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The serine-rich repeat glycoprotein Srr1 of Streptococcus agalactiae (GBS) is thought to be an important adhesin for the pathogenesis of meningitis. Although expression of Srr1 is associated with increased binding to human brain microvascular endothelial c

Staphylococcus aureus causes approximately 80% of skin and soft tissue infections (SSTIs). Collagen is the most abundant human extracellular matrix protein with critical roles in wound healing, and S. aureus encodes a collagen binding adhesin (Cna). The role of this protein during skin infections is unknown. Here we report that inability to bind collagen results in worsened pathology of intradermal Δ cna S. aureus infection. WT/Cna+ S. aureus showed reduced infection severity, aggregate formation, and significantly improved clearance of bacteria. Cna binds to the collagen-like domain of serum C1q protein to reduce its opsonophagocytic functions. We demonstrate that infection of C1qKO mice with WT bacteria show results similar to the Δ cna group. Conversely, inability to bind collagen resulted in an amplified inflammatory response caused in part by macrophage and neutrophil small molecule mediators released at the infection site (MMP-9, MMP-12, LTB 4 ), resulting in increased immune cell infiltration and death., Competing Interests: Declaration of interests. The authors declare no competing interests.

Group B Streptococcus (GBS) is a Gram-positive pathobiont that commonly colonizes the gastrointestinal and lower female genital tracts but can cause sepsis and pneumonia in newborns and is a leading cause of neonatal meningitis. Despite the resulting disease severity, the pathogenesis of GBS is not completely understood, especially during the early phases of infection. To investigate GBS factors necessary for blood stream survival, we performed a transposon (Tn) mutant screen in our bacteremia infection model using a GBS mariner transposon mutant library previously developed by our group. We identified significantly underrepresented mutations in 539 genes that contribute to survival in the blood, including those encoding known virulence factors such as capsule, the β-hemolysin, and inorganic metal ion transport systems. Most of the underrepresented genes have not been previously characterized or studied in GBS, including gloA and gloB, which are homologs for genes involved in methylglyoxal (MG) detoxification. MG is a byproduct of glycolysis and a highly reactive toxic aldehyde that is elevated in immune cells during infection. Here, we observed MG sensitivity across multiple GBS isolates and confirm that gloA contributes to MG tolerance and invasive GBS infection. We show specifically that gloA contributes to GBS survival in the presence of neutrophils and depleting neutrophils in mice abrogates the decreased survival and infection of the gloA mutant. The requirement of the glyoxalase pathway during GBS infection suggests that MG detoxification is important for bacterial survival during host-pathogen interactions.

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 L XG T oxin 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.IMPORTANCECompetition between neighboring, non-kin bacteria is essential for microbial niche establishment in mucosal environments. Gram-positive bacteria encoding T7SSb have been shown to engage in competition through the export of LXG-motif-containing toxins, but these have not been characterized in group B Streptococcus (GBS), an opportunistic colonizer of the polymicrobial female genital tract. Here, we show a role for GBS T7SS in competition with mucosal pathobiont Enterococcus faecalis , both in vitro and in vivo . We further find that a GBS LXG protein contributing to this antagonism is exported by the T7SS and is intracellularly toxic to other bacteria; therefore, we have named this protein group B streptococcal L XG T oxin A (BltA). Finally, we show that BltA and its associated chaperones promote persistence within female genital tract tissues, in vivo . These data reveal previously unrecognized mechanisms by which GBS may compete with other mucosal opportunistic pathogens to persist within the female genital tract., Competing Interests: The authors declare no conflict of interest.

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 potential to mitigate subsequent transmission and disease. Type VIIb secretion systems (T7SSb) are encoded by Firmicutes 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) that contributes to these phenotypes. We identify BltA as a T7SS substrate that is toxic to E. coli and S. aureus upon induction of 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., Competing Interests: Competing interests: The authors have declared that no competing interests exist.

Streptococcus agalactiae [group B Streptococcus (GBS)] is a leading cause of neonatal meningitis, with late-onset disease (LOD) occurring after gastrointestinal tract colonization in infants. Bacterial membrane lipids are essential for host-pathogen interactions, and the functions of glycolipids are yet to be fully elucidated. GBS synthesizes three major glycolipids: glucosyl-diacylglycerol (Glc-DAG), diglucosyl-DAG (Glc 2 -DAG), and lysyl-Glc-DAG (Lys-Glc-DAG). Here, we identify the enzyme, IagB, as responsible for biosynthesis of Glc-DAG, the precursor for the two other glycolipids in GBS. To examine the collective role of glycolipids to GBS virulence, we adapted a murine model of neonatal meningitis to simulate LOD. The GBS∆ iagB mutant traversed the gut-epithelial barrier comparable to wild type but was severely attenuated in bloodstream survival, resulting in decreased bacterial loads in the brain. The GBS∆ iagB mutant was more susceptible to neutrophil killing and membrane targeting by host antimicrobial peptides. This work reveals an unexplored function of GBS glycolipids with their ability to protect the bacterial cell from host antimicrobial killing.

Diabetic wound infections including diabetic foot ulcers (DFUs) are a major global health concern and a leading cause of non-traumatic amputations. Numerous bacterial species establish infection in DFUs, and treatment with antibiotics often fails due to widespread antibiotic resistance and biofilm formation. Determination of bacterial species that reside in DFU and their virulence potential is critical to inform treatment options. Here, we isolate bacteria from debridement tissues from patients with diabetes at the University of Colorado Anschutz Medical Center. The most frequent species were Gram-positive including Enterococcus faecalis, Staphylococcus aureus , and Streptococcus agalactiae , also known as Group B Streptococcus (GBS). Most tissues had more than one species isolated with E. faecalis and GBS frequently occurring in polymicrobial infection with S. aureus. S. aureus was the best biofilm producing species with E. faecalis and GBS isolates exhibiting little to no biofilm formation. Antibiotic susceptibility varied amongst strains with high levels of penicillin resistance amongst S. aureus , clindamycin resistance amongst GBS and intermediate vancomycin resistance amongst E. faecalis . Finally, we utilized a murine model of diabetic wound infection and found that the presence of S. aureus led to significantly higher recovery of GBS and E. faecalis compared to mice challenged in mono-infection., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Competing Interests: The authors have declared that no competing interests exist.

Group B Streptococcus (GBS) is known to colonize the female reproductive tract and causes adverse pregnancy outcomes and neonatal disease. DNA methylation is a common mechanism for both phage defense and transcriptional regulation. Here, we report the m6A and m4C methylomes of four clinical GBS isolates, CJB111, A909, COH1, and NEM316., Competing Interests: The authors declare no conflict of interest.

