No two are the same: Identification and characterization of novel hyperactive PrfA* variants in Listeria monocytogenes

Abstract

The virulence potential of the human pathogen Listeria monocytogenes is primarily controlled by the master virulence regulator PrfA. PrfA activity is tightly regulated to prevent virulence gene expression outside the host, while enabling their rapid induction upon host cell entry. One mechanism controlling PrfA activity is the sugar-dependent inhibition. Readily metabolizable sugars, such as glucose or cellobiose, inhibit PrfA; however, the precise mechanism and the factors involved in this regulation remain unknown. Here, a suppressor screen was performed to identify these factors. Although the screen did not reveal any factors mediating the sugar-dependent inhibition of PrfA, it resulted in the isolation of 13 novel hyperactive PrfA variants, termed PrfA*. All newly identified PrfA* variants were characterized using growth, PlcB activity and promoter activity assays. Interestingly, two of these PrfA* variants, PrfAY126F and PrfAV153M, were shown to function even in the absence of PrfAs cofactor glutathione. In addition, a subset of variants was analyzed for intracellular replication using in vitro infection assays. Across all assays, the effect of the PrfA* mutations were found to be highly variant- and gene-dependent, with no two variants exhibiting identical phenotypes. Importantly, these results demonstrate that the transcriptional and phenotypic consequences of PrfA* mutations cannot be reliably predicted from a single assay. Thus, comprehensive characterization using multiple complementary approaches is essential to determine the effects of PrfA* mutations on the expression of PrfA-regulated genes and the pathogenic potential of L. monocytogenes strains carrying these mutations.