sRNA-mediated crosstalk between cell wall stress and galactose metabolism in Staphylococcus aureus

Abstract

Staphylococcus aureus is an opportunistic pathogen responsible for a wide range of diseases in humans. During infections, this bacterium is exposed to various stresses that target its cell wall, such as oxidative or acid environments as well as various cell wall-acting antimicrobials. Staphylococcus aureus has effective regulatory systems for responding to environmental stresses, enabling the expression of factors necessary for its survival. Bacterial small RNAs (sRNAs) play a crucial role in this adaptation process. In this study, we show that RsaOI, an S. aureus sRNA, accumulates under acid stress conditions. This response is mediated via the two-component system VraSR, which is associated with the cell wall damage response. As a component of the VraSR regulon, RsaOI contributes to the survival of S. aureus under acid stress and affects its susceptibility to glycopeptide antibiotics. Our findings reveal that RsaOI targets the lacABCDFEG operon, which encodes components of tagatose pathway, a unique mechanism responsible for galactose metabolism in S. aureus. By antisense base pairing near the ribosome binding site of lacD, RsaOI inhibits the expression of this gene, encoding tagatose-6-phosphate aldolase. This regulation disrupts the tagatose pathway, impairing galactose utilization in S. aureus. These findings highlight the role of RsaOI in the mediation between cell wall stress responses and a specific metabolic pathway.

Graphical Abstract

Figure 1.

Induced by low pH, rsaOI contributes to survival under acidic stress. (A) Visualization of the most significantly accumulated mRNAs under acidic (pH 4) and neutral (pH 7) conditions using a volcano plot. The genes with the highest differential expression are highlighted and labeled. (B) Genomic localization of rsaOI gene in HG003 strain, situated between proP and vraABC operon. (C) RsaOI expression in response to pH range was analyzed by northern blot analysis using rsaOI-specific probe, with tmRNA used as loading control. (D) rsaOI expression in HG003 WT, ΔrsaOI (both carrying pICS3 plasmid; WT/p and ΔrsaOI/p), or complemented strain (ΔrsaOI, carrying pICS3-rsaOI plasmid) was analyzed by qPCR, normalized to the control gene gyrB, and calculated using 2^−ΔΔCt method for relative quantification. (E) Strains from pannel (D) were cultured in LB medium for 2 h, and then pelleted and resuspended in LB buffered to pH 5. After 2 h at pH 5, samples were collected and plated to determine CFU counts. Statistical analysis was conducted using Student’s t-test. Error bars represent the average of three independent experiments. Statistical significance is indicated by bars and asterisks as follows: *P< .01, **P< .05.

Figure 2.

VraR affects rsaOI expression. (A) Fluorescence levels of GFP under control of either tufA or rsaOI promoters. Cells were grown for 2 h in LB medium, followed by addition of HCl. All statistical analyses were performed using Student’s t-test. Error bars represent the average of three independent experiments. Statistical significance is indicated by bars and asterisks as follows: ***P< .001. (B) Analysis of rsaOI levels in HG003 and Sa564 WT strains (WT) and their isogenic mutants. Relative quantification of rsaOI expression levels was measured by qPCR, normalized to the control gene gyrB, and calculated using 2^−ΔΔCt method. (C) rsaOI expression in WT and in ΔvraR mutant according to pH. Cells were cultured in LB medium for 2 h, and then pelleted and resuspended in LB buffered with 100 mM HEPES at pH 5 or 7 for 30 min. (D) Fluorescence levels of GFP under control of rsaOI promoter in both HG003 WT and ΔvraR strains. Cells were grown for 2 h in LB medium, followed by HCl addition. Statistical analysis was performed using Student’s t-test. Error bars represent the average of three independent experiments. Statistical significance is indicated by bars and asterisks as follows: ***P< .001, ****P< .0001. (E) Illustration of the induction mediated by the TCS VraSR on the expression of the sRNA rsaOI during acid stress or in the presence of the glycopeptide antibiotic vancomycin.

Figure 3.

RsaOI modulates S. aureus resistance to vancomycin. Ten-fold serial dilutions of overnight cultures of WT HG003, ΔrsaOI, or complemented strain (ΔrsaOI/p-rsaOI) were plated and incubated for 24 h at 37°C. The 10⁻⁶ dilution are shown for TSA condition (A), and the 10⁻⁴ dilution for TSA supplemented with 1 μg/ml vancomycin (B). Diagrams show colony growth quantification. Statistical analysis was performed using Student’s t-test. Error bars represent the average of three independent experiments. Statistical significance is indicated by bars and asterisks as follows: ***P< .001.