Importance: Group B Streptococcus (GBS) colonizes the female reproductive tract (FRT) in one-third of women, and carriage leads to numerous adverse pregnancy outcomes including the preterm premature rupture of membranes, chorioamnionitis, and stillbirth. The presence of GBS in the FRT during pregnancy is also the largest predisposing factor for the transmission of GBS and invasive neonatal diseases, including pneumonia, sepsis, and meningitis. The factors contributing to GBS colonization are still being elucidated. Here, we show for the first time that GBS transcription is regulated by an orphan DNA cytosine methyltransferase (Dcm). Many GBS factors are regulated by Dcm, especially those involved in carbohydrate transport and metabolism. We show that GBS persistence in the FRT is dependent on the catabolism of sugars found on the vaginal mucin MUC5B. Collectively, this work highlights the regulatory importance of a DNA methyltransferase and identifies both host and bacterial factors required for GBS colonization., Competing Interests: The authors declare no conflict of interest.

Group B Streptococcus (GBS) is a Gram-positive pathobiont that can cause adverse health outcomes in neonates and vulnerable adult populations. GBS is one of the most frequently isolated bacteria from diabetic (Db) wound infections but is rarely found in the non-diabetic (nDb) wound environment. Previously, RNA sequencing of wound tissue from Db wound infections in lepr d b diabetic mice showed increased expression of neutrophil factors, and genes involved in GBS metal transport such as the zinc (Zn), manganese (Mn), and putative nickel (Ni) import systems. Here, we develop a Streptozotocin-induced diabetic wound model to evaluate the pathogenesis of two invasive strains of GBS, serotypes Ia and V. We observe an increase in metal chelators such as calprotectin (CP) and lipocalin-2 during diabetic wound infections compared to nDb. We find that CP limits GBS survival in wounds of non-diabetic mice but does not impact survival in diabetic wounds. Additionally, we utilize GBS metal transporter mutants and determine that the Zn, Mn, and putative Ni transporters in GBS are dispensable in diabetic wound infection but contributed to bacterial persistence in non-diabetic animals. Collectively, these data suggest that in non-diabetic mice, functional nutritional immunity mediated by CP is effective at mitigating GBS infection, whereas in diabetic mice, the presence of CP is not sufficient to control GBS wound persistence. IMPORTANCE Diabetic wound infections are difficult to treat and often become chronic due to an impaired immune response as well as the presence of bacterial species that establish persistent infections. Group B Streptococcus (GBS) is one of the most frequently isolated bacterial species in diabetic wound infections and, as a result, is one of the leading causes of death from skin and subcutaneous infection. However, GBS is notoriously absent in non-diabetic wounds, and little is known about why this species thrives in diabetic infection. The work herein investigates how alterations in diabetic host immunity may contribute to GBS success during diabetic wound infection., Competing Interests: The authors declare no conflict of interest.

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., (© 2023 John Wiley & Sons Ltd.)

The arachnoid barrier, a component of the blood-cerebrospinal fluid barrier (B-CSFB) in the meninges, is composed of epithelial-like, tight-junction-expressing cells. Unlike other central nervous system (CNS) barriers, its' developmental mechanisms and timing are largely unknown. Here, we show that mouse arachnoid barrier cell specification requires the repression of Wnt-β-catenin signaling and that constitutively active β-catenin can prevent its formation. We also show that the arachnoid barrier is functional prenatally and, in its absence, a small molecular weight tracer and the bacterium group B Streptococcus can cross into the CNS following peripheral injection. Acquisition of barrier properties prenatally coincides with the junctional localization of Claudin 11, and increased E-cadherin and maturation continues after birth, where postnatal expansion is marked by proliferation and re-organization of junctional domains. This work identifies fundamental mechanisms that drive arachnoid barrier formation, highlights arachnoid barrier fetal functions, and provides novel tools for future studies on CNS barrier development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Streptococcus agalactiae (Group B Streptococcus , GBS) is the leading cause of neonatal sepsis and meningitis but has been recently isolated from non-pregnant adults with underlying medical conditions like diabetes. Despite diabetes being a key risk factor for invasive disease, the pathological consequences during GBS infection remain poorly characterized. Here, we demonstrate the pathogenicity of the GBS90356-ST17 and COH1-ST17 strains in streptozotocin-induced diabetic mice. We show that GBS can spread through the bloodstream and colonize several tissues, presenting a higher bacterial count in diabetic-infected mice when compared to non-diabetic-infected mice. Histological sections of the lungs showed inflammatory cell infiltration, collapsed septa, and red blood cell extravasation in the diabetic-infected group. A significant increase in collagen deposition and elastic fibers were also observed in the lungs. Moreover, the diabetic group presented red blood cells that adhered to the valve wall and disorganized cardiac muscle fibers. An increased expression of KC protein, IL-1β, genes encoding immune cell markers, and ROS (reactive oxygen species) production was observed in diabetic-infected mice, suggesting GBS promotes high levels of inflammation when compared to non-diabetic animals. Our data indicate that efforts to reverse the epidemic of diabetes could considerably reduce the incidence of invasive infection, morbidity and mortality due to GBS.

The meninges are densely innervated by nociceptive sensory neurons that mediate pain and headache 1,2 . Bacterial meningitis causes life-threatening infections of the meninges and central nervous system, affecting more than 2.5 million people a year 3-5 . How pain and neuroimmune interactions impact meningeal antibacterial host defences are unclear. Here we show that Nav1.8 + nociceptors signal to immune cells in the meninges through the neuropeptide calcitonin gene-related peptide (CGRP) during infection. This neuroimmune axis inhibits host defences and exacerbates bacterial meningitis. Nociceptor neuron ablation reduced meningeal and brain invasion by two bacterial pathogens: Streptococcus pneumoniae and Streptococcus agalactiae. S. pneumoniae activated nociceptors through its pore-forming toxin pneumolysin to release CGRP from nerve terminals. CGRP acted through receptor activity modifying protein 1 (RAMP1) on meningeal macrophages to polarize their transcriptional responses, suppressing macrophage chemokine expression, neutrophil recruitment and dural antimicrobial defences. Macrophage-specific RAMP1 deficiency or pharmacological blockade of RAMP1 enhanced immune responses and bacterial clearance in the meninges and brain. Therefore, bacteria hijack CGRP-RAMP1 signalling in meningeal macrophages to facilitate brain invasion. Targeting this neuroimmune axis in the meninges can enhance host defences and potentially produce treatments for bacterial meningitis., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Competing Interests: The authors have declared that no competing interests exist.

