzweig, J and DaFonseca, CJ and Gustin, R and Wolf-Watz, H and Schesser, Kurt},
  issn         = {1096-1208},
  language     = {eng},
  number       = {5},
  pages        = {234--243},
  publisher    = {Academic Press},
  series       = {Microbial Pathogenesis},
  title        = {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},
  url          = {http://dx.doi.org/10.1016/j.micpath.2006.02.001},
  doi          = {10.1016/j.micpath.2006.02.001},
  volume       = {40},
  year         = {2006},
}