Abstract
Following entry into the host cytosol, the bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors. The expression of actA, whose protein product is required for L. monocytogenes actin-based intracellular motility, is increased by more than 200-fold in cytosolic bacteria in comparison to broth-grown cultures. Two distinct promoter elements have been reported to regulate actA expression. One promoter is located immediately upstream of actA coding sequences, while the second promoter is contributed by the upstream mpl gene via the generation of an mpl-actA-plcB transcript. A series of L. monocytogenes mutants were constructed to define the contributions of individual promoter elements to actA expression. The intracellular induction of actA expression was found to be dependent upon the actA proximal promoter; the mpl promoter appeared to contribute to the extracellular induction of actA but did not affect intracellular levels of expression. The actA promoter is dependent upon a regulatory factor known as PrfA for transcriptional activation; however, no increase in actA expression was detected following the introduction of a high-affinity PrfA binding site within the actA promoter. The presence of a mutationally activated form of PrfA, known as PrfA*, increased overall actA expression in broth-grown cultures of both wild-type and actA promoter mutant strains, but the levels of induction observed were still approximately 50-fold lower than those observed for intracellularly grown L. monocytogenes. Collectively, these results indicate that the dramatic induction of actA expression that occurs in the host cell cytosol is mediated through a single promoter element. Furthermore, intracellular induction of actA appears to require additional steps or factors beyond those necessary for the activation and binding of PrfA to the actA promoter.