Coordinated regulation of transcription by CcpA and the Staphylococcus aureus two-component system HptRS

Abstract

The success of Staphylococcus aureus as a pathogen is due in part to its ability to adapt to changing environmental conditions using signal transduction pathways, such as metabolite- responsive regulators and two-component systems. S. aureus has a two-component system encoded by the gene pair sav0224 (hptS) and sav0223 (hptR) that regulate the hexose phosphate transport (uhpT) system in response to extracellular glucose-6-phosphate. Glycolytic intermediates such as glucose-6-phosphate are important carbon sources that also modulate the activity of the global metabolite-responsive transcriptional regulator CcpA. Because uhpT has a putative CcpA binding site in its promoter and it is regulated by HptR, it was hypothesized the regulons of CcpA and HptR might intersect. To determine if the regulatory domains of CcpA and HptRS overlap, ccpA was deleted in strains SA564 and SA564-ΔhptRS and growth, metabolic, proteomic, and transcriptional differences were assessed. As expected, CcpA represses hptS and hptR in a glucose dependent manner; however, upon CcpA derepression, the HptRS system functions as a transcriptional activator of metabolic genes within the CcpA regulon. Importantly, inactivation of ccpA and hptRS altered sensitivity to fosfomycin and ampicillin in the absence of exogenous glucose-6-phosphate, indicating that both CcpA and HptRS modulate antibiotic susceptibility.

Fig 1. The growth and pH profile of SA564-ΔhptRS/ccpA is moderately different from SA564, SA564-ΔhptRS, and SA564-ΔccpA during the post-exponential phase.

The A₆₀₀ (A, B) and pH (C, D) were measured every hour for 12 hours. S. aureus strains SA564 (black symbols), SA564-ΔhptRS (red symbols), SA564-ΔccpA (green symbols), and SA564-ΔhptRS/ccpA (blue symbols) were cultivated in TSB (A, C) or TSB-dex (B, D). Data are representative of the mean of experiments performed in biological triplicate, with error bars representing the standard error of the mean. A statistically significant difference (p ≤ 0.05) between SA564-ΔccpA and SA564-ΔhptRS/ccpA is represented with an (*).

Fig 2. Metabolism of strain SA564-ΔhptRS/ccpA is different from strains SA564, SA564-ΔhptRS, and SA564-ΔccpA.

Ammonia accumulation (A, B), acetate accumulation and depletion (C, D), and glucose depletion (E) in the culture media for S. aureus strains SA564 (black symbols), SA564-ΔhptRS (red symbols), SA564-ΔccpA (green symbols), and SA564-ΔhptRS/ccpA (blue symbols) cultivated in TSB (A, C, E) and TSB-dex (B, D) are depicted. Data are representative of the mean metabolite concentrations plotted as a function of cell density (A₆₀₀) for experiments performed in biological triplicate, with error bars representing the standard error of the mean for absorbance (A₆₀₀) and [NH₃] or [Acetate] or [Glucose].

Fig 3. Model of strain SA564 exponential and post-exponential growth phase metabolism during cultivation in TSB, and proteins identified as significantly different during proteomic analysis.

Green arrows represent reactions or pathways that occur primarily during the exponential growth phase. Red arrows represent reactions or pathways that occur primarily during the post-exponential growth phase. Proteins within the depicted metabolic pathways, which had significant 3-way (s x m x h) interactions, 2-way (s x h |m) interactions or a main effect of strain are indicated using the protein abbreviations from the proteomic analysis.

Fig 4. Gene Ontology (GO) analysis of proteins with a significant interaction (s x m x h, s x h | m) or main effect of strain indicates that the majority of differential proteins are involved in catalytic activity.

GO analysis was performed by inputting gene symbol abbreviations (e.g. ADH, PflB, Dha1) into the gene list analysis tab within the PANTHER classification webpage (http://www.pantherdb.org/) and searching against the S. aureus database within the PANTHER Classification System Database [53].

Fig 5. CcpA and HptRS co-regulate transcription of genes during the exponential (2 h) and post-exponential (6 h) growth phases.

The relative mRNA abundance during the exponential (A, B) and post-exponential (C, D) growth phases for genes hptR, hptS, hptA, dha1, adh, ccpA, pflB, rocA, and uhpT was determined by RT-qPCR. Data are the mean and standard error of the mean for 3 biological replicates of SA564 (black bars), SA564-ΔhptRS (red bars), SA564-ΔccpA (green bars), and SA564-ΔhptRS/ccpA (blue bars) cultivated in TSB (A, C) and TSB-dex (B, D). The data were normalized using 16S rRNA and are plotted relative to SA564. A statistically significant difference (p ≤ 0.05) between SA564-ΔccpA and SA564-ΔhptRS/ccpA is represented with an (*).

Fig 6. Transcriptional regulation by CcpA and HptRS alter fosfomycin and ampicillin susceptibility.

Antibiotic susceptibility to fosfomycin (A) and ampicillin (B) was investigated using broth microdilution. Data are the mean and standard error of the mean for 3 biological replicates, each with 6 technical replicates. Significant differences (p ≤ 0.05) between SA564-ΔccpA and SA564-ΔhptRS/ccpA are represented with an (*).

Fig 7. Transcriptional regulation by CcpA and HptRS mediate antibiotic susceptibility.

Antibiotic susceptibility was investigated using antibiotic disks on TSA. Data represent the mean and standard error of the mean of the zone of inhibition (edge of the disk to the first colony) for 3 biological replicates. Significant differences (p ≤ 0.05) between SA564-ΔccpA and SA564-ΔhptRS/ccpA are represented with an (*).

Fig 8. Model for CcpA and HptRS coregulation of uhpT transcription and fosfomycin susceptibility.

In the absence of glucose, CcpA de-repression increases mRNA abundance of sav0225 (hptA), sav0224 (hptS), and sav0223 (hptR). Transcription is further increased by the presence of metabolites potentially derived from gluconeogensis. Upon activation by glucose-6-phosphate, or another unknown stimulus, the HptRS system functions to activate transcription of genes normally repressed by CcpA, including uhpT. Increased transcription of uhpT is correlated with increased transport of the antibiotic fosfomycin; hence, coregulation of transcription by CcpA and HptRS directly alter fosfomycin susceptibility, and susceptibility to other antimicrobials.