Modulation of cellular Pseudomonas exotoxin A sensitivity through induced changes in toxin receptor expression

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Laithwaite, James Edward
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University of Guelph

Pseudomonas' Exotoxin A (PEA) is an intracellular-acting bacterial exotoxin produced by the opportunistic pathogen 'Pseudomonas aeruginosa'. PEA intoxication is a multi-step process in which PEA exploits the cellular machinery of target cells in an effort to reach the cytoplasm in an activated state capable of inhibiting protein synthesis. We hypothesized that induced alterations in the expression levels of specific components of the cellular machinery exploited by PEA during intoxication represent mechanisms by which cellular sensitivity to PEA can be altered by the host. To test this hypothesis, we first investigated whether alterations in cellular sensitivity to PEA can be induced by diverse cellular events. Using a [3H]leucine incorporation assay to measure inhibition of protein synthesis, we demonstrated that primary rat hepatocytes cultured on a collagen type I matrix show markedly decreased sensitivity to PEA in a time-dependent fashion. PEA sensitivity is also markedly decreased in the rat macrophage-like cell line HS-P in a dose- and time-dependent manner after LPS treatment. We also demonstrated that normal and transformed murine liver cells exhibit divergent PEA sensitivities, with transformed cells demonstrating greater PEA sensitivity than their non-transformed counterparts. PEA intoxication begins when PEA binds the low-density lipoprotein receptor-related protein (LRP). PEA toxicity was decreased by a LRP antagonist, the receptor-associated protein, confirming the importance of the LRP in PEA intoxication in these three cell types. Induced alterations in cellular PEA sensitivity were positively correlated with changes in functional cell surface LRP expression, as measured by [alpha]2-macroglobulin internalization studies, and LRP mRNA levels, as determined by Northern blot analysis. The importance of LRP expression in mediating PEA sensitivity was also addressed with conjugate PEA toxins that do not utilize the LRP for cellular entry. We conclude that induced alterations in the expression levels of the LRP are an important mechanism by which cellular PEA sensitivity is altered by the host. These studies indicate that it is probable that the production of various LRP regulatory factors may be initiated in response to 'P. aeruginosa', consequently altering cellular and tissue PEA sensitivity during infection.

Pseudomonas exotoxin A, cellular sensitivity, induction, toxin receptor expression