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.

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