Glutathione Synthesis Contributes to Virulence of Streptococcus agalactiae in a Murine Model of Sepsis

Abstract

Streptococcus agalactiae, a leading cause of sepsis and meningitis in neonates, utilizes multiple virulence factors to survive and thrive within the human host during an infection. Unique among the pathogenic streptococci, S. agalactiae uses a bifunctional enzyme encoded by a single gene (gshAB) to synthesize glutathione (GSH), a major antioxidant in most aerobic organisms. Since S. agalactiae can also import GSH, similar to all other pathogenic streptococcal species, the contribution of GSH synthesis to the pathogenesis of S. agalactiae disease is not known. In the present study, gshAB deletion mutants were generated in strains representing three of the most prevalent clinical serotypes of S. agalactiae and were compared against isogenic wild-type and gshAB knock-in strains. When cultured in vitro in a chemically defined medium under nonstress conditions, each mutant and its corresponding wild type had comparable growth rates, generation times, and growth yields. However, gshAB deletion mutants were found to be more sensitive than wild-type or gshAB knock-in strains to killing and growth inhibition by several different reactive oxygen species. Furthermore, deletion of gshAB in S. agalactiae strain COH1 significantly attenuated virulence compared to the wild-type or gshAB knock-in strains in a mouse model of sepsis. Taken together, these data establish that GSH is a virulence factor important for resistance to oxidative stress and that de novo GSH synthesis plays a crucial role in S. agalactiae pathogenesis and further suggest that the inhibition of GSH synthesis may provide an opportunity for the development of novel therapies targeting S. agalactiae disease.IMPORTANCE Approximately 10 to 30% of women are naturally and asymptomatically colonized by Streptococcus agalactiae However, transmission of S. agalactiae from mother to newborn during vaginal birth is a leading cause of neonatal meningitis. Although colonized mothers who are at risk for transmission to the newborn are treated with antibiotics prior to delivery, S. agalactiae is becoming increasingly resistant to current antibiotic therapies, and new treatments are needed. This research reveals a critical stress resistance pathway, glutathione synthesis, that is utilized by S. agalactiae and contributes to its pathogenesis. Understanding the role of this unique bifunctional glutathione synthesis enzyme in S. agalactiae during sepsis may help elucidate why S. agalactiae produces such an abundance of glutathione compared to other bacteria.

Keywords: Streptococcus agalactiae; glutathione; hydrogen peroxide; hypochlorous acid; oxidative stress; virulence.

FIG 1

Deletion and reinsertion of gshAB only affects gshAB and not downstream genes. (A) Position of SAG_1821 gshAB in reference to its first downstream gene, SAG_1822 hyp, in strain 2603V/R as indicated. An identical genomic arrangement is present in A909 and COH1; therefore, only 2603V/R is shown as a reference. Filled block arrows represent individual open reading frames, dashed arrows indicate forward and reverse qPCR primers, and solid line arrows represent putative operons. gshAB and hyp are predicted to be encoded in independent operons (thin arrows above the block arrows) separated by 123 bp. Real-time qPCR was used to determine the relative transcript levels of gshAB and hyp in the ΔgshAB and gshAB⁺ mutants compared to their respective WT strain. (B to D) Results for A909 (B), COH1 (C), and 2603V/R (D) show that the hyp levels are approximately equivalent to that of the WT (∼1-fold change) for both ΔgshAB and gshAB⁺ mutants, whereas the gshAB levels are approximately equivalent for the gshAB⁺ mutant but were undetectable for the ΔgshAB mutant (indicated by an asterisk).

FIG 2

Growth curves and generation times of WT, ΔgshAB, and gshAB⁺ S. agalactiae for strains A909, COH1, and 2603V/R were assayed by using a standard 96-well plate assay. (A to C) A 200-μl portion of GSH-free CD medium was inoculated with one bacterial colony (n ≥ 8), and the OD₆₀₀ was measured every 15 min for 24 h to produce growth curves. (D) The generation time was determined using the formula for the indirect method of determining generation time [G = t_{(A 0.4)} – t_{(A 0.2)}]. Error bars represent the SEM, and no significant differences were observed between WT, ΔgshAB, and gshAB⁺ strains of any of the three serotypes, as determined by a Mann-Whitney rank sum test.

FIG 3

Glutathione production of WT, ΔgshAB, and gshAB⁺ S. agalactiae. WT, ΔgshAB, and gshAB⁺ S. agalactiae strains were grown overnight at 37°C in GSH-free CD medium prior to GSH analysis. Three serotypes were assayed: A909, COH1, and 2603V/R. A “#” symbol indicates that the ΔgshAB mutant was below limit of detection (1 nmol/mg), error bars represent the SEM, and an asterisk indicates a significant difference (P < 0.05). To determine the significance between ΔgshAB and WT strains and between ΔgshAB and gshAB⁺ strains, the limit of detection (1 nmol/mg) was assumed for all ΔgshAB values. There was no significant difference determined by the Mann-Whitney rank sum test between WT and gshAB⁺ strains for any of the three serotypes.

FIG 4

Bacterial growth restriction on solid media in response to H₂O₂, HOCl, and erythromycin. Bacterial growth restriction was assessed between WT, ΔgshAB, and gshAB⁺ strains grown in the presence of 750 mM H₂O₂ (A), 589 mM HOCl (B), or 15 μg of erythromycin (C). Error bars represent the SEM with n ≥ 3, and asterisks indicate a significant difference (*, P < 0.05; **, P < 0.01; ***, P < 0.001) between WT and ΔgshAB strains, as determined by a Mann-Whitney rank sum test.

FIG 5

MICs (A, C, and E) and MBCs (B, D, and F) of H₂O₂ and HOCl on S. agalactiae serotypes A909 (A and B), COH1 (C and D), and 2603V/R (E and F). Asterisks indicate a significant difference (*, P < 0.05; **, P < 0.01). WT and gshAB⁺ strains were not significantly different for any condition, except the H₂O₂ MIC for A909. Error bars represent the SEM, and significance was determined by a Mann-Whitney rank sum test (n ≥ 4).

FIG 6

GSH is essential for full virulence of S. agalactiae in mice. (A) WT, ΔgshAB, and gshAB⁺ S. agalactiae strains produce significantly different survival profiles depending on the GSH synthesis ability following intravenous challenge of C57BL/6 mice. Murine survival is significantly different (P < 0.0001) between WT/gshAB⁺ and ΔgshAB strains. (B) Bacterial loads (as measured in blood) are significantly higher at 4 h postinfection and at the time of death (TOD) for bacteria able to synthesize GSH. Significance for survival was determined by using Kaplan-Meier analysis, whereas the significance for the bacterial load was determined by using a Mann-Whitney rank sum test with n ≥ 10.