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen and frequent colonizer of human skin and mucosal membranes, including the vagina, with vaginal colonization reaching nearly 25% in some pregnant populations. MRSA vaginal colonization can lead to aerobic vaginitis (AV), and during pregnancy, bacterial ascension into the upper reproductive tract can lead to adverse birth outcomes. USA300, the most prominent MRSA lineage to colonize pregnant individuals, is a robust biofilm former and causative agent of invasive infections; however, little is known about how it colonizes and ascends in the female reproductive tract (FRT). Our previous studies showed that a MRSA mutant of seven fibrinogen-binding adhesins was deficient in FRT epithelial attachment and colonization. Using both monolayer and multilayer air-liquid interface cell culture models, we determine that one class of these adhesins, the fibronectin binding proteins (FnBPA and FnBPB), are critical for association with human vaginal epithelial cells (hVECs) and hVEC invasion through interactions with α 5 β 1 integrin. We observe that both FnBPs are important for biofilm formation as single and double fnbAB mutants exhibit reduced biofilm formation on hVECs. Using heterologous expression of fnbA and fnbB in Staphylococcus carnosus, FnBPs are also found to be sufficient for hVEC cellular association, invasion, and biofilm formation. In addition, we found that an Δ fnbAB mutant displays attenuated ascension in our murine vaginal colonization model. Better understanding of MRSA FRT colonization and ascension can ultimately inform treatment strategies to limit MRSA vaginal burden or prevent ascension, especially during pregnancy and in those prone to AV.

Competing Interests: The authors have declared that no competing interests exists.

Diabetic wounds have poor healing outcomes due to the presence of numerous pathogens and a dysregulated immune response. Group B Streptococcus (GBS) is commonly isolated from diabetic wound infections, but the mechanisms of GBS virulence during these infections have not been investigated. Here, we develop a murine model of GBS diabetic wound infection and, using dual RNA sequencing, demonstrate that GBS infection triggers an inflammatory response. GBS adapts to this hyperinflammatory environment by up-regulating virulence factors including those known to be regulated by the two-component system covRS , such as the surface protein pbsP , and the cyl operon, which is responsible for hemolysin/pigmentation production. We recover hyperpigmented/hemolytic GBS colonies from the murine diabetic wound, which we determined encode mutations in covR . We further demonstrate that GBS mutants in cylE and pbsP are attenuated in the diabetic wound. This foundational study provides insight into the pathogenesis of GBS diabetic wound infections.

Streptococcus agalactiae, otherwise known as Group B Streptococcus (GBS), is an opportunistic pathogen that vaginally colonizes approximately one third of healthy women. During pregnancy, this can lead to in utero infection, resulting in premature rupture of membranes, chorioamnionitis, and stillbirths. Furthermore, GBS causes serious infection in newborns, including sepsis, pneumonia, and meningitis. Previous studies have indicated that GBS antigen (Ag) I/II family proteins promote interaction with vaginal epithelial cells; thus, we hypothesized that the Ag I/II Group B streptococcal surface protein C (BspC) contributes to GBS colonization of the female reproductive tract (FRT). Here, we show that a Δ bspC mutant has decreased bacterial adherence to vaginal, ecto-, and endocervical cells, as well as decreased auto-aggregation and biofilm-like formation on cell monolayers. Using a murine model of vaginal colonization, we observed that the Δ bspC mutant strain exhibited a significant fitness defect compared to wild-type (WT) GBS and was less able to ascend to the cervix and uterus in vivo, resulting in reduced neutrophil chemokine signaling. Furthermore, we determined that BspC interacts directly with the host intermediate filament protein cytokeratin 19 (K19). Surface localization of K19 was increased during GBS infection, and interaction was mediated by the BspC variable (V) domain. Finally, mice treated with a drug that targets the BspC V-domain exhibited reduced bacterial loads in the vaginal lumen and reproductive tissues. These results demonstrate the importance of BspC in promoting GBS colonization of the FRT and that it may be targeted therapeutically to reduce GBS vaginal persistence and ascending infection. IMPORTANCE Group B Streptococcus (GBS) asymptomatically colonizes the female reproductive tract (FRT) of up to one third of women, but GBS carriage can lead to adverse pregnancy outcomes, including premature rupture of membranes, preterm labor, and chorioamnionitis. GBS colonization during pregnancy is also the largest predisposing factor for neonatal GBS disease, including pneumonia, sepsis, and meningitis. The molecular interactions between bacterial surface proteins and the host cell receptors that promote GBS colonization are vastly understudied, and a better understanding would facilitate development of novel therapeutics to prevent GBS colonization and disease. Here, we characterize the role of the GBS surface protein BspC in colonization of the FRT. We show for the first time that GBS infection induces cytokeratin 19 (K19) surface localization on vaginal epithelial cells; GBS then uses the BspC V-domain to interact with K19 to promote colonization and ascending infection. Furthermore, this interaction can be targeted therapeutically to reduce GBS carriage.

Group B Streptococcus (GBS) in the vaginal tract is a risk factor for preterm birth and adverse pregnancy outcomes. GBS colonization is also transient in nature, which likely reflects the contributions of pathogen determinants, interactions with commensal flora, and host factors, making this environment particularly challenging to understand. Additionally, dietary zinc deficiency is a health concern on the global scale that is known to be associated with recurrent bacterial infection and increased rate of preterm birth or stillbirth. However, the impact of zinc deficiency on vaginal health has not yet been studied. Here we use a murine model to assess the role of dietary zinc on GBS burden and the impact of GBS colonization on the vaginal microbiome. We show that GBS vaginal colonization is increased in a zinc-deficient host and that the presence of GBS significantly alters the microbial community structure of the vagina. Using machine learning approaches, we show that vaginal community turnover during GBS colonization is driven by computationally predictable changes in key taxa, including several organisms not previously described in the context of the vaginal microbiota, such as Akkermansia muciniphila. We observed that A. muciniphila increases GBS vaginal persistence and, in a cohort of human vaginal microbiome samples collected throughout pregnancy, we observed an increased prevalence of codetection of GBS and A. muciniphila in patients who delivered preterm compared to those who delivered at full term. These findings reveal the importance and complexity of both host zinc availability and native microbiome to GBS vaginal persistence. IMPORTANCE The presence of group B Streptococcus (GBS) in the vaginal tract, perturbations in the vaginal microbiota, and dietary zinc deficiency are three factors that are independently known to be associated with increased risk of adverse pregnancy outcomes. Here, we developed an experimental mouse model to assess the impact of dietary zinc deficiency on GBS vaginal burden and persistence and to determine how changes in GBS colonization impact vaginal microbial structure. We have employed unique animal, in silica metabolic, and machine learning models, paired with analyses of human cohort data, to identify taxonomic biomarkers that contribute to host susceptibility to GBS vaginal persistence. Collectively, the data reported here identify that both dietary zinc deficiency and the presence of A. muciniphila could perpetuate an increased GBS burden and prolonged exposure in the vaginal tract, which potentiate the risk of invasive infection in utero and in the newborn.

