Staphylococcus aureus Stress Response to Bicarbonate Depletion

Abstract

Bicarbonate and CO₂ are essential substrates for carboxylation reactions in bacterial central metabolism. In Staphylococcus aureus, the bicarbonate transporter, MpsABC (membrane potential-generating system) is the only carbon concentrating system. An mpsABC deletion mutant can hardly grow in ambient air. In this study, we investigated the changes that occur in S. aureus when it suffers from CO₂/bicarbonate deficiency. Electron microscopy revealed that ΔmpsABC has a twofold thicker cell wall thickness compared to the parent strain. The mutant was also substantially inert to cell lysis induced by lysostaphin and the non-ionic surfactant Triton X-100. Mass spectrometry analysis of muropeptides revealed the incorporation of alanine into the pentaglycine interpeptide bridge, which explains the mutant's lysostaphin resistance. Flow cytometry analysis of wall teichoic acid (WTA) glycosylation patterns revealed a significantly lower α-glycosylated and higher ß-glycosylated WTA, explaining the mutant's increased resistance towards Triton X-100. Comparative transcriptome analysis showed altered gene expression profiles. Autolysin-encoding genes such as sceD, a lytic transglycosylase encoding gene, were upregulated, like in vancomycin-intermediate S. aureus mutants (VISA). Genes related to cell wall-anchored proteins, secreted proteins, transporters, and toxins were downregulated. Overall, we demonstrate that bicarbonate deficiency is a stress response that causes changes in cell wall composition and global gene expression resulting in increased resilience to cell wall lytic enzymes and detergents.

Keywords: MpsABC; Staphylococcus aureus; bicarbonate transporter; cell wall; peptidoglycan; transcriptome.

Figure 1

Deletion of mpsABC causes severe growth delay under ambient air. (a) Illustration of the construction of ΔmpsABC deletion mutant in the parent strain S. aureus JE2 (locus tag prefix: B7H15). For homologous recombination using allelic exchange, plasmid pBASE6-ΔmpsABC containing approximately 2 kb upstream and downstream DNA sequences of mpsABC was used. (b) Growth rates of ΔmpsABC were significantly lower than those of the parent JE2 strain (** p < 0.01 as determined by unpaired two-sided t-test). Arrows indicate the time points at 4 and 8 h when the samples were collected for transcriptomic analysis. Each time point in the graph represents the mean ± standard deviation (SD) from three independent biological replicates.

Figure 2

Cell wall (CW) of the JE2ΔmpsABC grown in ambient air is thicker than that of the parent strain. Transmission electron microscopy (TEM) images of JE2 parent and JE2ΔmpsABC. Cells were grown under ambient air and 5% CO₂ at a starting A₅₇₈ of 0.1, harvested at A₅₇₈ 0.5 and washed with PBS pH 7.3 before fixation with 4% formaldehyde, 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4. The CW of JE2ΔmpsABC showed increased thickness. Scale bar is 200 nm.

Figure 3

JE2ΔmpsABC cells are more resistant to lysis. (a) Concentration of genomic DNA (gDNA) released upon cell lysis from JE2 parent and its ΔmpsABC grown under ambient air and 5% CO₂ following treatment with lysostaphin (Lss). Genomic DNA was isolated using the commercial Quick-DNATM Microprep Kit (ZYMO Research, Germany) according to the manufacturer’s protocol. Significantly less gDNA was isolated from the mutant than from the parent strain. Each bar in the chart shows the mean ± standard deviations (SD) from three independent biological replicates. Significance was calculated by two-way ANOVA with (** p < 0.01). The concentration of released gDNA upon cell lysis is listed in Table S1. (b) Triton X-100 induced autolysis assay. Triton X-100 (0.05% in PBS) was added at 0 h to mid-exponential phase cells. Autolysis was monitored by measuring the decrease in absorption (A₅₇₈) every 30 min for 6 h (** p < 0.01 as determined by unpaired two-sided t-test). Triton X-100 alone served as negative control. Each value in the graph represents the mean ± standard deviations (SD) from three independent biological replicates.

Figure 4

Muropeptides identified from lysostaphin-cellosyl PG digestion of JE2ΔmpsABC indicate the incorporation of L-alanine into the pentaglycine bridge. (a) Purified PG was digested with lysostaphin and cellosyl and the resulting profile of the JE2ΔmpsABC strain revealed distinct peaks at ~ 75–80 min retention time, which were absent in the PG digestion profile of the parent strain. (b) Collected peaks for lysostaphin-cellosyl-digested PG of JE2ΔmpsABC via LC-MS and LC-MS/MS analysis show incorporation of L-alanine into the pentaglycine bridge in the red highlighted peaks. The calculated masses are summarized in Table S3.

Figure 5

JE2ΔmpsABC has less α- and more ß-glycosylated GlcNAc WTA. Flow cytometry analysis of GlcNAc WTA (N-acetylglucosamine wall teichoic acid). Cells were grown in ambient air (A) or 5% CO₂ (CO₂) overnight, adjusted to A₅₇₈ 0.4 and treated with 200 µg/mL proteinase K. Isotype control was anti-HIV protein gp120 (b12-IgG) Fab fragment (5 μg/mL). Secondary antibody was fluorescein isothiocyanate-labeled goat anti-human IgG F(ab′)2 FITC conjugate (2 μg/mL). (a) mAb (monoclonal antibody) 4461 Fab fragment against TarM dependent α-GlcNAc WTA. (b) mAb 4497 Fab fragment against TarS dependent ß-GlcNAc WTA. MFI: mean fluorescence intensity. Each value in the graph shows the mean ± standard error of the mean (SEM) from at least three independent biological replicates. Significance was calculated by two-way ANOVA with (**** p < 0.05).