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Regulation of the prfA transcriptional activator of Listeria monocytogenes: multiple promoter elements contribute to intracellular growth and cell-to-cell spread.

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 10104-6076.

ABSTRACT

The prfA gene product is a transcriptional activator of Listeria monocytogenes determinants of pathogenicity. In this study, we provide direct evidence that the PrfA protein is a site-specific DNA-binding protein. Additionally, we describe the characterization of two classes of L. monocytogenes mutants which contain transposon insertions either in the prfA structural gene (exemplified by strain DP-L1075) or within the prfA promoter region (exemplified by strain DP-L973). Both mutants are completely avirulent and secrete greatly reduced levels of listeriolysin O and phosphatidylinositol-specific phospholipase C, and both are fully complemented by the introduction of prfA on a multicopy plasmid. The behaviors of the two mutants differ markedly within cultured macrophages. Following infection, no cytoplasmic growth was observed for DP-L1075 whereas DP-L973 escaped from the phagosome and grew in the cell cytoplasm. However, DP-L973 was defective in nucleation of actin filaments and spread to adjacent cells. Transcription of prfA in DP-L973 was directed from a single, previously unidentified promoter (prfAp2) located close to the prfA initiation codon. This promoter is therefore capable of providing sufficient prfA expression for escape from the host cell vacuole but is insufficient for wild-type levels of bacterially induced actin polymerization and cell-to-cell spread. Transcription directed from both prfAp1 and prfAp2 promoters was increased in the absence of a functional prfA gene product, suggesting that PrfA protein contributes to down-regulating its own expression.

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