Abstract
The Yersinia protein kinase A (YpkA) and outer protein J (YopJ) are co-expressed from a single transcript and are injected directly into eukaryotic cells by the plague bacterium Yersinia pestis. When overexpressed in vertebrate or yeast cells, YpkA disrupts the actin-based cytoskeletal system by an unknown mechanism, whereas YopJ obstructs inductive chemokine expression by inhibiting MAPK and NF-kappaB signaling. Previously, we showed that the fission yeast Schizosaccharomyces pombe was sensitive to the kinase activity of YpkA. Here, we screened yeast for cellular processes important for YpkA activity and found that the eIF2alpha kinases mollify the toxicity imparted by the kinase activity of YpkA. Specifically, strains lacking the eIF2alpha kinase Hri2 were particularly sensitive to YpkA. Unexpectedly, the activity of YopJ, which conferred a phenotype consistent with its inhibitory effect on MAPK signaling, was also found to be dependent on Hri2. When expressed in S. pombe, YopJ sensitized cells to osmotic and oxidative stresses through a Hri2-dependent mechanism. However, when co-expressed with YpkA, YopJ protected cells from YpkA-mediated toxicity, and this protection was entirely dependent on Hri2. In contrast, YopJ did not confer protection against the toxic effects of the Yersinia virulence factor YopE. These findings are the first to functionally link YpkA and YopJ and suggest that eIF2alpha kinases, which are critically important in antiviral defenses and protection against environmental stresses, also play a role in bacterial virulence.
Highlights
Health Service Grant AI53459. 1 To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of Miami Miller School of Medicine, 1600 NW 10th Ave, Miami, FL 33136
Yersinia protein kinase A (YpkA) and its associated kinase activity is necessary for the immediate survival of Yersinia following attachment to host cells; this was suggested by animal infection experiments and confirmed at the cellular level in a function-based “infectivity” assay that measures both survival and growth of macrophage-associated Yersinia [3, 4]
YpkA has been widely characterized as a “cytoskeletal disruptor” based, again, on either transfection systems or infection studies in which YpkA is overexpressed in trans in the absence of the other 5 Yops; predictably this activity is primarily dependent on the GTPase-binding domain, and, to a much lesser degree, on its kinase activity [12, 13, 16]
Summary
Health Service Grant AI53459. 1 To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of Miami Miller School of Medicine, 1600 NW 10th Ave, Miami, FL 33136. For the elongation assay measuring YopJ activity, cells type), putative YpkAR clones were crossed with the parental were propagated and induced as described for the ghost assay wild-type strain, and the resulting diploids were selected and except that after 16 h of induction cultures were diluted to A595 maintained by intra-allelic complementation.
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