A new PrfA‐regulated gene of Listeria monocytogenes encoding a small, secreted protein which belongs to the family of internalins

Abstract

A mutant of Listeria monocytogenes EGD was constructed that carries an extended deletion removing the entire PrfA-regulated gene cluster from plcA to plcB and a second deletion inactivating the inlA gene. Upon supplementation of this mutant with multiple gene copies of prfA, a protein of 30 kDa was detected in the supernatant of the mutant strain. The gene encoding this protein was obtained by direct and inverse polymerase chain reaction using oligonucleotide primers that were deduced from partial amino acid sequences of the purified 30 kDa protein. The amino acid sequence of the gene product revealed a protein of 297 amino acids that carried eight repeat units with high homology to those of the two known internalin proteins A and B. This secretory protein, termed internalin C, is much smaller than InlA or InlB and its complete sequence is related to the two known internalins. The gene inlC is transcribed into a monocistronic mRNA from a single promoter which shows a typical consensus sequence for PrfA-binding at the position −40. In contrast to the transcription of the inlAB operon, which is downregulated after shift of an L. monocytogenes EGD culture from brain–heart infusion into minimum essential medium (MEM), transcription of inlC is induced in MEM like most of the other known PrfA-regulated virulence genes. In addition, inlC is strongly transcribed in the cytoplasm of phagocytic J774 cells whereas inlA is poorly transcribed under these conditions, suggesting that internalin C may play a role in a late stage of L. monocytogenes infection rather than in the uptake of L. monocytogenes by non-professional phagocytic cells. An inlC deletion mutant shows reduced virulence when tested in an intravenous mouse model, but intracellular replication of the mutant in Caco-2 and J774 cells appears to be comparable with that of the wild-type strain.