Biology subjects

Doran, K. S.

Publications and source records attributed to Doran, K. S..

16 recordsLinked to original sources

bioRxiv2026

Meningeal inflammation and arachnoid barrier breakdown in a mouse model of neonatal bacterial meningitis

Newborns are especially susceptible to bacterial meningitis, primarily caused by Group B Streptococcus (GBS), due to incomplete maturation of immune and barrier systems. While meningitis is well known to break down the blood-brain barrier (BBB), how the meningeal arachnoid barrier, a critical component of the blood-cerebrospinal fluid barrier (B-CSFB), responds to infection is poorly understood. Using a neonatal mouse model of bacterial meningitis, we demonstrate that GBS infection significantly increases arachnoid barrier permeability, coinciding with alterations in Claudin-11 tight junction distribution and elevated meningeal production of proinflammatory cytokines (IL-6, TNF-, CXCL1). CD206+/Lyve1+ border-associated macrophages (BAMs) undergo significant morphological and molecular activation post-infection, but their depletion prior to GBS infection did not attenuate arachnoid barrier leakage or inflammatory cytokine levels during infection. We show that meningeal fibroblasts are a main source of proinflammatory cytokines in response to GBS infection and that exposure to the inflammatory cytokine TNF- alone is sufficient to induce neonatal arachnoid barrier breakdown. These results support neonatal arachnoid barrier is vulnerable to cytokine-induced breakdown in bacterial infection and highlight the role of non-immune meningeal cells like fibroblasts during bacterial infection.

neuroscience↗

bioRxiv2025

A murine model of adult gastrointestinal colonization by Group B Streptococcus

Group B Streptococcus (Streptococcus agalactiae, GBS) is a leading cause of invasive infections in neonates and adults. The adult gastrointestinal (GI) tract represents an understudied site of asymptomatic carriage with potential relevance for both transmission and disease. Here, we establish a murine model of GBS colonization in the adult GI tract, which provides a tractable system for probing host-microbe interactions within this niche. Using this model, we establish that GI carriage is generalizable to diverse GBS isolates and leverage transposon sequencing (Tn-Seq) to identify candidate GBS factors important for GI colonization. Informed by these TN-Seq data, we identify GBS capsule as a critical colonization factor of the adult murine GI tract. Taken together, this work highlights the GI tract as a reservoir for GBS and introduces a new experimental framework for investigating the bacterial and host determinants of GBS GI carriage.

microbiology↗

bioRxiv2025

Akkermansia muciniphila Impacts Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae or Group B Streptococcus (GBS) is an opportunistic pathogen that asymptomatically colonizes the vaginal tract of up to 30% of healthy individuals. However, during pregnancy it is associated with adverse pregnancy outcomes, and GBS can be transmitted to the fetus in utero, or the newborn during vaginal birth, resulting in invasive neonatal disease. Previously we identified that Akkermansia muciniphila alters the dynamics of GBS vaginal colonization. However, the global effect of A. muciniphila in the female genital tract remains largely unknown, as it has predominantly been studied in the context of gastrointestinal health. Here we determine that A. muciniphila promotes GBS aggregation and attachment to human vaginal epithelial cells (hVECs). RNA-sequencing analysis revealed that A. muciniphila changed expression of 258 unique GBS genes during hVEC colonization, with many involved in cell wall/membrane/envelope biogenesis. We demonstrate that A. muciniphila-mediated increases in GBS aggregation and attachment to hVECs are dependent on GBS capsule and pili, respectively. Lastly, we found that A. muciniphila promoted GBS aggregation in the murine vaginal lumen and continual treatment with A. muciniphila reduced GBS vaginal persistence. Our results highlight the dynamic impact of A. muciniphila on GBS gene expression and vaginal colonization, as well as demonstrate the probiotic potential of A. muciniphila in the vaginal environment. IMPORTANCEGroup B Streptococcus (GBS) is a frequent colonizer of the vaginal tract of healthy people, however during pregnancy, maternal colonization is associated with adverse pregnancy outcomes. GBS is a leading cause of neonatal sepsis and meningitis and can be transmitted to a newborn during vaginal delivery or by ascension into the uterus during pregnancy. Influence of the vaginal microbiota on GBS pathogenesis remains greatly underappreciated. We have found that GBS is associated with the mucin-degrading intestinal commensal Akkermansia muciniphila, a newly identified colonizer of the vaginal tract. Significance of our research is in identifying the mechanistic impact of this commensal organism on GBS aggregation, cell adherence, and gene expression, as well as its therapeutic potential during GBS vaginal colonization. Unraveling relationships between GBS and the vaginal microbiota will improve maternal and fetal health and may enterprise the development of alternative methods to reduce GBS in utero complications and neonatal disease.