Competing Interests: The authors have declared that no competing interests exist.

Streptococcus spp. are an important genus of Gram-positive bacteria, many of which are opportunistic pathogens that are capable of causing invasive disease in a wide range of populations. Metals, especially transition metal ions, are an essential nutrient for all organisms. Therefore, to survive across dynamic host environments, Streptococci have evolved complex systems to withstand metal stress and maintain metal homeostasis, especially during colonization and infection. There are many different types of transport systems that are used by bacteria to import or export metals that can be highly specific or promiscuous. Focusing on the most well studied transition metals of zinc, manganese, iron, nickel, and copper, this review aims to summarize the current knowledge of metal homeostasis in pathogenic Streptococci, and their role in virulence.

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 (Mn 2+ )-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 Δ mtsA mutant strain and found by using inductively coupled plasma mass spectrometry that it exhibited decreased concentrations of intracellular Mn 2+ , confirming its involvement in Mn 2+ acquisition. The Δ mtsA mutant was significantly more susceptible to the metal chelator calprotectin and to oxidative stressors, including both H 2 O 2 and paraquat, than wild-type (WT) GBS. We further observed that the Δ mtsA 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 Mn 2+ 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 (Mn 2+ ) transport, were significantly underrepresented in all in vivo samples collected. We also show that mtsA contributes to Mn 2+ 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 H 2 O 2 and paraquat and has a severe fitness defect compared to WT GBS in the murine vaginal tract. This work reveals the importance of Mn 2+ homeostasis at the host-pathogen interface in the FRT.

The female reproductive tract (FRT) is a complex environment, rich in mucin glycoproteins that form a dense network on the surface of the underlying epithelia. Group B Streptococcus (GBS) asymptomatically colonizes 25-30% of healthy women, but during pregnancy can cause ascending infection in utero or be transmitted to the newborn during birth to cause invasive disease. Though the cervicovaginal mucosa is a natural site for GBS colonization, the specific interactions between GBS and mucins remain unknown. Here we demonstrate for the first time that MUC5B interacts directly with GBS and promotes barrier function by inhibiting both bacterial attachment to human epithelial cells and ascension from the vagina to the uterus in a murine model of GBS colonization. RNA sequencing analysis of GBS exposed to MUC5B identified 128 differentially expressed GBS genes, including upregulation of the pilus island-2b (PI-2b) locus. We subsequently show that PI-2b is important for GBS attachment to reproductive cells, binding to immobilized mucins, and vaginal colonization in vivo . Our results suggest that while MUC5B plays an important role in host defense, GBS upregulates pili in response to mucins to help promote persistence within the vaginal tract, illustrating the dynamic interplay between pathogen and host. IMPORTANCE Mucin glycoproteins are a major component that contributes to the complexity of the female reproductive tract (FRT). Group B Streptococcus (GBS) is present in the FRT of 25-30% of healthy women, but during pregnancy can ascend to the uterus to cause preterm birth and fetal infection in utero. Here we show that a prominent mucin found in the FRT, MUC5B, promotes host defense by inhibiting GBS interaction with epithelial cells found in the FRT and ascension from the vagina to the uterus in vivo . In response to MUC5B, GBS induces the expression of surface expressed pili, which in turn contributes to GBS persistence within the vaginal lumen. These observations highlight the importance and complexity of GBS-mucin interactions that warrant further investigation.

Bacterial infections are a major cause of morbidity and mortality worldwide and the rise of antibiotic resistance necessitates development of alternative treatments. Pathogen adhesins that bind to host cells initiate disease pathogenesis and represent potential therapeutic targets. We have shown previously that the BspC adhesin in Group B Streptococcus (GBS), the leading cause of bacterial neonatal meningitis, interacts with host vimentin to promote attachment to brain endothelium and disease development. Here we determined that the BspC variable (V-) domain contains the vimentin binding site and promotes GBS adherence to brain endothelium. Site directed mutagenesis identified a binding pocket necessary for GBS host cell interaction and development of meningitis. Using a virtual structure-based drug screen we identified compounds that targeted the V-domain binding pocket, which blocked GBS adherence and entry into the brain in vivo. These data indicate the utility of targeting the pathogen-host interface to develop anti-virulence therapeutics., Competing Interests: The authors have declared that no competing interests exist.

Group B Streptococcus (GBS) is a Gram-positive bacterium that colonizes the lower gastrointestinal tract, and in females, the urogenital tract, in up to 30% of healthy adults. However, GBS is a leading cause of mortality and morbidity in newborns due to ascending infection of the womb or by neonatal acquisition during vaginal passage. GBS neonatal disease manifests as pneumonia, sepsis, or meningitis, and an estimated 4 million newborns die each year globally. This commentary reflects on recent work by Mejia and colleagues (M. E. Mejia, S. Ottinger, A. Vrbanac, P. Babu, et al., mSphere 6:e00885-21, 2022, https://doi.org/10.1128/msphere.00885-21) that has examined human milk oligosaccharides (HMOs) as a natural product with anti-GBS activity. They show that HMOs reduce GBS vaginal colonization without impacting the normal vaginal microbiota. This study advances the possibility of using novel therapeutics to limit GBS maternal colonization and subsequent neonatal disease.

Bacterial membrane lipids are critical for membrane bilayer formation, cell division, protein localization, stress responses, and pathogenesis. Despite their critical roles, membrane lipids have not been fully elucidated for many pathogens. Here, we report the discovery of a novel cationic glycolipid, lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), which is synthesized in high abundance by the bacterium Streptococcus agalactiae (Group B Streptococcus, GBS). To our knowledge, Lys-Glc-DAG is more positively charged than any other known lipids. Lys-Glc-DAG carries 2 positive net charges per molecule, distinct from the widely described lysylated phospholipid lysyl-phosphatidylglycerol (Lys-PG) that carries one positive net charge due to the presence of a negatively charged phosphate moiety. We use normal phase liquid chromatography (NPLC) coupled with electrospray ionization (ESI) high-resolution tandem mass spectrometry (HRMS/MS) and genetic approaches to determine that Lys-Glc-DAG is synthesized by the enzyme MprF in GBS, which covalently modifies the neutral glycolipid Glc-DAG with the cationic amino acid lysine. GBS is a leading cause of neonatal meningitis, which requires traversal of the endothelial blood-brain barrier (BBB). We demonstrate that GBS strains lacking mprF exhibit a significant decrease in the ability to invade BBB endothelial cells. Further, mice challenged with a GBSΔmprF mutant developed bacteremia comparably to wild-type (WT) infected mice yet had less recovered bacteria from brain tissue and a lower incidence of meningitis. Thus, our data suggest that Lys-Glc-DAG may contribute to bacterial uptake into host cells and disease progression. Importantly, our discovery provides a platform for further study of cationic lipids at the host-pathogen interface., Competing Interests: The authors have declared that no competing interests exist

