PBP4 is required for serum-induced cell wall thickening and antibiotic tolerance in Staphylococcus aureus

Abstract

The bacterial pathogen Staphylococcus aureus responds to the host environment by synthesizing a thick peptidoglycan cell wall, which protects the bacterium from membrane-targeting antimicrobials and the immune response. However, the proteins required for this response were previously unknown. Here, we demonstrate by three independent approaches that the penicillin-binding protein PBP4 is crucial for serum-induced cell wall thickening. First, mutants lacking various non-essential cell wall synthesis enzymes were tested, revealing that a mutant lacking pbp4 was unable to generate a thick cell wall in serum. This resulted in reduced serum-induced tolerance of the pbp4 mutant toward the last resort antibiotic daptomycin relative to wild-type cells. Second, we found that serum-induced cell wall thickening occurred in each of a panel of 134 clinical bacteremia isolates, except for one strain with a naturally occurring mutation that results in an S140R substitution in the active site of PBP4. Finally, inhibition of PBP4 with cefoxitin prevented serum-induced cell wall thickening and the resulting antibiotic tolerance in the USA300 strain and clinical MRSA isolates. Together, this provides a rationale for combining daptomycin with cefoxitin, a PBP4 inhibitor, to potentially improve treatment outcomes for patients with invasive MRSA infections.

Keywords: PBP4; Staphylococcus aureus; antibiotic tolerance; cell wall; daptomycin; peptidoglycan.

Fig 1

Non-essential cell wall biosynthetic proteins are required for serum-induced cell wall thickening and daptomycin tolerance. (A) TSB-grown and serum-incubated cultures of Col WT and MIN and Newman WT and MIN were generated in the presence of HADA and de novo cell wall synthesis determined by fluorescence microscopy. Scale bars, 5 µm. (B) Fluorescence at the peripheral cell wall of 50 cells per condition per replicate of panel A was determined. TSB-grown and serum-incubated cultures of (C) Col WT and MIN and (D) Newman WT and MIN were exposed to 80 µg mL⁻¹ daptomycin for 6 h and survival determined by CFU counts. Data in B represent the fluorescence of 50 cells per replicate (150 cells total per condition). *, P ≤ 0.05 as determined by two-way ANOVA with Tukey’s post hoc test. Data in C and D represent the geometric mean ± geometric standard deviation of three independent replicates. *, P ≤ 0.05 as determined by two-way ANOVA with Tukey’s post hoc test; serum-incubated WT vs serum-incubated MIN at indicated time points.

Fig 2

PBP4 is required for serum-induced cell wall thickening and daptomycin tolerance. (A) Cell wall thickening measured by HADA fluorescence (RFU) divided by OD₆₀₀ of serum-incubated cultures of JE2 WT and indicated NARSA transposon mutants. (B) Survival presented as log₁₀ CFU mL⁻¹ of serum-incubated cultures of JE2 WT and indicated NARSA transposon mutants during a 6-h exposure to 80 µg mL⁻¹ daptomycin. (C) Cell wall thickening measured by HADA fluorescence (RFU) divided by OD₆₀₀ of serum-incubated cultures of JE2 WT, pbp4::Tn mutant, and pbp4::Tn mutant complemented with empty pCN34 or pCN34 containing a WT copy of pbp4. (D) Survival presented as log₁₀ CFU mL⁻¹ of serum-incubated cultures of JE2 WT, pbp4::Tn mutant, and pbp4::Tn mutant complemented with empty pCN34 or pCN34 containing a WT copy of pbp4 during exposure to 80 µg mL⁻¹ daptomycin for 6 h. Data in A and C represent the mean ± standard deviation of three independent repeats. Data in B and D represent the geometric mean ± geometric standard deviation of at least three independent repeats. Data in A and C were analyzed by one-way ANOVA with Dunnett’s post hoc test. Data in B and D were analyzed by two-way ANOVA with Dunnett’s post hoc test. *, P ≤ 0.05, ***, P ≤ 0.0001, WT vs mutant or complemented mutant at indicated time points.

