Intracellular Accumulation of Staphylopine Can Sensitize Staphylococcus aureus to Host-Imposed Zinc Starvation by Chelation-Independent Toxicity

Abstract

The host restricts the availability of zinc to prevent infection. To overcome this defense, Staphylococcus aureus and Pseudomonas aeruginosa rely on zincophore-dependent zinc importers. Synthesis of the zincophore staphylopine by S. aureus and its import are both necessary for the bacterium to cause infection. In this study, we sought to elucidate how loss of zincophore efflux impacts bacterial resistance to host-imposed zinc starvation. In culture and during infection, mutants lacking CntE, the staphylopine efflux pump, were more sensitive to zinc starvation imposed by the metal-binding immune effector calprotectin than those lacking the ability to import staphylopine. However, disruption of staphylopine synthesis reversed the enhanced sensitivity phenotype of the ΔcntE mutant to calprotectin, indicating that intracellular toxicity of staphylopine is more detrimental than the impaired ability to acquire zinc. Unexpectedly, intracellular accumulation of staphylopine does not increase the expression of metal importers or alter cellular metal concentrations, suggesting that, contrary to prevailing models, the toxicity associated with staphylopine is not strictly due to intracellular chelation of metals. As P. aeruginosa and other pathogens produce zincophores with similar chemistry, our observations on the crucial importance of zincophore efflux are likely to be broadly relevant.IMPORTANCEStaphylococcus aureus and many other bacterial pathogens rely on metal-binding small molecules to obtain the essential metal zinc during infection. In this study, we reveal that export of these small molecules is critical for overcoming host-imposed metal starvation during infection and prevents toxicity due to accumulation of the metal-binding molecule within the cell. Surprisingly, we found that intracellular toxicity of the molecule is not due to chelation of cellular metals.

Keywords: Staphylococcus aureus; calprotectin; nutritional immunity; pseudopaline; staphylopine; zincophore.

FIG 1

CntE promotes resistance to calprotectin-imposed metal starvation. (A) Wild-type S. aureus and ΔcntA, ΔcntE, ΔcntA ΔadcA, and ΔcntE ΔadcA mutants were incubated in the presence of increasing concentrations of CP, and growth was assessed by measuring optical density at 600 nm (OD₆₀₀) at 12 h. (B) Wild-type S. aureus and ΔcntA, ΔcntE, ΔcntKLM ΔcntE, ΔcntA ΔadcA, ΔcntE ΔadcA, and ΔcntKLM ΔcntE ΔadcA mutants were incubated in the presence of increasing concentrations of CP, and the growth was assessed by measuring the OD₆₀₀ at 12 h. (C) Wild-type S. aureus and ΔcntE and ΔcntE ΔadcA mutants with the indicated plasmid were incubated in the presence of increasing concentrations of CP, and the growth was assessed by measuring the OD₆₀₀ at 12 h. (A and B) *, P < 0.05 compared to wild type at the same CP concentration via two-way analysis of variance (ANOVA) with Dunnett’s posttest; #, P < 0.05 compared to the ΔcntA (A) or ΔcntKLM (B) mutant at the same CP concentration via two-way ANOVA with Dunnett’s posttest. n ≥ 3. (C) *, P < 0.05 compared to wild-type vector control (VC) at the same CP concentration via two-way ANOVA with Dunnett’s posttest. Error bars in all panels are standard errors of the mean, and each panel represents an independent series of experiments performed the indicated number (n) of times on independent days. See also Fig. S1 in the supplemental material.

