The Two-Component System ArlRS and Alterations in Metabolism Enable Staphylococcus aureus to Resist Calprotectin-Induced Manganese Starvation

Abstract

During infection the host imposes manganese and zinc starvation on invading pathogens. Despite this, Staphylococcus aureus and other successful pathogens remain capable of causing devastating disease. However, how these invaders adapt to host-imposed metal starvation and overcome nutritional immunity remains unknown. We report that ArlRS, a global staphylococcal virulence regulator, enhances the ability of S. aureus to grow in the presence of the manganese-and zinc-binding innate immune effector calprotectin. Utilization of calprotectin variants with altered metal binding properties revealed that strains lacking ArlRS are specifically more sensitive to manganese starvation. Loss of ArlRS did not alter the expression of manganese importers or prevent S. aureus from acquiring metals. It did, however, alter staphylococcal metabolism and impair the ability of S. aureus to grow on amino acids. Further studies suggested that relative to consuming glucose, the preferred carbon source of S. aureus, utilizing amino acids reduced the cellular demand for manganese. When forced to use glucose as the sole carbon source S. aureus became more sensitive to calprotectin compared to when amino acids are provided. Infection experiments utilizing wild type and calprotectin-deficient mice, which have defects in manganese sequestration, revealed that ArlRS is important for disease when manganese availability is restricted but not when this essential nutrient is freely available. In total, these results indicate that altering cellular metabolism contributes to the ability of pathogens to resist manganese starvation and that ArlRS enables S. aureus to overcome nutritional immunity by facilitating this adaptation.

Fig 1. The S. aureus two-component system ArlRS plays a role in resisting calprotectin-imposed Mn starvation.

Growth assays were performed in rich medium in the presence of increasing concentrations of CP for (A) WT (Newman) and the arlRS:erm derivative (Tn 917 transposon mutant), (B) WT (Newman), ΔarlRS and arlR:erm derivatives, (C) WT (Newman) and ΔarlRS containing either pOS1 plgt or pOS1 plgt:arlRS, (D) WT (USA300) and the arlR:erm derivative and (E) WT (SH1000) and the arlR:erm derivative. (F) Growth of WT S. aureus (Newman) and ΔarlRS, pre-cultured in NRPMI, in the presence of 480 μg/ml of WT CP, as well as the ΔMn/Zn, ΔZn and ΔMn/ZnΔZn site mutants. * = p≤0.05 by two-way ANOVA with Tukey’s posttests of selected means. n≥3. Error bars indicate SD. See also S1 Fig.

Fig 2. Autolysis does not contribute to increased sensitivity to metal starvation.

The growth of (A) WT (Newman), ΔarlRS, ΔarlRS/lytM:erm, ΔarlRS/atl:erm and ΔarlRS/lytN:erm and (B) WT (Newman), lytM:erm, atl:erm, and lytN:erm were assessed in rich medium in the presence of increasing concentrations of CP. * = p≤0.05 by two-way ANOVA with Tukey’s posttests of selected means. n≥3. Error bars indicate SD.

Fig 3. Loss of ArlRS does not alter Mn and Zn import.

WT S. aureus (Newman) and ΔarlRS were grown in rich medium in the presence and absence of 240 μg/ml of CP. (A) Transcript levels of mntA and mntH were assessed by qRT-PCR. Expression was compared to wild type bacteria grown in the absence of CP. * = p≤0.05 by two-way ANOVA on log-transformed values with Sidak’s multiple comparisons test. n≥3. (B) WT S. aureus (Newman), arlR:erm, ΔmntC ΔmntH and ΔmntC ΔmntH arlR:erm were grown in the presence of increasing concentrations of CP. * = p≤0.05 by two-way ANOVA with Tukey’s posttests of selected means. n≥3. Strains are normalized to growth of parental strains, i.e., arlR:erm is normalized to WT and ΔmntC ΔmntH arlR:erm is normalized to ΔmntC ΔmntH. (C) Intracellular Mn, Zn and Fe levels were determined by ICP-OES. * = p≤0.05 by Student’s t-test. n≥3 Error bars indicate SD. See also S2 Fig.

