A Peptide Motif in Two Distinct Gram-positive Virulence Regulators Links Carbon Catabolite Repression to Virulence Gene Expression

Jerry Woo, Michael Federle, Nancy Freitag

Carbon catabolite repression (CCR) is a conserved phenomenon in bacterial species, where genes involved in the transport and catabolism of less preferred sugars are repressed in the presence of a preferred sugar. In some pathogens, such as Streptococcus pyogenes and Listeria monocytogenes, it has been observed that the expression of virulence factors is also subject to CCR. In S. pyogenes, the master regulator of virulence, Mga, is a unique transcription factor that harbors two Phosphotransferase Recognition domains (PRD) in tandem, followed by an EIIBGat domain. The PRDs interact with the Histidyl-phosphorylated phosphor-carrier protein HPr (HPr-His15~P), a crucial member of the Phosphotransferase System (PTS), a set of proteins dedicated for the import of sugars for cellular metabolism. The HPr-His15~P phosphorylates the conserved histidine residues within the PRDs to exert CCR on Mga regulation of virulence and Rgg2/3 quorum-sensing (QS) genes. Previous studies suggested that the Mga C-terminus EIIBGat domain is only involved in Mga oligomerization; however, our results indicate this domain also plays a role in CCR of the Rgg2/3 QS system. A cysteine residue conserved within the EIIBGat domain in other structurally similar transcriptional regulators is also present within the EIIBGat domain of Mga but instead it is uniquely gated by three aromatic amino acids (YCFF), analogous to a motif solely found within glycerol kinase that has a histidine residue instead (YHFF) and is phosphorylated by PTS components. We hypothesized that this motif in Mga is phosphorylated by ManL, the PTS for mannose transport, which we have previously demonstrated is essential for the suppression of Rgg2/3 QS during growth in sucrose. We substituted the cysteine residue to a serine (MgaC388S) or glutamic acid (MgaC388E) to simulate a phosphoablative or phosphomimetic conformation of Mga, respectively and observed that the former resulted in an inactive state while the latter behaves just like WT in suppressing the Rgg2/3 QS. However, in a ΔmanL background, we observed that the MgaC388E variant suppresses Rgg2/3 QS, strongly suggesting that this motif could potentially be phosphorylated by ManL to modulate the activity of Mga in a carbohydrate dependent manner. We extended our search for other transcriptional regulators that could harbor this novel motif and observed that PrfA, the master virulence regulator of L. monocytogenes contains a degenerate motif (YCIF). Using the same strategy, we substituted the cysteine residue within this motif in PrfA to a serine (PrfAC38S) or glutamic acid (PrfAC38E) and observed that the former variant behaves similarly to WT in terms of virulence factor synthesis in a carbohydrate-dependent manner and ability to initiate cell-cell spreading in various mammalian cell-lines in vitro. Interestingly, the PrfAC38E variant demonstrated dysfunctional regulation of virulence genes independent of carbohydrate and impaired cell-cell spreading but remained capable of proliferating intracellularly, suggesting that the cysteine residue is critical for optimal PrfA activity and could potentially be involved in CCR of PrfA activity. Collectively, these results strongly suggest that this novel conserved motif is central to the regulation of virulence gene expression by CCR in at least two important Gram-positive pathogens.