Small, noncoding RNAs (sRNAs) are being increasingly identified as important regulatory molecules in prokaryotes. Due to the prevalence of next-generation sequencing-based techniques, such as RNA sequencing (RNA-seq), there is potential for increased discovery of sRNAs within bacterial genomes; however, these elements are rarely included in annotation files. Consequently, expression values for sRNAs are omitted from most transcriptomic analyses, and mechanistic studies have lagged behind those of protein regulators in numerous bacteria. Two previous studies have identified sRNAs in the human pathogen group B Streptococcus (GBS). Here, we utilize the data from these studies to create updated genome annotation files for the model GBS strains NEM316 and COH1. Using the updated COH1 annotation file, we reanalyze publicly available GBS RNA-seq whole-transcriptome data from GenBank to monitor GBS sRNA expression under a variety of conditions and genetic backgrounds. This analysis generated expression values for 232 putative sRNAs that were overlooked in previous transcriptomic analyses in 21 unique comparisons. To demonstrate the utility of these data, we identify an sRNA that is upregulated during vaginal colonization and demonstrate that overexpression of this sRNA leads to increased bacterial invasion into host epithelial cells. Finally, to monitor RNA degradation, we perform a transcript stability assay to identify highly stable sRNAs and compare stability profiles of sRNA- and protein-coding genes. Collectively, these data provide a wealth of transcriptomic data for putative sRNAs in GBS and a platform for future mechanistic studies. IMPORTANCE In recent years, sRNAs have emerged as potent regulatory molecules in bacteria, including numerous streptococcal species, and contribute to diverse processes, including stress response, metabolism, housekeeping, and virulence regulation. Improvements in sequencing technologies and in silico analyses have facilitated identification of these regulatory molecules as well as improved attempts to determine the location of sRNA genes on the genome. However, despite these advancements, sRNAs are rarely included in genome annotation files. Consequently, these molecules are often omitted from transcriptomic data analyses and are commonly repeat identified across multiple studies. Updating current genomes to include sRNA genes is therefore critical for better understanding bacterial regulation.

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., Competing Interests: The authors have declared that no competing interests exist.

Streptococcus agalactiae (group B Streptococcus, or GBS) is an opportunistic pathogen capable of causing invasive disease in susceptible individuals, including the newborn. Currently, GBS is the leading cause of meningitis in the neonatal period. We have recently shown that GBS interacts directly with host type III intermediate filament vimentin to gain access to the central nervous system. This results in characteristic meningeal inflammation and disease progression; however, the specific role of vimentin in the inflammatory process is unknown. Here, we investigate the contribution of vimentin to the pathogenesis of GBS meningitis. We show that a CRISPR-targeted deletion of vimentin in human cerebral microvascular endothelial cells (hCMEC) reduced GBS induction of neutrophil attractants interleukin-8 (IL-8) and CXCL-1 as well as NF-κB activation. We further show that inhibition of vimentin localization also prevented similar chemokine activation by GBS. One known chemokine regulator is the nucleotide-binding oligomerization domain containing protein 2 (NOD2), which is known to interact directly with vimentin. Thus, we hypothesized that NOD2 would also promote GBS chemokine induction. We show that GBS infection induced NOD2 transcription in hCMEC comparably to the muramyl dipeptide (MDP) NOD2 agonist, and the chemokine induction was reduced in the presence of a NOD2 inhibitor. Using a mouse model of GBS meningitis, we also observed increased NOD2 transcript and NOD2 activation in brain tissue of infected mice. Lastly, we show that NOD2-mediated IL-8 and CXCL1 induction required vimentin, further indicating the importance of vimentin in mediating inflammatory responses in brain endothelium.

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is the major cause of neonatal sepsis in humans. A critical step to infection is adhesion of bacteria to epithelial surfaces. GBS adhesins have been identified to bind extracellular matrix components and cellular receptors. However, several putative adhesins have no host binding partner characterised. We report here that surface-expressed β protein of GBS binds to human CEACAM1 and CEACAM5 receptors. A crystal structure of the complex showed that an IgSF domain in β represents a novel Ig-fold subtype called IgI3, in which unique features allow binding to CEACAM1. Bioinformatic assessment revealed that this newly identified IgI3 fold is not exclusively present in GBS but is predicted to be present in adhesins from other clinically important human pathogens. In agreement with this prediction, we found that CEACAM1 binds to an IgI3 domain found in an adhesin from a different streptococcal species. Overall, our results indicate that the IgI3 fold could provide a broadly applied mechanism for bacteria to target CEACAMs., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

The COVID-19 pandemic has forced academic research communities to develop online means of learning, networking, and engaging in new research. To allow increased interaction and engagement of the streptococcal research community during the COVID-19 shutdown, we organized the Virtual Streptococcal Seminar Series and Virtual Streptococcal Trainee Symposium and advertised via e-mail and social media outlets. The seminar series initially met weekly on Thursdays at 12 pm Eastern Daylight Time and transitioned to monthly seminars, while the trainee symposium spanned 3 days in September 2020. In this study, we analyzed seminar attendance data and online recording accesses from the first 20 seminars and found community engagement to be independent of speaker gender, career stage, geographic location, and organism of interest, with an average of 124 live attendees and 1,683 recording accesses per seminar. We also report attendance and speaker statistics from the 3-day Virtual Streptococcal Trainee Symposium, which hosted a total of 38 trainees from five continents presenting on Streptococcus pneumoniae , Streptococcus agalactiae , Streptococcus pyogenes , Streptococcus suis , oral streptococci, or Enterococcus faecalis . The Virtual Streptococcal Trainee Symposium averaged 119 live attendees per session, with a total of 220 unique attendees from six continents across the 3-day event. We conclude that while online platforms do not replace in-person conferences, the seminar and symposium successfully engaged the streptococcal research community and have provided a forum for scientific sharing during the COVID-19 crisis., (©2021 Author(s). Published by the American Society for Microbiology.)

