Species-Specific Differences in the Activity of PrfA,the Key Regulator of Listerial VirulenceGenes

Abstract

ABSTRACT PrfA, the master regulator of LIPI-1, is indispensable for the pathogenesis of the human pathogen Listeria monocytogenes and the animal pathogen Listeria ivanovii . PrfA is also present in the apathogenic species Listeria seeligeri , and in this study, we elucidate the differences between PrfA proteins from the pathogenic and apathogenic species of the genus Listeria . PrfA proteins of L. monocytogenes (PrfA Lm and PrfA* Lm ), L. ivanovii (PrfA Li ), and L. seeligeri (PrfA Ls ) were purified, and their equilibrium constants for binding to the PrfA box of the hly promoter (P hly Lm ) were determined by surface plasmon resonance. In addition, the capacities of these PrfA proteins to bind to the PrfA-dependent promoters P hly and P actA and to form ternary complexes together with RNA polymerase were analyzed in electrophoretic mobility shift assays, and their abilities to initiate transcription in vitro starting at these promoters were compared. The results show that PrfA Li resembled the constitutively active mutant PrfA* Lm more than the wild-type PrfA Lm , whereas PrfA Ls showed a drastically reduced capacity to bind to the PrfA-dependent promoters P hly and P actA . In contrast, the efficiencies of PrfA Lm , PrfA* Lm , and PrfA Li forming ternary complexes and initiating transcription at P hly and P actA were rather similar, while those of PrfA Ls were also much lower. The low binding and transcriptional activation capacities of PrfA Ls seem to be in part due to amino acid exchanges in its C-terminal domain (compared to PrfA Lm and PrfA Li ). In contrast to the significant differences in the biochemical properties of PrfA Lm , PrfA Li , and PrfA Ls , the PrfA-dependent promoters of hly (P hly Lm , P hly L i , and P hly L s ) and actA (P actA Lm , P actA L i , and P actA L s ) of the three Listeria species did not significantly differ in their binding affinities to the various PrfA proteins and in their strengths to promote transcription in vitro. The allelic replacement of prfA Lm with prfA Ls in L. monocytogenes leads to low expression of PrfA-dependent genes and to reduced in vivo virulence of L. monocytogenes , suggesting that the altered properties of PrfA Ls protein are a major cause for the low virulence of L. seeligeri .