Novel motif associated with carbon catabolite repression in two major Gram-positive pathogen virulence regulatory proteins

Abstract

Carbon catabolite repression (CCR) is a widely conserved regulatory process that ensures enzymes and transporters of less-preferred carbohydrates are transcriptionally repressed in the presence of a preferred carbohydrate. This phenomenon can be regulated via a CcpA-dependent or CcpA-independent mechanism. The CcpA-independent mechanism typically requires a transcriptional regulator harboring a phosphotransferase regulatory domain (PRD) that interacts with phosphotransferase system (PTS) components. PRDs contain a conserved histidine residue that is phosphorylated by the PTS-associated HPr-His15~P protein. PRD-containing regulators often harbor additional domains that resemble PTS-associated EIIB protein domains with a conserved cysteine residue that can be phosphorylated by cognate PTS components. We noted that Mga, the PRD-containing central virulence regulator of Streptococcus pyogenes, has an EIIB^Gat domain containing a cysteine that, based on the presence of a similar motif in glycerol kinase, could be a target for phosphorylation. Using site-directed mutagenesis, we constructed phospho-ablative and phospho-mimetic substitutions of this cysteine and found that these substitutions modify the CCR of the Rgg2/3 quorum-sensing system. Moreover, we provide genetic evidence that the phospho-donor of this cysteine residue is likely to be ManL, the EIIA/B subunit of the mannose PTS system. Interestingly, a structurally distinct virulence gene regulator, PrfA of Listeria monocytogenes, harbors a similar cysteine-containing motif, and phospho-ablative and phospho-mimetic substitutions of the cysteine-altered CCR of PrfA-dependent virulence gene expression. Collectively, our data suggest that phosphorylation of a cysteine within the shared novel motif in Mga and PrfA may be a heretofore missing link between cellular metabolism and virulence.IMPORTANCEIn this study, we identified a novel cysteine-containing motif within the amino acid sequence of two structurally distinct transcriptional regulators of virulence in two Gram-positive pathogens that appears to link carbon metabolism with virulence gene expression. The results also highlight the potential post-translational modification of cysteine in bacterial species, a rare and understudied modification.

Keywords: CCR; Listeria monocytogenes; Mga; PrfA; Streptococcus; virulence.

Fig 1

The structural domains of Mga and amino acid sequence alignment of EIIB^Gat domain between Type I and Type II lineages. The structural domains of both Type-I and Type-II Mga are highly conserved, but the amino acid alignment of the EIIB^Gat domain between them (383–493 a.a) revealed the presence of a cysteine residue in the latter lineage. The residue is gated by a triad of aromatic amino acids.

Fig 2

The cysteine residue in Type II Mga harbors a regulatory role in modulating the Rgg2/3 QS system during growth in different carbohydrates. Induction of the Rgg2/3 QS circuitry is monitored by the expression of luciferase genes driven by the promoter of the pheromone encoding gene, shp3 in chemically defined media supplemented with glucose (A and D), mannose (B and E), and sucrose (C and E). RLUs were obtained by normalizing the abundance of luminescence units per second to the OD₆₀₀ of the corresponding sample and plotted on the Y axis on a logarithmic scale, while the X axis is the OD₆₀₀ value of the sample. Results shown are representative of at least three biological replicates.

Fig 3

Amino acid sequence alignment of Mga and PrfA reveals the conservation of this novel motif. The amino acid sequence alignment of Type II Mga EIIB^Gat domain (382–477 a.a) from GAS, Mga EIIB^Gat domain (385–479 a.a) from SDSE and the N-terminus of PrfA (32–117 a.a). Highlighted in red is the presence and near conservation of this Aro-Cys-(Aro/Xle)-Aro motif between Mga and PrfA.

Fig 4

The phosphomimetic variant of the cysteine residue in the novel motif of PrfA showed dysregulation of virulence factors. Cells were grown to the stationary phase in LB or LB supplemented with 10 mM glucose and (A and B) the supernatant was used to determine the amount of hemolysin secreted by each strain, while (C and D) whole cell lysate was used to determine the amount of β-glucuronidase, which is a proxy for actA expression. The results shown is the mean of at least three biological replicates ± SD. **P < 0.001 and *P < 0.05.

Fig 5

The phosphomimetic variant of the cysteine residue in the novel motif of PrfA is attenuated in virulence in vitro. L. monocytogenes WT and their respective variants were used to determine their plaque-forming capabilities in (A) L2 fibroblast cells and (B) intracellular growth capabilities in Ptk2 cells. Cells were infected with the respective samples at an MOI of 1:100. For plaque assay, plaques were stained with neutral red 3 dpi for easier visualization and enumeration. For intracellular growth in Ptk2 cells, coverslips (n = 3) were removed and resuspended in 1 mL of sterile water, serially diluted, and spot plated (10 µL) onto BHI plates to enumerate colony-forming units (CFUs). Each experiment was performed at least three times, and the results shown are representative of one experiment.

Fig 6

The phosphomimetic variant of the cysteine residue in the novel motif of PrfA showed a cell-cell spreading defect. L. monocytogenes WT and their following mutants: ΔprfA, ΔactA, C38S, or C38E were used to infect PTK2 cells at an MOI of 1:40. Cells were fixed after 24 h p.i. and subsequently probed with anti-Lm antibody, followed by Texas Red-conjugated secondary antibody, phalloidin to stain actin (green), or DAPI to stain nuclei (blue). Red channel: L. monocytogenes (LMO), green channel: actin, and blue channel: DNA. Scale bar = 10 µm.

Fig 7

A proposed model theorizing how the novel motif impacts the activity of PrfA in Listeria monocytogenes. Demarcated lines indicate the flow of phosphate (yellow circle). (A) In Lm, the expression of virulence factors is repressed when cells are grown in glucose, due to the inactivation of PrfA, possibly through the phosphorylation of the cysteine residue within this novel motif by ManL, the EIIA/B subunit of PTS^mpt. (B) Upon growth in less-preferred carbon source, such as glucose-6-phosphate, ManL expression is suppressed and consequently the lack of phosphorylation of the cysteine residue results in the activation of PrfA, therefore upregulating the expression of virulence genes.