Streptococcus agalactiae is a recognized pathogen associated with infections in neonates, elderly, and immunocompromised adults, particularly those with cancer. In the present investigation, clinical-epidemiological features, multidrug resistance profiles, and virulence genes of S. agalactiae strains isolated from cancer patients were investigated. S. agalactiae capsular distribution assays demonstrated that Ia (43.6%) and V (23.6%) types were predominantly detected among 55 clinical isolates tested; only one strain (GBS1428) was capsular type III/ST-17. The fbsB and hylB genes were detected in all isolates, while the iag, lmb, and fbsA genes were detected in 94.5%, 91%, and 91% of oncological isolates, respectively. The combination of PI-1 and PI-2a was the most common (60%) among S. agalactiae strains isolated from oncologic patients. S. agalactiae strains were resistant to tetracycline (85.5%), erythromycin (9%), and clindamycin (5.5%). Norfloxacin non-susceptible was detected in 7.3% of S. agalactiae strains. Our findings reinforce the need for S. agalactiae control measures in Brazil, including cancer patients.

Group B Streptococcus (GBS) is an asymptomatic colonizer of the female reproductive tract but can cause maternal and neonatal infections and adverse pregnancy outcomes. Here, we closed the genome sequence of strain CJB111, a neonatal GBS clinical isolate from a case of late-onset bacteremia without focus (Houston, TX; 1990)., (Copyright © 2021 Spencer et al.)

Streptococci are Gram-positive bacteria that belong to the natural microbiota of humans and animals. Certain streptococcal species are known as opportunistic pathogens with the potential to cause severe invasive disease. Antigen I/II (AgI/II) family proteins are sortase anchored cell surface adhesins that are nearly ubiquitous across streptococci and contribute to many streptococcal diseases, including dental caries, respiratory tract infections, and meningitis. They appear to be multifunctional adhesins with affinities to various host substrata, acting to mediate attachment to host surfaces and stimulate immune responses from the colonized host. Here we will review the literature including recent work that has demonstrated the multifaceted nature of AgI/II family proteins, focusing on their overlapping and distinct functions and their important contribution to streptococcal colonization and disease., (Copyright © 2020 Manzer, Nobbs and Doran.)

Nutritional immunity is an elegant host mechanism used to starve invading pathogens of necessary nutrient metals. Calprotectin, a metal-binding protein, is produced abundantly by neutrophils and is found in high concentrations within inflammatory sites during infection. Group B Streptococcus (GBS) colonizes the gastrointestinal and female reproductive tracts and is commonly associated with severe invasive infections in newborns such as pneumonia, sepsis, and meningitis. Although GBS infections induce robust neutrophil recruitment and inflammation, the dynamics of GBS and calprotectin interactions remain unknown. Here, we demonstrate that disease and colonizing isolate strains exhibit susceptibility to metal starvation by calprotectin. We constructed a mariner transposon ( Krmit ) mutant library in GBS and identified 258 genes that contribute to surviving calprotectin stress. Nearly 20% of all underrepresented mutants following treatment with calprotectin are predicted metal transporters, including known zinc systems. As calprotectin binds zinc with picomolar affinity, we investigated the contribution of GBS zinc uptake to overcoming calprotectin-imposed starvation. Quantitative reverse transcriptase PCR (qRT-PCR) revealed a significant upregulation of genes encoding zinc-binding proteins, adcA , adcAII , and lmb , following calprotectin exposure, while growth in calprotectin revealed a significant defect for a global zinc acquisition mutant (Δ adcA Δ adcAII Δ lmb ) compared to growth of the GBS wild-type (WT) strain. Furthermore, mice challenged with the Δ adcA Δ adcAII Δ lmb mutant exhibited decreased mortality and significantly reduced bacterial burden in the brain compared to mice infected with WT GBS; this difference was abrogated in calprotectin knockout mice. Collectively, these data suggest that GBS zinc transport machinery is important for combatting zinc chelation by calprotectin and establishing invasive disease. IMPORTANCE Group B Streptococcus (GBS) asymptomatically colonizes the female reproductive tract but is a common causative agent of meningitis. GBS meningitis is characterized by extensive infiltration of neutrophils carrying high concentrations of calprotectin, a metal chelator. To persist within inflammatory sites and cause invasive disease, GBS must circumvent host starvation attempts. Here, we identified global requirements for GBS survival during calprotectin challenge, including known and putative systems involved in metal ion transport. We characterized the role of zinc import in tolerating calprotectin stress in vitro and in a mouse model of infection. We observed that a global zinc uptake mutant was less virulent than the parental GBS strain and found calprotectin knockout mice to be equally susceptible to infection by wild-type (WT) and mutant strains. These findings suggest that calprotectin production at the site of infection results in a zinc-limited environment and reveals the importance of GBS metal homeostasis to invasive disease., (Copyright © 2020 Burcham et al.)

Enterococcus faecalis is a Gram-positive commensal bacterium native to the gastrointestinal tract and an opportunistic pathogen of increasing clinical concern. E. faecalis also colonizes the female reproductive tract, and reports suggest vaginal colonization increases following antibiotic treatment or in patients with aerobic vaginitis. Currently, little is known about specific factors that promote E. faecalis vaginal colonization and subsequent infection. We modified an established mouse vaginal colonization model to explore E. faecalis vaginal carriage and demonstrate that both vancomycin-resistant and -sensitive strains colonize the murine vaginal tract. Following vaginal colonization, we observed E. faecalis in vaginal, cervical, and uterine tissue. A mutant lacking endocarditis- and biofilm-associated pili (Ebp) exhibited a decreased ability to associate with human vaginal and cervical cells in vitro but did not contribute to colonization in vivo Thus, we screened a low-complexity transposon (Tn) mutant library to identify novel genes important for E. faecalis colonization and persistence in the vaginal tract. This screen revealed 383 mutants that were underrepresented during vaginal colonization at 1, 5, and 8 days postinoculation compared to growth in culture medium. We confirmed that mutants deficient in ethanolamine catabolism or in the type VII secretion system were attenuated in persisting during vaginal colonization. These results reveal the complex nature of vaginal colonization and suggest that multiple factors contribute to E. faecalis persistence in the reproductive tract., (Copyright © 2020 American Society for Microbiology.)

