INTRACELLULAR TARGETING OF EXOENZYME-S OF PSEUDOMONAS-AERUGINOSA VIA TYPE-III-DEPENDENT TRANSLOCATION INDUCES PHAGOCYTOSIS RESISTANCE, CYTOTOXICITY AND DISRUPTION OF ACTIN MICROFILAMENTS

Exoenzyme S (ExoS) is an ADP-ribosyltransferase secreted by the opport unistic pathogen Pseudomonas aeruginosa. The amino-terminal half of Ex oS exhibits homology to the YopE cytotoxin of pathogenic Yersinia. Rec ently, YopE was found to be translocated into the host cell by a bacte ria-cell contact-dependent mechanism involving the ysc-encoded type II I secretion system, By using an approach in which exoS was expressed i n different strains of Yersinia, including secretion and translocation mutants, we could demonstrate that ExoS was secreted and translocated into HeLa cells by a similar mechanism to that described previously f or YopE, Similarly to YopE, the presence of ExoS in the host cell elic ited a cytotoxic response, correlating with disruption of the actin mi crofilament structure. A similar cytotoxic response was also induced b y a mutated form of ExoS with a more than 2000-fold reduced ADP-ribosy ltransferase activity. However, the enzymatically active ExoS elicited a more definite rounding up of the HeLa cells, which also correlated with decreased Viability of the cells after prolonged infection compar ed with cells infected with strains expressing mutated ExoS or YopE. T his suggests that ExoS can act through two different mechanisms on the host cell, The expression of ExoS by Yersinia also mediated an anti-p hagocytic effect on macrophages. In addition, we present evidence that extracellularly located P. aeruginosa is able to target ExoS into euk aryotic cells. Taken together, our data suggest that P. aeruginosa, by analogy with Yersinia, targets virulence proteins into the eukaryotic cytosol via a type III secretion-dependent mechanism as part of an an ti-phagocytic strategy.