FIG 2

Zinc and iron reverse the growth defect of strains lacking CntE. (A) Wild-type S. aureus and ΔcntA, ΔcntE, ΔcntA ΔadcA, and ΔcntE ΔadcA mutants were grown in medium containing 480 μg/ml of wild type, ΔS1, or ΔS2 CP. Growth was assessed by measuring the OD₆₀₀ at 12 h. *, P < 0.05 compared to wild-type bacteria grown in the same variant of CP via two-way ANOVA with Dunnett’s posttest. #, P < 0.05 compared to the ΔcntA mutant grown at the same CP concentration via two-way ANOVA with Dunnett’s posttest. n ≥ 3. (B) Wild-type S. aureus and ΔcntA, ΔcntE, ΔcntA ΔadcA, and ΔcntE ΔadcA mutants were grown in NRPMI containing 25 μM ZnSO₄, MnCl₂, or FeSO₄ as indicated. Growth was assessed by measuring the OD₆₀₀ at 7 h. *, P < 0.05 compared to wild type in the same medium via two-way ANOVA with Dunnett’s posttest. n ≥ 3. Error bars in all panels are standard errors of the mean. See also Fig. S2 in the supplemental material.

FIG 3

Over accumulation of staphylopine is toxic in metal-replete medium. S. aureus ΔcntKLM and ΔcntKLM ΔcntE mutants containing the indicated plasmids were grown in medium supplemented with anhydrotetracycline (Atc). (A) Growth was assessed by measuring the OD₆₀₀. *, P < 0.05 compared to the same strain carrying the vector control (VC) plasmid at the same concentration of Atc via two-way ANOVA with Tukey’s posttest. #, P < 0.05 compared to the ΔcntKLM pCntKLM mutant via two-way ANOVA with Tukey’s posttest. n ≥ 3. (B) Bacteria were grown in the presence of 6.25 ng/ml Atc, and gene expression was assessed via qRT-PCR. n ≥ 3. (C to G) The growth medium was supplemented with Zn (C), Fe (D), Co (E), Mn (F), and Ni (G). Growth was assessed by measuring the OD₆₀₀ at 8 h. *, P < 0.05 compared to the same strain grown in the absence of additional metals via two-way ANOVA with Dunnett’s posttest. Error bars in all panels are standard errors of the mean. See also Fig. S3 in the supplemental material.

FIG 4

Intracellular staphylopine does not lead to accumulation of metals. S. aureus ΔcntKLM and ΔcntKLM ΔcntE mutants containing the indicated plasmids were grown in the presence of 6.25 ng/ml of anhydrotetracycline (Atc) with or without 400 μM ZnSO₄. The intracellular metal concentration was assessed using ICP-MS. (A) Zn; (B) Mn; (C) Fe; (D) Co; (E) Ni; (F) Cu. *, P < 0.05 compared to the same strain grown with no Zn added via one-way ANOVA using Tukey’s posttest. n ≥ 3. Error bars in all panels are standard errors of the mean.

FIG 5

Accumulation of staphylopine intermediates are toxic to S. aureus. S. aureus ΔcntKLM (A) and ΔcntKLM ΔcntE (B) mutants containing the indicated plasmids were grown in increasing concentrations of anhydrotetracycline (Atc). Growth was assessed by measuring the OD₆₀₀ at 8 h. *, P < 0.05 compared to vector control at the same Atc concentration via two-way ANOVA using Dunnett’s posttest. n ≥ 3. Error bars in all panels are standard errors of the mean.

FIG 6

ΔcntE is more attenuated than ΔcntA during infection. C57BL/6J mice were retro-orbitally infected with 1 × 10⁷ CFU of wild-type S. aureus or ΔcntA, ΔcntE, ΔcntA adcA, and ΔcntE ΔadcA mutants. The weights of the mice (A) as well as the bacterial burdens in the heart (B), liver (C), and kidney (D) were assessed. (A) *, #, and ^, P < 0.05 for the ΔcntE mutant, the ΔcntA ΔadcA mutant, and the ΔcntE ΔadcA mutant, respectively, compared to mice infected with the wild type via two-way ANOVA with Dunnett’s posttest. (B to D) *, P < 0.05 compared to mice infected with the wild type via Mann-Whitney U test. #, P < 0.05 compared to the ΔcntA mutant via Mann-Whitney U test. Error bars in panel A are standard errors of the mean.