Crucial Role for Lipoteichoic Acid Assembly in the Metabolic Versatility and Antibiotic Resistance of Staphylococcus aureus

Abstract

Staphylococcus aureus is a public health threat due to the prevalence of antibiotic resistance and the capacity of this organism to infect numerous organs in vertebrates. To generate energy needed to proliferate within tissues, S. aureus transitions between aerobic respiration and fermentation. Fermentation results in a distinct colony morphology called the small-colony variant (SCV) due to decreased membrane potential and ATP production. These traits promote increased resistance to aminoglycoside antibiotics. Consequently, SCVs are associated with persistent infections. We hypothesize that dedicated physiological pathways support fermentative growth of S. aureus that represent potential targets for treatment of resistant infections. Lipoteichoic acid (LTA) is an essential component of the Gram-positive cell envelope that functions to maintain ion homeostasis, resist osmotic stress, and regulate autolytic activity. Previous studies revealed that perturbation of LTA reduces viability of metabolically restricted S. aureus, but the mechanism by which LTA supports S. aureus metabolic versatility is unknown. Though LTA is essential, the enzyme that synthesizes the modified lipid anchor, YpfP, is dispensable. However, ypfP mutants produce altered LTA, leading to elongation of the polymer and decreased cell association. We demonstrate that viability of ypfP mutants is significantly reduced upon environmental and genetic induction of fermentation. This anaerobic viability defect correlates with decreased membrane potential and is restored upon cation supplementation. Additionally, ypfP suppressor mutants exhibiting restored anaerobic viability harbor compensatory mutations in the LTA biosynthetic pathway that restore membrane potential. Overall, these results demonstrate that LTA maintains membrane potential during fermentative proliferation and promotes S. aureus metabolic versatility.

Keywords: Staphylococcus aureus; cation; glycolipid anchor; ion homeostasis; lipoteichoic acid; membrane potential; metabolism; small-colony variant; ypfP.

FIG 1

Respiration-arresting conditions impair S. aureus ypfP proliferation. (A) CFU of WT or ypfP mutant cells grown on TSA supplemented with 5 μg mL⁻¹ of gentamicin (gent) or tobramycin (tob) were enumerated after 24 h at 37°C. Values are percentages of the WT value, where the number of CFU of the ypfP mutant was divided by the number of CFU of the WT and the WT value was set to 100% (dotted line). Data are means from at least five independent experiments. Error bars represent one standard deviation from the mean. (B) Zones of inhibited growth generated by colonies of P. aeruginosa spotted on top of S. aureus WT or ypfP mutant lawns were measured after 24 h at 37°C. Data are means from three independent experiments performed in triplicate. Statistical significance was determined by an unpaired two-tailed t test for unequal variance. P < 0.0001. Error bars represent one standard deviation from the mean. (C) The zone of reduced growth generated by HQNO on lawns of WT or ypfP mutant cells was measured after 24 h at 37°C. Data are means from three independent experiments performed in triplicate. Error bars represent one standard deviation from the mean. Statistical significance was determined by an unpaired two-tailed t test for unequal variance. P < 0.01. (D) CFU of WT and ypfP, gtrR or gtrR ypfP mutant strains were quantified after 24 h (for respiring colonies) or 48 h (for respiration arrest colonies) of growth on TSA or TSA supplemented with ALA at 37°C. Error bars represent one standard deviation for three independent experiments. Statistical significance was determined by two-way analysis of variance (ANOVA) with a Šidák method for multiple comparisons. **, P < 0.01.

FIG 2

ypfP mutant cells demonstrate reduced anaerobic proliferation. (A) CFU of WT or ypfP mutant bacteria generated after incubation under aerobic (+O₂) or anaerobic (−O₂) conditions were enumerated after 24 h at 37°C. Potassium nitrate (KNO₃; 100 mM) was used to induced anaerobic respiration. Data are means from three independent experiments performed in triplicate. Error bars represent one standard deviation from the mean. Statistical significance was determined by a two-way ANOVA with Tukey’s multiple-comparison test. ****, P < 0.0001. (B) The WT and the ypfP mutant were subcultured 1:100 from an overnight culture and grown anaerobically at 37°C. Growth was measured at the indicated time points by monitoring OD₆₀₀. The experiment was performed in triplicate. Error bars represent standard deviations from the mean.

