doi: 10.1101/2025.01.29.635275

Spencer BL, Nguyen DT, Marroquin SM, Gapin L, O'Brien RL, Doran KS

Introduction: Group B Streptococcus (GBS) asymptomatic colonizes the female genital tract (FGT) but can contribute to adverse pregnancy outcomes including pre-term birth, chorioamnionitis, and neonatal infection. We previously observed that GBS elicits FGT cytokine responses, including IL-17, during murine vaginal colonization; yet the anti-GBS cellular immune response during colonization remained unknown. We hypothesized that GBS may induce cellular immunity, resulting in FGT clearance.

Methods: Herein, we utilize depleting antibodies and knockout mice and performed flow cytometry to investigate cellular immunes responses during GBS colonization.

Results: We found that neutrophils (effectors of the IL-17 response) are important for GBS mucosal control as neutrophil depletion promoted increased GBS burdens in FGT tissues. Flow cytometric analysis of immune populations in the vagina, cervix, and uterus revealed, however, that GBS colonization did not induce a marked increase in FGT CD45+ immune cells. We also found that that Vγ6+ γδ T cells comprise a primary source of FGT IL-17. Finally, using knockout mice, we observed that IL-17-producing γδ T cells are important for the control of GBS in the FGT during murine colonization.

Conclusions: Taken together, this work characterizes FGT cellular immunity and suggests that GBS colonization does not elicit a significant immune response, which may be a bacterial directed adaptive outcome. However, certain FGT immune cells, such as neutrophils and ɣδ T cells, contribute to host defense and control of GBS colonization.

doi: 10.1128/mbio.02088-24

Job AM, Doran KS, Spencer BL.

Group B Streptococcus (GBS) asymptomatically colonizes the vagina but can opportunistically ascend to the uterus and be transmitted vertically during pregnancy, resulting in neonatal pneumonia, bacteremia, and meningitis. GBS is a leading etiologic agent of neonatal infection and understanding the mechanisms by which GBS persists within the polymicrobial female genital mucosa has the potential to mitigate subsequent transmission and disease. Type VIIb secretion systems (T7SSb) are encoded by Bacillota and often mediate interbacterial competition using LXG toxins that contain conserved N-termini important for secretion and variable C-terminal toxin domains that confer diverse biochemical activities. Our recent work characterized a role for the GBS T7SSb in vaginal colonization and ascending infection but the mechanisms by which the T7SSb promotes GBS persistence in this polymicrobial niche remain unknown. Herein, we investigate the GBS T7SS in interbacterial competition and GBS niche establishment in the female genital tract. We demonstrate GBS T7SS-dependent inhibition of mucosal pathobiont Enterococcus faecalis both in vitro using predator-prey assays and in vivo in the murine genital tract and found that a GBS LXG protein encoded within the T7SS locus (herein named group B streptococcal LXG Toxin A) contributes to these phenotypes. We identify BltA as a T7SS substrate that is toxic to E. coli and S. aureus upon induction of intracellular expression along with associated chaperones. Finally, we show that BltA and its chaperones contribute to GBS vaginal colonization. Altogether, these data reveal a role for a novel T7b-secreted toxin in GBS mucosal persistence and competition.

doi: 10.1111/mmi.15115

Spencer BL, Job AM, Robertson CM, Hameed ZA, Serchejian C, Wiafe-Kwakye CS, Mendonça JC, Apolonio MA, Nagao PE, Neely MN, Korotkova N, Korotkov KV, Patras KA, Doran KS

