Genomic Analyses Identify Manganese Homeostasis as a Driver of Group B Streptococcal Vaginal Colonization

Abstract

Group B Streptococcus (GBS) is associated with severe infections in utero and in newborn populations, including pneumonia, sepsis, and meningitis. GBS vaginal colonization of the pregnant mother is an important prerequisite for transmission to the newborn and the development of neonatal invasive disease; however, our understanding of the factors required for GBS persistence and ascension in the female reproductive tract (FRT) remains limited. Here, we utilized a GBS mariner transposon (Krmit) mutant library previously developed by our group and identified underrepresented mutations in 535 genes that contribute to survival within the vaginal lumen and colonization of vaginal, cervical, and uterine tissues. From these mutants, we identified 47 genes that were underrepresented in all samples collected, including mtsA, a component of the mtsABC locus, encoding a putative manganese (Mn²⁺)-dependent ATP-binding cassette transporter. RNA sequencing analysis of GBS recovered from the vaginal tract also revealed a robust increase of mtsA expression during vaginal colonization. We engineered an ΔmtsA mutant strain and found by using inductively coupled plasma mass spectrometry that it exhibited decreased concentrations of intracellular Mn²⁺, confirming its involvement in Mn²⁺ acquisition. The ΔmtsA mutant was significantly more susceptible to the metal chelator calprotectin and to oxidative stressors, including both H₂O₂ and paraquat, than wild-type (WT) GBS. We further observed that the ΔmtsA mutant strain exhibited a significant fitness defect in comparison to WT GBS in vivo by using a murine model of vaginal colonization. Taken together, these data suggest that Mn²⁺ homeostasis is an important process contributing to GBS survival in the FRT. IMPORTANCE Morbidity and mortality associated with GBS begin with colonization of the female reproductive tract (FRT). To date, our understanding of the factors required for GBS persistence in this environment remain limited. We identified several necessary systems for initial colonization of the vaginal lumen and penetration into the reproductive tissues via transposon mutagenesis sequencing. We determined that mutations in mtsA, the gene encoding a protein putatively involved in manganese (Mn²⁺) transport, were significantly underrepresented in all in vivo samples collected. We also show that mtsA contributes to Mn²⁺ acquisition and GBS survival during metal limitation by calprotectin, a metal-chelating protein complex. We further demonstrate that a mutant lacking mtsA is hypersusceptible to oxidative stress induced by both H₂O₂ and paraquat and has a severe fitness defect compared to WT GBS in the murine vaginal tract. This work reveals the importance of Mn²⁺ homeostasis at the host-pathogen interface in the FRT.

Keywords: Group B Streptococcus; intrauterine infection; manganese; vaginal colonization.

FIG 1

Determinants of GBS fitness in the female vaginal tract. (A) Model of transposon mutagenesis sample collection. (B to F) Volcano plots of underrepresented (red), overrepresented (blue), or unchanged (black) gene insertion mutations in the vaginal lumen at days 1 (B) and 3 (C) post-colonization or in vaginal (D), cervical (E), or uterine (F) tissues. (G and H) Venn diagram (G) and clusters of orthologous groupings (COGs) (H) as determined by EggNOG 5.0 for underrepresented mutations from all sample locations of underrepresented mutations.

FIG 2

Important GBS factors for colonization of the vaginal lumen or uterus (A to C). In vivo Tn-seq identified (A) 94 unique genes and corresponding COGs that are important for colonization of the (B) FRT or (C) uterus. (D) Several systems involved in metal transport mutations were underrepresented and important for colonization of the FRT and uterus.

FIG 3

GBS mtsA contributes to Mn²⁺ import and survival during calprotectin stress in vitro. (A) Intracellular Mn₅₅ was determined for GBS CJB111 WT and ΔmtsA by ICP-MS. (B) Quantitative RT-PCR was used to assess expression of mtsA following exposure to 960 μg/mL calprotectin with or without supplementation with 50 μM MnCl₂. Fold change was calculated by ΔΔC_T analysis, with gyrA serving as the internal control. Data are displayed as the average fold change in expression from three independent experiments. (C and D) Growth of GBS WT and ΔmtsA strains in the presence of 720 to 960 μg/mL calprotectin (C) or 720 to 960 μg/mL calprotectin with 10 μM MnCl₂ supplementation (D). Significance was determined by an unpaired two-tailed Student's t test (A), one-way ANOVA with Tukey’s multiple-comparison test (B), or two-way ANOVA with Tukey’s multiple-comparison test (C and D). *, P < 0.05; ****, P < 0.0001; ns, not significant.

FIG 4

GBS Mn²⁺ homeostasis contributes to vaginal colonization in vivo. Female CD-1 mice were colonized with 1 × 10⁷ CFU of both GBS WT and ΔmtsA strains. WT and mutant strains were tested in both the CJB111, serotype V (A to D), and A909, serotype Ia (E to H), genetic lineages. Recovered CFU were quantified from vaginal lavage fluid over time (A and E) or from vaginal (B and F), cervical (C and G), or uterine (D and H) tissue homogenates harvested on day 6 post-colonization. Significance was determined by two-way ANOVA with Sídák’s multiple-comparison test (A and E) or an unpaired two-tailed Student's t test (B to D and F to H). **, P < 0.01; ****, P < 0.0001.

FIG 5

GBS Mn²⁺ homeostasis is independent of host calprotectin stress in the FRT. Female C57BL/6 and S100A9^−/− mice were colonized with 1 × 10⁷ CFU of GBS CJB111 WT and ΔmtsA strains in competition (A to D) or as single challenge (E to H). Recovered CFU were quantified from vaginal lumen (A and E) or vaginal (B and F), cervical (C and G), or uterine (D and H) tissue homogenates. Significance was determined by two-way ANOVA with Sídák’s multiple-comparison test (A and E) or one-way ANOVA with Sídák’s multiple-comparison test (B to D and F to H). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

FIG 6

Role of Mn²⁺ homeostasis in GBS oxidative stress resistance. Growth of GBS WT and ΔmtsA strains in the presence of increasing concentrations of H₂O₂ (A) and paraquat (B). Significance was determined by two-way ANOVA with Sídák’s multiple-comparison test. ****, P < 0.0001.