Fig 4. ArlRS is necessary for establishing invasive S. aureus infection and for resisting Mn sequestration during infection.

Wild type C57BL/6 (C57) and CP-deficient C57BL/6 S100A9-/- (CP-/-) mice were infected with either S. aureus Newman (wild type) or ΔarlRS and (A) mean weight loss and (B-C) bacterial burdens in the (B) liver and (C) heart and kidneys were assessed after four days of infection. (A) See S3 Fig for pair wise comparisons that include error bars. * = p≤ 0.05 relative to C57BL/6 mice infected with wild type S. aureus and # = p≤ 0.05 relative to C57BL/6 mice infected with ΔarlRS by two-way ANOVA with Tukey’s posttest corrected for repeated measurements. (B-C) * = p≤0.05 as determined by Mann-Whitney test. The lines indicate the mean. The data are the results from three independent experiments. See also S3 Fig.

Fig 5. ArlRS enhances gene expression in response to calprotectin.

WT S. aureus (Newman) and ΔarlRS were grown in rich medium in the presence and absence of 240 μg/ml of CP and transcript levels of the threonine/serine deaminase (NWMN_1348) was assessed by qRT-PCR. Expression was compared to wild type bacteria grown in the absence of CP. * = p≤0.05 by one-way ANOVA with Sidak’s multiple comparisons test. Error bars indicate SD. n≥3.

Fig 6. Alteration in carbon source utilization promotes resistance to Mn starvation.

(A) Growth of S. aureus WT (Newman) and ΔarlRS in defined medium lacking a carbon source (DM) and medium supplemented with glucose (glc) or casamino acids (AA). (B-D) Growth assays were performed in defined medium containing either glucose (glc) or casamino acids (AA) as the sole carbon source in the presence of increasing concentrations of CP for (B) Newman, (C) USA300 (JE2), and (D) Newman with 120 μg/ml of WT CP, the ΔMn/Zn mutant or the ΔZn mutant. * = p≤0.05 by two-way ANOVA with Bonferroni’s posttest corrected for repeated measurements. n≥3. (E) WT (Newman) was grown in defined medium containing either glucose (glc) or casamino acids (AA) as a carbon source in the presence and absence of 120 μg/ml of CP and in the presence and absence of 500 μM MnCl₂. (F) WT (Newman) was grown in defined medium containing either glucose (glc) or casamino acids (AA) as a carbon source in the presence and absence of 120 μg/ml of CP and transcript levels of mntA were assessed by qRT-PCR. Expression was compared to wild type bacteria grown in glucose-containing medium in the absence of CP. * = p≤0.05 by one-way ANOVA on log-transformed values with Dunnett’s multiple comparisons test. n≥3. L-Lactate production was measured for bacteria pre-cultured in NRPMI and assayed in defined medium containing both glucose and casamino acids in the presence of increasing concentrations of CP for (G) WT (Newman) and ΔarlRS and (H) WT (USA300 (JE2)) and arlR:erm. ND = not determined due to insufficient growth. * = p≤0.05 by one-way ANOVA on log-transformed values with Dunnett’s multiple comparisons test. n≥3. Error bars indicate SD for all panels except panels G and H, in which error bars indicate SEM. See also S4 Fig.

Fig 7. Working model of how calprotectin impacts staphylococcal metabolism and the contribution of ArlRS to resisting Mn limitation.

In manganese (Mn)-replete environments (A) sugars serve as the primary energy source for S. aureus. However, in Mn-poor environments (B), such as in the presence of the Mn-binding protein calprotectin (CP), glycolysis is inhibited. ArlRS appears to enhance the ability of S. aureus to resist Mn limitation in part by increasing amino acid (AA) catabolic pathways and reducing the expression of alternative sugar importers.