Type VIIb secretion systems (T7SSb) in Gram-positive bacteria facilitate physiology, interbacterial competition, and/or virulence via EssC ATPase-driven secretion of small ɑ-helical proteins and toxins. Recently, we characterized T7SSb in group B Streptococcus (GBS), a leading cause of infection in newborns and immunocompromised adults. GBS T7SS comprises four subtypes based on variation in the C-terminus of EssC and the repertoire of downstream effectors; however, the intraspecies diversity of GBS T7SS and impact on GBS-host interactions remains unknown. Bioinformatic analysis indicates that GBS T7SS loci encode subtype-specific putative effectors, which have low interspecies and inter-subtype homology but contain similar domains/motifs and therefore may serve similar functions. We further identify orphaned GBS WXG100 proteins. Functionally, we show that GBS T7SS subtype I and III strains secrete EsxA in vitro and that in subtype I strain CJB111, esxA1 appears to be differentially transcribed from the T7SS operon. Furthermore, we observe subtype-specific effects of GBS T7SS on host colonization, as CJB111 subtype I but not CNCTC 10/84 subtype III T7SS promotes GBS vaginal colonization. Finally, we observe that T7SS subtypes I and II are the predominant subtypes in clinical GBS isolates. This study highlights the potential impact of T7SS heterogeneity on host-GBS interactions.

doi: 10.1371/journal.ppat.1010680

Spencer BL, Doran KS

Secretion systems are commonly used by bacteria to export proteins to their extracellular environment and are integral in bacterial niche adaptation. While most secretion systems are encoded by Gram-negative organisms, the type VII secretion system was discovered in Actinobacteria and then in Firmicutes (designated T7SSa and T7SSb, respectively) and has been implicated in development, interbacterial competition, nutrient acquisition, and virulence. Broadly, the T7SS is characterized by ATPase-driven export of approximately 100-residue alpha-helical proteins that lack traditional signal sequences but contain a central WXG motif, or “WXG100” proteins, with EsxA being the first identified T7SS substrate. The T7SSb core machinery required for substrate secretion has been best characterized in Staphylococcus aureus and includes cytoplasmic EsaB as well as membrane-bound EsaA, EssA, EssB, and EssC (the ATPase that powers substrate translocation and confers substrate specificity. Interestingly, WXG100 protein EsxA has been hypothesized to perform dual roles as both a secreted effector and as a core T7SSb component, as it may associate with T7SSb membrane proteins and/or may chaperone other T7SSb substrates (as proposed for WXG100-like proteins). However, further experimental investigation is needed to confirm these putative EsxA functions. Translocation of S. aureus T7SS substrates also requires peptidoglycan hydrolase EssH, which may facilitate apparatus assembly. However, while a T7SSb membrane complex has been observed, the biochemical functions of these proteins (aside from EssC) are still being elucidated. The structure of these components has been nicely reviewed elsewhere. Homologs of these T7SSb core components have now been described in numerous other Gram-positive organisms, including Bacillus, Listeria, Enterococcus, and Streptococcus. Here, we review the known and recently discovered functions of the T7SSb in host and bacterial interactions as well as discuss hurdles in the study of T7SSb biology.

doi: 10.1371/journal.ppat.1010121

Spencer BL, Tak U, Mendonça JM, Nagao, PE, Niederweis MN, Doran KS

Type VII secretion systems (T7SS) have been identified in Actinobacteria and Firmicutes and have been shown to secrete effector proteins with functions in virulence, host toxicity, and/or interbacterial killing in a few genera. Bioinformatic analysis indicates that isolates of Group B Streptococcus (GBS) encode at least four distinct subtypes of T7SS machinery, three of which encode adjacent putative T7SS effectors with WXG and LXG motifs. However, the function of T7SS in GBS pathogenesis is unknown. Here we assessed the role of the most abundant GBS T7SS subtype during GBS pathogenesis. In a murine model of hematogenous meningitis, mice infected with GBS lacking a functional T7SS or lacking the secreted WXG100 effector EsxA exhibited less mortality, lower bacterial burdens in tissues, and decreased inflammation in the brain compared to mice infected with the parental GBS strain. We further showed that this T7SS induces cytotoxicity in brain endothelium and that EsxA contributes to these cytotoxicity phenotypes in a WXG motif-dependent manner. Finally, we determined that EsxA is a pore-forming protein, thus demonstrating the first role for a non-mycobacterial EsxA homolog in pore formation. This work reveals the importance of a T7SS in host-GBS interactions and has implications for T7SS effector function in other Gram-positive bacteria.