Complementation of Listeria monocytogenes Null Mutants with Selected Listeria seeligeri Virulence Genes Suggests Functional Adaptation of Hly and PrfA and Considerable Diversification of prfA Regulation in L. seeligeri

Abstract

ABSTRACT While Listeria seeligeri and L. monocytogenes contain the main Listeria virulence gene cluster, only L. monocytogenes is considered an intracellular pathogen. Initial evolutionary analyses showed that the virulence genes prfA , hly , and plcA are conserved in L. seeligeri , with specific Hly and PrfA amino acid residues showing evidence for positive selection in L. seeligeri . Our data also show that temperature-dependent transcript patterns for prfA , which encodes a transcriptional regulator of virulence genes, differed between L. monocytogenes and L. seeligeri . To further investigate the divergence of virulence gene function and regulation, L. seeligeri prfA ( prfA LS ), hly ( hly LS ), and plcA ( plcA LS ), as well as prfA LS constructs with different prfA promoter regions, were introduced into appropriate L. monocytogenes null mutants. Only when prfA LS was under the control of the L. monocytogenes prfA promoters (P1- and P2 prfA ) (P1P2 LM prfA LS ) was prfA LS able to fully complement the Δ prfA LM deletion. hly LS introduced into an L. monocytogenes background under its native promoter showed transcript levels similar to those of hly LM and was able to partially restore L. monocytogenes wild-type-level hemolysis and intracellular growth, even though Hly LM and Hly LS showed distinct patterns of cell- and supernatant-associated hemolytic activities. Our data indicate that (i) regulation of prfA expression differs between L. monocytogenes and L. seeligeri , although hly transcription is temperature dependent in both species, and (ii) PrfA and Hly functions are largely, but not fully, conserved between L. seeligeri and L. monocytogenes . Virulence gene homologues and their expression thus appear to have adapted to distinct but possibly related functions in these two species.