Manganese uptake by MtsABC contributes to the pathogenesis of human pathogen group A streptococcus by resisting host nutritional immune defenses

Abstract

The interplay between host nutritional immune mechanisms and bacterial nutrient uptake systems has a major impact on the disease outcome. The host immune factor calprotectin (CP) limits the availability of essential transition metals, such as manganese (Mn) and zinc (Zn), to control the growth of invading pathogens. We previously demonstrated that the competition between CP and the human pathogen group A streptococcus (GAS) for Zn impacts GAS pathogenesis. However, the contribution of Mn sequestration by CP in GAS infection control and the role of GAS Mn acquisition systems in overcoming host-imposed Mn limitation remain unknown. Using a combination of in vitro and in vivo studies, we show that GAS-encoded mtsABC is a Mn uptake system that aids bacterial evasion of CP-imposed Mn scarcity and promotes GAS virulence. Mn deficiency caused by either the inactivation of mtsC or CP also impaired the protective function of GAS-encoded Mn-dependent superoxide dismutase. Our ex vivo studies using human saliva show that saliva is a Mn-scant body fluid, and Mn acquisition by MtsABC is critical for GAS survival in human saliva. Finally, animal infection studies using wild-type (WT) and CP-/- mice showed that MtsABC is critical for GAS virulence in WT mice but dispensable in mice lacking CP, indicating the direct interplay between MtsABC and CP in vivo. Together, our studies elucidate the role of the Mn import system in GAS evasion of host-imposed metal sequestration and underscore the translational potential of MtsABC as a therapeutic or prophylactic target.

Keywords: manganese; metal uptake; nutritional immunity; pathogenesis; streptococcus.

Fig 1

MtsABC is critical for GAS Mn acquisition. (A) Genetic organization of the operon-encoding mtsABC genes and the divergently transcribed mtsR gene encoding Mn-sensing transcription regulator in GAS genome is shown. The bent arrows above and below the line indicate the putative transcription start sites of mtsABC and mtsR, respectively. (B) Intracellular metal content of the indicated GAS strains as assessed using total-reflection X-ray fluorescence (TXRF) analyses. GAS strains were grown to mid-exponential growth phase (A₆₀₀ 0.8) in Todd-Hewitt broth (THY) supplemented with 2 µM Mn (THY-M). Cell pellets were washed twice in phosphate-buffered saline (PBS) containing 1 mM nitrilotriacetic acid, followed by two washes in chelexed PBS, and suspended in chelexed PBS. The metal content in the clarified cell lysate was analyzed using TXRF analyses. Data graphed are mean ± standard deviation for three biological replicates. P values (**P < 0.01) were determined by comparison to the respective WT GAS control and were derived from the Kruskal–Wallis test. (C) WT GAS cells were grown to mid-exponential growth phase (A₆₀₀ ~0.8) in the presence or absence of indicated concentrations of recombinant WT or mutant CP proteins. GAS grown in the absence of CP was used as the reference bacterial growth. Transcript levels of mtsC was assessed by reverse transcriptase quantitative PCR (qRT-PCR). The fold change in transcript levels relative to the reference is shown. Data graphed are mean ± standard deviation for three biological replicates grown on separate occasions. P values (**P < 0.01) were determined by comparison to GAS grown in the absence of CP and were derived from the Kruskal–Wallis test.

Fig 2

Gene encoding mtsC is critical for GAS defense against CP-mediated Mn limitation. The survival of GAS grown in the presence or absence of recombinant CP, as assessed using CFU analyses, is shown. GAS was inoculated in THY-CP medium [38% (vol/vol) THY medium and 62% (vol/vol) CP buffer containing 20 mM Tris-HCl pH 7.5, 0.1 M NaCl, 10 mM β-mercaptoethanol, 3 mM CaCl₂] supplemented with 2 µM MnCl₂ and indicated concentrations of recombinant WT (A), ∆His₃Asp site (Zn) (B), ∆His₆ site (C), or ∆His₃Asp site/∆His₆ site (D) mutant CP. After 6 h of incubation, cells were serially diluted, plated, and GAS CFUs were enumerated. Three biological replicates were grown on separate occasions, and the mean ± standard deviation is shown. P values (**P < 0.01, ***P < 0.001) were determined by comparison to respective WT GAS control and were derived from the Kruskal−Wallis test.

Fig 3

Mn-dependent activity of SodA is sensitive to Mn scarcity caused by mtsC inactivation or CP. (A) Native-PAGE analyses of recombinant SodA for SOD activity. Purified recombinant SodA was incubated with or without 5 µM of indicated metals, and the reaction mixture was resolved on a 10% native polyacrylamide gel. The presence of a band corresponding to SodA indicates SOD activity. (B) Indicated strains were grown in THY + CP buffer supplemented with 2 µM Mn to mid-exponential phase of growth (A₆₀₀ ~1.0) and incubated with 2 mM paraquat for 15 min. Cell lysates were assessed for SOD activity. (C) The WT GAS was grown in THY + CP buffer supplemented with 2 µM Mn with or without 250 µg/mL of CP. To assess Mn-dependent restoration of SOD activity, 10 µM Mn was added. Cells were grown to mid-exponential phase of growth (A₆₀₀ ~1.0) and incubated with 2 mM paraquat for 15 min. Cell lysates were assessed for SOD activity. P values (*P < 0.05) were determined by comparison to respective WT GAS control and were derived from the Kruskal–Wallis test.

Fig 4

MtsABC is critical for GAS survival in human saliva ex vivo. (A) TXRF analyses of metal content in human saliva collected from three healthy donors. Samples were analyzed in triplicate, and the mean ± standard deviation is shown. (B) WT GAS was grown in saliva in the presence or absence of 2 µM of indicated metals. GAS grown in THY-M was used as the reference bacterial growth. Transcript levels of the mtsC was assessed using qRT-PCR. The fold change in transcript levels relative to reference is shown. Data graphed are mean ± standard deviation for three biological replicates grown on separate occasions. (C) The human saliva was inoculated with 10³ CFUs of each indicated GAS strain. Samples were collected at the indicated time points, serially diluted, plated, and GAS CFUs were enumerated. Three biological replicates grown on separate occasion were used, and the mean ± standard deviation is shown. P values (*P < 0.05 **P < 0.01) were determined by comparison to respective WT GAS control and were derived from Kruskal–Wallis test.

Fig 5

Gene encoding mtsC is critical for GAS resistance against CP-mediated Mn limitation in vivo and contributes significantly to GAS virulence. The S100a9^+/+ (WT) (A) or S100a9^−/− (B) mice (n = 10 per group) were infected intramuscularly with 1 × 10⁸ CFUs of each indicated bacterial strain. Kaplan–Meier survival curve with P-values derived by log-rank test is shown.