FIG 3

ypfP, not ltaA, is responsible for alterations in viability and LTA production under anaerobic conditions. (A) The LTA-biosynthetic pathway in S. aureus. YpfP uses DAG and two UDP glucose (UDP-Glc) molecules to generate the lipid anchor Glc₂DAG. Glc₂-DAG is then flipped to the outer leaflet of the membrane by LtaA, and glycerol phosphate (GroP) taken from phosphatidylglycerol (PG) is added directly to the anchor by LtaS. Illustration created using Biorender.com. (B) Illustration of the ypfP ltaA operon demonstrating the 41-bp ypfP-ltaA overlap. Locations of transposon insertions are indicated with arrowheads. (C) WT and ypfP mutant NWMN cells harboring the S. aureus expression vector pOS containing the indicated genes were spot plated on TSA and incubated in an anaerobic chamber for 24 h of growth at 37°C prior to CFU enumeration. Data are means from three independent experiments performed in triplicate. Error bars represent one standard deviation from the mean. Significance was determined via two-tailed t test. ****, P < 0.0001; ***, P < 0.001. (D) Representative immunoblot using a monoclonal anti-LTA antibody and a secondary antibody conjugated with horseradish peroxidase shows the altered LTA profiles of NWMN ypfP and ltaA mutants cultured under aerobic (+O₂) and anaerobic (−O₂) conditions compared to the WT. Differences in the LTA profile are indicated with arrowheads.

FIG 4

Cation supplementation restores anaerobic viability of ypfP mutants undergoing respiration arrest. (A) Membrane potential of the WT and ypfP mutant. The y axis shows the quotient of the 610 nm and 530 nm emission spectra. Data are means from three independently performed experiments performed in technical triplicate. Error bars represent one standard deviation from the mean. Significance was determined using two-way ANOVA. ****, P < 0.0001. (B) The antimicrobial activity of the potassium-specific ionophore valinomycin was determined for WT and ypfP mutant cells. The WT and the ypfP mutant were normalized to an OD₆₀₀ of 0.5 and were allowed to grow in liquid medium in the presence of various concentrations of valinomycin anaerobically for 16 h. Cells were suspended by pipetting, and the OD₆₀₀ was measured. Results are percent OD₆₀₀ compared to that of the untreated control. The mean for five independent experiments performed in triplicate is shown. The regression line was mapped using GraphPad Prism. Error bars represent one standard deviation from the mean. (C) Anaerobic viability of the WT and the ypfP mutant on medium supplemented with various concentrations of KCl, NaCl, or the osmoprotectant GB. CFU were enumerated after 24 h of growth at 37°C. Data are means from three independent experiments performed in triplicate. Error bars represent one standard deviation from the mean. (D) Anaerobic viability of the WT or the ypfP mutant on medium buffered to a pH of 7 or 6. CFU were enumerated after 48 h of growth at 37°C. Data are means from three independent experiments performed in triplicate. Error bars represent one standard deviation from the mean. (E) The WT and the ypfP mutant were subcultured 1:100 in TSB, and the OD₆₀₀ and pH were measured at the indicated time points. The data are averages from three independent experiments. Error bars represent one standard deviation from the mean. (B and C) Significance was determined using one-way ANOVA with multiple comparisons. ****, P < 0.0001; **, P <0.005.

FIG 5

Passaged ypfP mutants harbor suppressor mutations that lead to phenotypic differences in membrane potential, gentamicin resistance and LTA. (A) Immunoblot of LTA isolated from cells grown aerobically to late exponential phase. Passaged ypfP mutants are named to reflect the lineage number and the passage at which they reached viability equal to that of the WT. For example, S1P4 represents passaged lineage 1, which reached WT-like viability after four passes under anaerobic conditions. (B) Anaerobic membrane potential was measured using the fluorescent dye DiOC₂. Data are means from three independent experiments performed in technical triplicate. Error bars represent one standard deviation from the mean. Significance was determined using one-way ANOVA with multiple comparisons. ****, P < 0.0001; **, P < 0.01. (C) Percent gentamicin-resistant colonies relative to the WT (dotted line). Data represent 3 independent experiments. Error bars represent one standard deviation from the mean. Significance was determined using one-way ANOVA with multiple comparisons. ****, P < 0.0001; ***, P < 0.001; **, P < 0.001. (D) Strains were serially diluted, spot plated onto TSA, and incubated anaerobically and CFU were enumerated for the WT and the parental ypfP mutant complemented with empty pOS (EV), WT ltaS or ltaA (ltaS_WT and ltaA_WT), mutated ltaS from S1P4 (ltaS_S1P4), or mutated ltaA from S2P3 or S4P3 (ltaA_S2P3 and ltaA_S4P3). Data are means from 3 independent experiments. Error bars represent one standard deviation from the mean. Significance was determined using a two-tailed t test. ****, P < 0.0001; **, P < 0.01; *, P < 0.05.