Streptococcus pyogenes Hijacks Host Glutathione for Growth and Innate Immune Evasion

Abstract

The nasopharynx and the skin are the major oxygen-rich anatomical sites for colonization by the human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]). To establish infection, GAS must survive oxidative stress generated during aerobic metabolism and the release of reactive oxygen species (ROS) by host innate immune cells. Glutathione is the major host antioxidant molecule, while GAS is glutathione auxotrophic. Here, we report the molecular characterization of the ABC transporter substrate binding protein GshT in the GAS glutathione salvage pathway. We demonstrate that glutathione uptake is critical for aerobic growth of GAS and that impaired import of glutathione induces oxidative stress that triggers enhanced production of the reducing equivalent NADPH. Our results highlight the interrelationship between glutathione assimilation, carbohydrate metabolism, virulence factor production, and innate immune evasion. Together, these findings suggest an adaptive strategy employed by extracellular bacterial pathogens to exploit host glutathione stores for their own benefit. IMPORTANCE During infection, microbes must escape host immune responses and survive exposure to reactive oxygen species produced by immune cells. Here, we identify the ABC transporter substrate binding protein GshT as a key component of the glutathione salvage pathway in glutathione-auxotrophic GAS. Host-acquired glutathione is crucial to the GAS antioxidant defense system, facilitating escape from the host innate immune response. This study demonstrates a direct link between glutathione assimilation, aerobic metabolism, and virulence factor production in an important human pathogen. Our findings provide mechanistic insight into host adaptation that enables extracellular bacterial pathogens such as GAS to exploit the abundance of glutathione in the host cytosol for their own benefit.

Keywords: Streptococcus pyogenes; glutathione uptake; immune evasion; oxidative stress; redox homeostasis; virulence regulation.

FIG 1

GshT is required for glutathione import and aerobic growth. (a) Schematic of the gshT open reading frame in GAS. The accession number of the reference serotype M1 strain MGAS5005 (GenBank accession no. NC_007297.2) is given for each gene. The predicted gshT promoter is indicated (arrow) (17). (b) Colony morphology of HKU16 and HKU16ΔgshT on 5% horse blood agar after overnight incubation at 37°C. (c) Intracellular accumulation of glutathione in cell lysates of indicated strains (n = 6) from overnight growth on horse blood plates. Total GSH is presented as the sum of GSSG plus GSH. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparisons post hoc test against the HKU16 wild-type control group (****, P < 0.0001 for HKU16ΔgshT). Similar results were obtained from E. coli lysates under the same growth conditions (n = 3), which served as a control group for glutathione measurements. (d) Growth curves of indicated strains in THY medium supplemented with 0.0625, 0.25 and 2 mM GSH (n = 3). (e) Growth curves of HKU16ΔgshT in THY medium supplemented with 1 mM indicated reducing agents (n = 3). All data are presented as mean values ± SD.

FIG 2

Role of glutathione in metabolic redox changes. (a) Schematic representation of the sampling time points at early logarithmic (EL), mid-logarithmic (ML), and late logarithmic (LL) growth phases to measure intracellular redox couple abundances. (b) Quantification of intracellular levels of total GSH and NADP(H) in indicated strains (n = 3). Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparisons post hoc test against the HKU16 wild-type control group (*, P < 0.0; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). All data are presented as mean values ± SD. Source data are provided as a source data file. (c) The Embden-Meyerhof-Parnas (EMP) glycolytic pathway in GAS. Branching points for the oxidative pentose phosphate pathway (ox-PPP) and the tricarboxylic acid (TCA) cycle are shown. Enzymes of interest, namely, GapA (NAD⁺-dependent GAPDH [glyceraldehyde-3-phosphate dehydrogenase]; M5005_RS01330), PGK (phosphoglycerate kinase; M5005_RS07900), GapN (NADP⁺-dependent GAPDH; M5005_RS05535), LDH (lactate dehydrogenase; M5005_RS04370), and LctO (lactate oxidase; M5005_RS01875) are shown.

FIG 3

Global transcriptional response of GAS to glutathione depletion. (a) MA plot of the log₂ fold change of all genes in HKU16ΔgshT compared to the HKU16 wild-type strain (n = 3). Green points indicate genes with a log₂ fold change greater than 1.0 or less than −1.0 and P value of <0.05. (b) RNA-seq expression profile of the speB-prsA operon region in indicated strains. The plots illustrate the overall coverage distribution displaying the total number of sequenced reads. The two speB promoters P and P1, as well as the endoribonuclease Y (RNase Y) processing site, are indicated by black arrows (86). (c) Quantitative real-time PCR of select metabolic and virulence genes in indicated strains (n = 3). Data are presented as mean values ± SD. (d) Immunoblot detection of SpeB in culture supernatants of indicated strains. Band intensities of the zymogen (pro-SpeB) and mature (m-SpeB) form of SpeB were quantified with ImageJ. Data are presented as mean values ± SD. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple-comparison post hoc test against the HKU16 wild-type control group (***, P < 0.001 for HKU16ΔgshT) (n = 3).

FIG 4

Glutathione regulates resistance to oxidative stress and killing by human neutrophils. (a) Survival of mice following intraperitoneal challenge. Groups of 10 C57BL/6J mice were challenged intraperitoneally with 1.1 × 10⁷ CFU of HKU16 wild type and 9.8 × 10⁶ CFU of HKU16ΔgshT. Survival of mice was monitored daily for 10 days. Data are presented as a Kaplan-Meier plot. (b) Growth curves of indicated HKU16 strains, with or without the addition of 2 mM hydrogen peroxide (H₂O₂) (n = 3). Data are presented as mean values ± SD. Growth curves of the wild-type and complemented mutant strain are very similar with substantial overlap. (c) Human neutrophil killing assay (normal, control) showing the percent survival of indicated strains following coculture with human neutrophils in vitro for 30 min at a multiplicity of infection of 0.1 (neutrophil/bacterial CFU) (n = 4). Cytochalasin D (Cyt D) is a potent inhibitor of actin polymerization, preventing phagocytosis and intracellular uptake of bacteria (extracellular, +Cyt D) (n = 3). Data are presented as mean values ± SD. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple-comparison post hoc test against the HKU16 wild-type control group (****, P < 0.0001; *, P < 0.05 for HKU16ΔgshT).

FIG 5

Role for glutathione in host colonization and innate immune evasion. After initial adherence to host epithelial cells, GAS secrete the pore-forming toxin SLO, which binds to host cell membranes and then oligomerizes to form large pores inducing the release of glutathione from perforated host cells due to the significant concentration gradient (~1,000-fold) across the plasma membrane of eukaryotic cells (16). GshT then facilitates the import of extracellular host-derived glutathione enabling aerobic growth of GAS and triggering niche adaptation associated with an altered gene expression profile. Damaged tissues and cells release danger signals to recruit innate immune cells such as neutrophils (87). Intracellular glutathione protects GAS from reactive oxygen species (ROS) produced from infiltrating immune cells in different ways, such as (i) GSH directly and nonenzymatically reduces radical forms of oxygen, while (ii) GSSG is predominantly produced by the enzymatic catalysis of hydroperoxides by the glutathione peroxidase GpoA (12, 88). Glutathione reductase (GR) restores intracellular levels of GSH by reducing GSSG using NADPH as an electron donor, thereby maintaining the cellular supply of GSH. This figure was created with BioRender.com.