Streptococcus agalactiae are important pathogenic bacteria that cause severe infections in humans, especially neonates. The mechanism by which ST-17 causes invasive infections than other STs is not well understood. In this study, we sequenced the first genome of a S. agalactiae ST-17 strain isolated in Brazil using the Illumina HiSeq 2500 technology. S. agalactiae GBS90356 ST-17 belongs to the capsular type III and was isolated from a neonatal with a fatal case of meningitis. The genome presented a size of 2.03 Mbp and a G + C content of 35.2%. S. agalactiae has 706 genes in its core genome and an open pan-genome with a size of 5.020 genes, suggesting a high genomic plasticity. GIPSy software was used to identify 10 Pathogenicity islands (PAIs) which corresponded to 15% of the genome size. IslandViewer4 corroborated the prediction of six PAIs. The pathogenicity islands showed important virulence factors genes for S. agalactiae e.g. neu, cps, dlt, fbs, cfb, lmb. SignalP detected 20 proteins with signal peptides among the 352 proteins found in PAIs, which 60% were located in the SagPAI_5. SagPAI_2 and 5 were mainly detected in ST-17 strains studied. Moreover, we identified 51 unique genes, 9 recombination regions and a large number of SNPs with an average of 760.3 polymorphisms, which can be related with high genomic plasticity and virulence during host-pathogen interactions. Our results showed implications for pathogenesis, evolution, concept of species and in silico analysis value to understand the epidemiology and genome plasticity of S. agalactiae., Competing Interests: Declaration of Competing Interest The authors declared no potential conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is an important pathogen responsible for nosocomial and community-acquired infections in humans, and methicillin-resistant S. aureus (MRSA) infections have continued to increase despite widespread preventative measures. S. aureus can colonize the female vaginal tract, and reports have suggested an increase in MRSA infections in pregnant and postpartum women as well as outbreaks in newborn nurseries. Currently, little is known about specific factors that promote MRSA vaginal colonization and subsequent infection. To study S. aureus colonization of the female reproductive tract in a mammalian system, we developed a mouse model of S. aureus vaginal carriage and demonstrated that both hospital-associated and community-associated MRSA isolates can colonize the murine vaginal tract. Immunohistochemical analysis revealed an increase in neutrophils in the vaginal lumen during MRSA colonization. Additionally, we observed that a mutant lacking fibrinogen binding adhesins exhibited decreased persistence within the mouse vagina. To further identify novel factors that promote vaginal colonization, we performed RNA sequencing to determine the transcriptome of MRSA growing in vivo during vaginal carriage at 5 h, 1 day, and 3 days postinoculation. Over 25% of the bacterial genes were differentially regulated at all time points during colonization compared to laboratory cultures. The most highly induced genes were those involved in iron acquisition, including the Isd system and siderophore transport systems. Mutants deficient in these pathways did not persist as well during in vivo colonization. These results reveal that fibrinogen binding and the capacity to overcome host nutritional limitation are important determinants of MRSA vaginal colonization. IMPORTANCE Staphylococcus aureus is an opportunistic pathogen able to cause a wide variety of infections in humans. Recent reports have suggested an increasing prevalence of MRSA in pregnant and postpartum women, coinciding with the increased incidence of MRSA infections in neonatal intensive care units (NICUs) and newborn nurseries. Vertical transmission from mothers to infants at delivery is a likely route of MRSA acquisition by the newborn; however, essentially nothing is known about host and bacterial factors that influence MRSA carriage in the vagina. Here, we established a mouse model of vaginal colonization and observed that multiple MRSA strains can persist in the vaginal tract. Additionally, we determined that MRSA interactions with fibrinogen and iron uptake can promote vaginal persistence. This study is the first to identify molecular mechanisms which govern vaginal colonization by MRSA, the critical initial step preceding infection and neonatal transmission., (Copyright © 2019 Deng et al.)

Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a Gram-positive bacterium isolated from the vaginal tract of approximately 25% of women. GBS colonization of the female reproductive tract is of particular concern during pregnancy as the bacteria can invade gestational tissues or be transmitted to the newborn during passage through the birth canal. Infection of the neonate can result in life-threatening pneumonia, sepsis and meningitis. Thus, surveillance of GBS strains and corresponding virulence potential during colonization is warranted. Here we describe a panel of GBS isolates from the vaginal tracts of a cohort of pregnant women in Michigan, USA. We determined that capsular serotypes III and V were the most abundant across the strain panel, with only one isolate belonging to serotype IV. Further, 12.8% of strains belonged to the hyper-virulent serotype III, sequence type 17 (ST-17) and 15.4% expressed the serine rich repeat glycoprotein-encoding gene srr2. Functional assessment of the colonizing isolates revealed that almost all strains exhibited some level of β-hemolytic activity and that ST-17 strains, which express Srr2, exhibited increased bacterial adherence to vaginal epithelium. Finally, analysis of strain antibiotic susceptibility revealed the presence of antibiotic resistance to penicillin (15.4%), clindamycin (30.8%), erythromycin (43.6%), vancomycin (30.8%), and tetracycline (94.9%), which has significant implications for treatment options. Collectively, these data provide important information on vaginal GBS carriage isolate virulence potential and highlight the value of continued surveillance., Competing Interests: The authors have declared that no competing interests exist.

Bacterial meningitis is a serious life threatening infection of the CNS. To cause meningitis, blood-borne bacteria need to interact with and penetrate brain endothelial cells (BECs) that comprise the blood-brain barrier. BECs help maintain brain homeostasis and they possess an array of efflux transporters, such as P-glycoprotein (P-gp), that function to efflux potentially harmful compounds from the CNS back into the circulation. Oftentimes, efflux also serves to limit the brain uptake of therapeutic drugs, representing a major hurdle for CNS drug delivery. During meningitis, BEC barrier integrity is compromised; however, little is known about efflux transport perturbations during infection. Thus, understanding the impact of bacterial infection on P-gp function would be important for potential routes of therapeutic intervention. To this end, the meningeal bacterial pathogen, Streptococcus agalactiae, was found to inhibit P-gp activity in human induced pluripotent stem cell-derived BECs, and live bacteria were required for the observed inhibition. This observation was correlated to decreased P-gp expression both in vitro and during infection in vivo using a mouse model of bacterial meningitis. Given the impact of bacterial interactions on P-gp function, it will be important to incorporate these findings into analyses of drug delivery paradigms for bacterial infections of the CNS.