Figure 4.

Investigation of the potential direct targets of RsaOI based on in vivo proteomic data and in silico computational approaches. (A) Volcano plot showing the protein relative abundances between the strains ΔrsaOI/p (ΔrsaOI containing a pICS3 vector) versus ΔrsaOI/p-ΔrsaOI (complemented strain) in relation to the t-test P-value. VraR and LacD are significantly more abundant in ΔrsaOI strain. (B) Venn diagram illustrating the overlap between experimentally identified and in silico predicted RsaOI target candidates. Experimentally identified candidates, representing proteins with significantly different abundances between rsaOI mutant and complemented strain, the top 100 predicted targets from CopraRNA [46] and the top 100 predicted by RNA predator [47] are shown. Shared targets are represented in the overlapping regions. (C) Organization of the lac operon in the HG003 strain and the predicted base-pairing interaction between RsaOI and lac operon mRNA, generated using IntraRNA software [49]. The start codon of lacD is highlighted in red, and position +1 corresponds to the transcription start site for RsaOI.

Figure 5.

RsaOI inhibits lacD expression. (A) Complex formation between RsaOI and lacD mRNA was analyzed by native gel retardation assays. Gel shift assays of purified, labeled RsaOI and RsaOI-mut (0.1 pmol) were performed with increasing concentrations of lacD mRNA. (B) Quantification of complex formation from panel (A) was performed by ImageQuant Tools 7.0. (C) The effect of RsaOI on lacD expression was examined using gfp gene reporter assay. Staphylococcus aureus WT and ΔrsaOI strains containing the pCN33-PtufA-lacD-gfp fusion plasmid were co-transformed with pICS3, pICS3 expressing rsaOI or rsaOI-mut under control of its endogenous promoter, or rsaOI under constitutive promoter amiA. The fluorescent intensity was measured after 12 h of growth in LB medium adjusted to pH 6. Statistical analysis was conducted using a Kruskal–Wallis non-parametric test across all conditions, followed by Student’s t-test to determine significant differences among conditions. Error bars represent the average of four independent experiments. Statistical significance is indicated by bars and asterisks as follows: **P< .01, ***P< .001. (D) In parallel with the panel (C) experiment, strains were collected and pelleted, and relative quantification of lacD–gfp fusion mRNA expression levels was measured by qPCR, normalized to the control gene gyrB, and calculated using 2^−ΔΔCt method.

Figure 6.

rsaOI overexpression impairs using of galactose as an external source of carbons. (A) Illustration of the tagatose pathway, a metabolic pathway that catabolizes galactose or lactose through the lac operon. (B) Growth curves of S. aureus HG003 WT, ΔrsaOI, or complemented strain expressing rsaOI under constitutive promoter amiA were cultured in NZM media supplemented with glucose or galactose at 11 mM. Growth was followed during 16 h using a Biotek microplate reader. Error bars represent the average of three independent experiments.

Figure 7.

RsaOI acts as a mediator between cell wall stress and the tagatose metabolic pathway. In response to stress generated by exposure to glycopeptides such as vancomycin or to acid stress, the VraSR system, involved in resistance to cell wall-targeting antibiotics, is activated. The sensor unit VraS phosphorylates VraR, thereby activating it, while it was initially kept inactive through phosphorylation by the Stk1 kinase [62]. Once activated, VraR induces the transcription of genes belonging to the CWSS. This stimulon can be divided into two subsets: the first includes genes induced exclusively upon glycopeptide exposure, primarily involved in cell wall synthesis and dynamics (e.g. pbp2, fmtA, sgtB,andmurZ); the second subset comprises genes induced both by glycopeptides and acidic conditions (e.g. cwrA,vraX, and rsaOI). Under these stress conditions, RsaOI represses the expression of its target genes: atl,ptsH (Hpr), and lacD. LacD encodes the tagatose pathway aldolase, which is essential for galactose metabolism. RsaOI represses lacD expression through base pairing at the RBS, thereby reducing tagatose pathway activity and fine-tuning sugar metabolism in response to environmental stressors.