IreK-Mediated, Cell Wall-Protective Phosphorylation in Enterococcus faecalis

Abstract

Enterococcus faecalis is a Gram-positive bacterium that is a major cause of hospital-acquired infections due, in part, to its intrinsic resistance to cell wall-active antimicrobials. One critical determinant of this resistance is the transmembrane kinase IreK, which belongs to the penicillin-binding protein and serine/threonine kinase-associated kinase family of bacterial signaling proteins involved with the regulation of cell wall homeostasis. The activity of IreK is enhanced in response to cell wall stress, but direct substrates of IreK phosphorylation, leading to antimicrobial resistance, are largely unknown. To better understand stress-modulated phosphorylation events contributing to antimicrobial resistance, wild type E. faecalis cells treated with cell wall-active antimicrobials, chlorhexidine or ceftriaxone, were examined via phosphoproteomics. Among the most prominent changes was increased phosphorylation of divisome components after both treatments, suggesting that E. faecalis modulates cell division in response to cell wall stress. Phosphorylation mediated by IreK was then determined via a similar analysis with a E. faecalis ΔireK mutant strain, revealing potential IreK substrates involved with the regulation of peptidoglycan biosynthesis and within the E. faecalis CroS/R two-component system, another signal transduction pathway that promotes antimicrobial resistance. These results reveal critical insights into the biological functions of IreK.

Keywords: Enterococcus faecalis; IreK; PASTA kinase; antimicrobial resistance; cell wall stress; mass spectrometry; phosphoproteomics.

Figure 1.

General workflow for label-free quantitative phosphoproteomics. Proteins were extracted from untreated, ceftriaxone-, or chlorhexidine-treated E. faecalis wild type and ΔireK strains. Following extraction, proteins were reduced with DTT, alkylated with IAM, and trypsin digested. Before TiO₂ phosphopeptide enrichment, an aliquot was taken from each sample for global proteome analysis and phosphopeptide abundance normalization. Global proteome and phosphopeptide-enriched samples were analyzing using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Figure 2.

Unsupervised hierarchical clustering of the 87 phosphopeptides significantly changing across untreated, chlorhexidine-, and ceftriaxone-treated wild type E. faecalis after a one-way ANOVA (FDR-adjusted p-value < 0.05), displaying cell wall-active antimicrobial-modulated E. faecalis phosphorylation events. Cluster A contains peptides with increased phosphorylation within both the chlorhexidine- and ceftriaxone-treated samples compared to the untreated samples. Cluster B and C contain peptides with increased phosphorylation specifically after chlorhexidine or ceftriaxone treatment, respectively. Cluster D contains peptides with decreased phosphorylation within both the chlorhexidine- and ceftriaxone-treated samples compared to the untreated samples.

Figure 3.

The seven GpsB phosphopeptides clustered with the peptides increasing in abundance in the chlorhexidine- and ceftriaxone-treated wild type E. faecalis samples compared to the untreated samples after a one-way ANOVA (FDR-adjusted p-value < 0.05). The phosphosites within each phosphopeptide are labeled.

Figure 4.

Differential analysis of the E. faecalis phosphoproteome. This phosphoproteome data was normalized by dividing the phosphopeptide abundances with their protein abundances from the global proteome data in each replicate. Red circles represent significantly changing phosphopeptides (FC ≥ 2, FDR-adjusted p-value < 0.05) in the wild type strain after a two-sided, equal variance t-test, representing potential IreK phosphorylation substrates. (A) Comparison of untreated strains. (B) Comparison of chlorhexidine-treated strains. (C) Comparison of ceftriaxone-treated strains.

Figure 5.

IreK-directed phosphorylation of GpsB. (A) Differential analysis of GpsB phosphopeptides. Red circles represent significantly changing GpsB phosphopeptides (FC ≥ 2, FDR-adjusted p-value < 0.05) in the wild type strain after a two-sided, equal variance t-test, representing potential IreK phosphorylation substrates. The phosphosites within each phosphopeptide are labeled. From left to right: comparison of untreated, chlorhexidine-, and ceftriaxone-treated E. faecalis strains. (B) GpsB-His₆ was enriched from lysates of indicated exponentially growing E. faecalis cells (“input”) using immobilized metal affinity chromatography (“elution”). SDS-PAGE and immunoblotting were performed using anti-GpsB antisera (which detect GpsB and GpsB-His₆) or anti-pThreonine antibody (to detect phosphorylated GpsB, [P-GpsB-His₆]). (C) In vitro kinase assays contained purified recombinant GpsB, ATP, and either no kinase (none), wild type His₆-IreK-n catalytic domain (WT), or catalytically impaired His₆-IreK-n K41R mutant (K41R). Data is representative of a minimum of 3 independent replicates.

Figure 6.

Effect of IreK on MltG phosphorylation in vivo. (A) Differential analysis of MltG phosphopeptides. Red circles represent significantly changing MltG phosphopeptides (FC ≥ 2, FDR-adjusted p-value < 0.05) in the wild type strain after a two-sided, equal variance t-test, representing potential IreK phosphorylation substrates. The phosphosites within each phosphopeptide are labeled. From left to right: comparison of untreated, chlorhexidine-, and ceftriaxone-treated E. faecalis strains. (B) Immunoblot analysis was performed to analyze phosphorylation of MltG in exponentially growing E. faecalis cells. RpoA was used as a loading control. Asterisks indicate potential multiply phosphorylated MltG proteoforms present in the ΔireP mutant but not wild type. Each image is representative of three independent biological replicates.