Group B Streptococcus (GBS) is a major opportunistic pathogen in certain adult populations, including pregnant women, and remains a leading etiologic agent of newborn disease. During pregnancy, GBS asymptomatically colonizes the vaginal tract of 20-30% of healthy women, but can be transmitted to the neonate in utero or during birth resulting in neonatal pneumonia, sepsis, meningitis, and subsequently 10-15% mortality regardless of antibiotic treatment. While various GBS virulence factors have been implicated in vaginal colonization and invasive disease, the regulation of many of these factors remains unclear. Recently, CRISPR-associated protein-9 (Cas9), an endonuclease known for its role in CRISPR/Cas immunity, has also been observed to modulate virulence in a number of bacterial pathogens. However, the role of Cas9 in GBS colonization and disease pathogenesis has not been well-studied. We performed allelic replacement of cas9 in GBS human clinical isolates of the hypervirulent sequence-type 17 strain lineage to generate isogenic Δ cas9 mutants. Compared to parental strains, Δ cas9 mutants were attenuated in murine models of hematogenous meningitis and vaginal colonization and exhibited significantly decreased invasion of human brain endothelium and adherence to vaginal epithelium. To determine if Cas9 alters transcription in GBS, we performed RNA-Seq analysis and found that 353 genes (>17% of the GBS genome) were differentially expressed between the parental WT and Δ cas9 mutant strain. Significantly dysregulated genes included those encoding predicted virulence factors, metabolic factors, two-component systems (TCS), and factors important for cell wall formation. These findings were confirmed by qRT-PCR and suggest that Cas9 may regulate a significant portion of the GBS genome. We studied one of the TCS regulators, CiaR, that was significantly downregulated in the Δ cas9 mutant strain. RNA-Seq analysis of the WT and Δ ciaR strains demonstrated that almost all CiaR-regulated genes were also significantly regulated by Cas9, suggesting that Cas9 may modulate GBS gene expression through other regulators. Further we show that CiaR contributes to GBS vaginal colonization and persistence. Altogether, these data highlight the potential complexity and importance of the non-canonical function of Cas9 in GBS colonization and disease.

The central nervous system (CNS) barriers are highly specialized cellular barriers that promote brain homeostasis while restricting pathogen and toxin entry. The primary cellular constituent regulating pathogen entry in most of these brain barriers is the brain endothelial cell (BEC) that exhibits properties that allow for tight regulation of CNS entry. Bacterial meningoencephalitis is a serious infection of the CNS and occurs when bacteria can cross specialized brain barriers and cause inflammation. Models have been developed to understand the bacterial - BEC interaction that lead to pathogen crossing into the CNS, however, these have been met with challenges due to these highly specialized BEC phenotypes. This perspective provides a brief overview and outlook of the in vivo and in vitro models currently being used to study bacterial brain penetration, and opinion on improved models for the future.

Streptococcus agalactiae (Group B Streptococcus, GBS) normally colonizes healthy adults but can cause invasive disease, such as meningitis, in the newborn. To gain access to the central nervous system, GBS must interact with and penetrate brain or meningeal blood vessels; however, the exact mechanisms are still being elucidated. Here, we investigate the contribution of BspC, an antigen I/II family adhesin, to the pathogenesis of GBS meningitis. Disruption of the bspC gene reduced GBS adherence to human cerebral microvascular endothelial cells (hCMEC), while heterologous expression of BspC in non-adherent Lactococcus lactis conferred bacterial attachment. In a murine model of hematogenous meningitis, mice infected with ΔbspC mutants exhibited lower mortality as well as decreased brain bacterial counts and inflammatory infiltrate compared to mice infected with WT GBS strains. Further, BspC was both necessary and sufficient to induce neutrophil chemokine expression. We determined that BspC interacts with the host cytoskeleton component vimentin and confirmed this interaction using a bacterial two-hybrid assay, microscale thermophoresis, immunofluorescent staining, and imaging flow cytometry. Vimentin null mice were protected from WT GBS infection and also exhibited less inflammatory cytokine production in brain tissue. These results suggest that BspC and the vimentin interaction is critical for the pathogenesis of GBS meningitis., Competing Interests: The authors have declared that no competing interests exist.

Background: Group B Streptococcus (GBS) or Streptococcus agalactiae are β-hemolytic gram-positive bacteria that colonize the lower genital tracts of women and are frequently associated with infections during pregnancy. Innate immune defenses are critical for controlling GBS dissemination and systemic infection. Mast cells are resident sentinel cells that come into contact with pathogens early during colonization and infection., Objective: We aimed to investigate the contribution of chymase to systemic GBS infection and rates of preterm birth., Methods: Pharmacologic and genetic approaches using mice deficient in mast cell protease (MCPT) 4, the mouse functional homologue of human chymase, were used., Results: Our studies show that mast cells release a protease with chymotrypsin-like cleavage specificity in response to GBS. Additionally, increased GBS systemic infection and preterm births were observed in MCPT4-deficient mice versus MCPT4-sufficient mice. Furthermore, we observed that proteolytic cleavage of the host extracellular matrix protein fibronectin by peritoneal cell-derived mast cell lysates diminished GBS adherence. Consistent with this observation, the increase in GBS dissemination and preterm births observed in MCPT4-deficient mice was abolished when GBS was deficient in expression of the fibronectin-binding protein SfbA., Conclusions: Taken together, our results suggest that the protective effect of MCPT4 against GBS dissemination and preterm labor can be attributed in part to MCPT4-mediated proteolysis of fibronectin. Our studies reveal a novel role of mast cells in defense against bacterial infections., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Streptococcus agalactiae (group B Streptococcus [GBS]) is often a commensal bacterium that colonizes healthy adults asymptomatically and is a frequent inhabitant of the vaginal tract in women. However, in immunocompromised individuals, particularly the newborn, GBS may transition to an invasive pathogen and cause serious disease. Despite the use of the currently recommended intrapartum antibiotic prophylaxis for GBS-positive mothers, GBS remains a leading cause of neonatal septicemia and meningitis. To adapt to the various host environments encountered during its disease cycle, GBS possesses multiple two-component regulatory systems (TCSs). Here we investigated the contribution of a transcriptional regulator containing a LytTR domain, LtdR, to GBS pathogenesis. Disruption of the ltdR gene in the GBS chromosome resulted in a significant increase in bacterial invasion into human cerebral microvascular endothelial cells (hCMEC) in vitro as well as the greater penetration of the blood-brain barrier (BBB) and the development of meningitis in vivo Correspondingly, infection of hCMEC with the Δ ltdR mutant resulted in increased secretion of the proinflammatory cytokines interleukin-8 (IL-8), CXCL-1, and IL-6. Further, using a mouse model of GBS vaginal colonization, we observed that the Δ ltdR mutant was cleared more readily from the vaginal tract and also that infection with the Δ ltdR mutant resulted in increased cytokine production from human vaginal epithelial cells. RNA sequencing revealed global transcriptional differences between the Δ ltdR mutant and the parental wild-type GBS strain. These results suggest that LtdR regulates many bacterial processes that can influence GBS-host interactions to promote both bacterial persistence and disease progression., (Copyright © 2018 American Society for Microbiology.)