Fig 3

Serum-induced cell wall thickening occurs in clinical isolates. (A) HADA fluorescence (RFU) divided by OD₆₀₀ of serum-incubated cultures of a panel of clinical bacteremia isolates. Each data point represents the mean of three independent repeats of one clinical isolate. The arrow indicates ASARM124, the strain with a significantly reduced cell wall thickness in serum. TSB-grown and serum-incubated cultures of (B) ASARM141, (C) ASARM179, (D) ASARM184, (E) ASARM208, and (F) ASARM124 were generated in the presence of HADA, and de novo cell wall synthesis determined by fluorescence microscopy. Scale bars, 5 µm. (G) Fluorescence at the peripheral cell wall of 50 cells per condition per replicate of panels B–F was determined. ****, P ≤ 0.0001; ns, P > 0.05 as determined by two-way ANOVA with Sidak’s post hoc test.

Fig 4

Serum-induced cell wall thickening and daptomycin tolerance in clinical isolates are PBP4 dependent. (A) HADA fluorescence (RFU) divided by OD₆₀₀ of serum-incubated cultures of the JE2 pbp4::Tn mutant complemented with empty pCN34, pCN34 containing a WT copy of pbp4 (pCC22-WT), or pCN34 containing a copy of pbp4 containing the SNP (pCC22-pbp4_S140R). (B) Log₁₀ CFU mL⁻¹ of serum-incubated cultures of the JE2 pbp4::Tn mutant complemented with empty pCN34, pCN34 containing a WT copy of pbp4 (pCC22-WT), or pCN34 containing a copy of pbp4 containing the SNP (pCC22-pbp4_S140R) after a 6-h exposure to 80 µg mL⁻¹ daptomycin. Data in A represent the mean ± standard deviation of three independent repeats, and data in B represent the geometric mean ± geometric standard deviation of three independent repeats. Data in A were analyzed by one-way ANOVA with Dunnett’s post hoc test (**, P ≤ 0.01), and data in B were analyzed by two-way ANOVA with Dunnett’s post hoc test (*, P ≤ 0.05; pCN34 vs pCC22 at indicated time points).

Fig 5

Inhibition of PBP4 with cefoxitin reduces cell wall thickening and daptomycin tolerance in JE2. (A) Cell wall thickening measured by HADA fluorescence (RFU/OD₆₀₀) of JE2 WT after a 16-h incubation in serum supplemented with the indicated concentration of cefoxitin (FOX) or the pbp4::Tn mutant incubated in serum alone. (B and C) JE2 WT or the pbp4::Tn mutant were incubated in serum with or without 2 µg mL⁻¹ cefoxitin (FOX) for 16 h before they were incubated (B) without or (C) with daptomycin at 80 µg mL⁻¹ and survival measured over 6 h. Data in A represent the mean ± standard deviation of three independent replicates. Data in B and C represent the geometric mean ± geometric standard deviation of three independent replicates. Data in A were analyzed by one-way ANOVA with Dunnett’s post hoc test (*, P ≤ 0.05; pbp4::Tn vs other conditions). Data in B and C were analyzed by two-way ANOVA with Dunnett’s post hoc test (*, P ≤ 0.05; WT vs other conditions at indicated time points).

Fig 6

Inhibition of PBP4 with cefoxitin reduces cell wall thickening and daptomycin tolerance in clinical isolates. Cell wall thickening as measured by HADA fluorescence (RFU/OD₆₀₀) of (A) ASARM141, (B) ASARM179, (C) ASARM184, (D) ASARM208, and (E) ASARM124 after a 16-h incubation in serum supplemented with the indicated concentration of cefoxitin. Survival as measured by log₁₀ CFU mL⁻¹ of (F) ASARM141, (G) ASARM179, (H) ASARM184, (I) ASARM208, and (J) ASARM124 following incubation for 16 h in serum supplemented or not with 2 µg mL⁻¹ cefoxitin and the addition, or not, of 80 µg mL⁻¹ daptomycin for 6 h. Data in A–E represent the mean ± standard deviation of three independent replicates. Data in F–J represent the geometric mean ± geometric standard deviation of three independent replicates. Data in A–E were analyzed by one-way ANOVA with Dunnett’s post hoc test (*, P ≤ 0.05; serum alone vs serum supplemented with cefoxitin). Data in F–J were analyzed by two-way ANOVA with Sidak’s post hoc test (*, P ≤ 0.05; daptomycin survival in serum alone vs serum supplemented with cefoxitin).