No two are the same: Identification and characterization of novel hyperactive PrfA* variants in Listeria monocytogenes
Year: 2026
Type: preprint
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, PrfA Y126F and PrfA V153M , were shown to function even in the absence of PrfA’s 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. Author summary The activity of the main virulence regulator PrfA of the human pathogen L. monocytogenes is regulated at multiple levels. One intriguing mechanism is the sugar-dependent inhibition of PrfA, which was identified more than three decades ago but still remains poorly understood. We used a suppressor screen to identify factors involved in this regulation. Surprisingly, the screen identified 13 novel hyperactive PrfA variants that were less inhibited by cellobiose. In-depth characterization revealed that although all variants increased PrfA activity, each variant produced distinct phenotypes, with effects that depended on both the specific amino acid substitution and the PrfA-regulated gene. Our findings further indicate that individual PrfA mutations can alter functional properties of the regulator, including its structural state and its cofactor dependence. Remarkably, enhanced PrfA activity has now been associated with mutations at 24 of its 237 amino acid positions. This broad spectrum of activating mutations may explain why genetic screens aimed at identifying factors involved in sugar-dependent PrfA inhibition repeatedly yielded hyperactive PrfA variants rather than the factors underlying this regulation.
Institutions: University of Göttingen, University of Southern Denmark
Language: English
Subfield: Food Science
Domain: Life Sciences
Open Access status: green
Funder: Deutsche Forschungsgemeinschaft
Award: G3803127787
OpenAlex ID: W7222173267