microbiology↗

bioRxiv2025

A fungal pathobiont promotes Streptococcus agalactiae vaginal persistence and pathogenesis through physical and metabolic interactions

Complex polymicrobial interactions at the host interface can shape the mucosal landscape and tip the scales between commensalism and pathogenicity. Here, we use a newly adapted murine model of vaginal colonization to show that the human pathobiont Candida albicans (Ca) supports Group B Streptococcus (GBS) fitness in the vaginal tract and ascension to the uterus. GBS frequently colonizes the vagina asymptomatically; however, during pregnancy, colonization can lead to adverse outcomes and neonatal invasive infection. Using human vaginal isolates of Ca and GBS, we demonstrate that physical interactions contribute to persistence. Triple RNA sequencing of Ca, GBS, and a physiologically relevant model of the human vaginal epithelium reveals that GBS induces arginine biosynthesis in Ca. This drives the expression of bacterial virulence factors and primes GBS for adhesion to the epithelium. We show that interkingdom nutrient exchange can increase GBS pathogenic potential and identify a new target for preventative therapies.

microbiology↗

bioRxiv2025

Global genome analysis identifies glycolipids and lipoteichoic acid alanylation as contributors to Group B streptococcal diabetic wound infection

Individuals with diabetes frequently develop chronic, hyper-inflammatory, non-healing wounds, which are the leading cause of all non-traumatic amputations. Group B Streptococcus (GBS) is a prominent bacterium isolated from diabetic wound infections and in a murine model of diabetic wound infection, GBS stimulates an influx of neutrophils into the wound. Utilizing a transposon mutant screen, we identified 291 bacterial genes required for fitness during diabetic wound infection including enzymes involved in glycolipid biosynthesis and lipoteichoic acid (LTA) alanylation. GBS mutants lacking either LTA alanylation ({Delta}dltA) or all glycolipids ({Delta}iagB) are attenuated in a murine diabetic wound infection. GBS induces primary and secondary degranulation in primary human neutrophils and the {Delta}iagB mutant is significantly more susceptible to neutrophil killing by cationic antimicrobial peptides and reactive oxygen species than {Delta}dltA. Finally, we found that depletion of neutrophils led to significantly greater bacterial recovery, highlighting the importance of neutrophil defense during diabetic wound infection.

microbiology↗

bioRxiv2025

Characterization of the Cellular Immune Response to Group B Streptococcal Vaginal Colonization

IntroductionGroup 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. MethodsHerein, we utilize depleting antibodies and knockout mice and performed flow cytometry to investigate cellular immunes responses during GBS colonization. ResultsWe 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 V{gamma}6+ {gamma}{delta} T cells comprise a primary source of FGT IL-17. Finally, using knockout mice, we observed that IL-17-producing {gamma}{delta} T cells are important for the control of GBS in the FGT during murine colonization. ConclusionsTaken 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 {gamma}{delta} T cells, contribute to host defense and control of GBS colonization.

immunology↗

bioRxiv2024

Collagen binding adhesin restricts Staphylococcus aureus skin infection

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 {Delta}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 {Delta}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, LTB4), resulting in increased immune cell infiltration and death.

microbiology↗

bioRxiv2024

Identification of Glyoxalase A in Group B Streptococcus and its contribution to methylglyoxal tolerance and virulence

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 623 genes that contribute to survival in the blood, including those encoding known virulence factors such as capsule, the {beta}-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.

microbiology↗

bioRxiv2024

A group B streptococcal type VII secreted LXG toxin mediates interbacterial competition and colonization of the female genital tract

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. 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 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 LXG Toxin 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.

microbiology↗

bioRxiv2023

Heterogeneity of the group B streptococcal type VII secretion system and influence on colonization of the female genital tract

Type VIIb secretion systems (T7SSb) in Gram-positive bacteria facilitate physiology, interbacterial competition, and/or virulence via EssC ATPase-driven secretion of small [a]-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 intra-species 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 inter-species 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. Further, we observe subtype-specific effects of GBS T7SS on host colonization, as subtype I but not subtype III T7SS promotes GBS vaginal persistence. 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.

microbiology↗

bioRxiv2022

Group B Streptococcus Adaptation Promotes Survival in a Hyper-inflammatory Diabetic Wound Environment

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 upregulating 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. TeaserThe Fight for Survival by the Bacterium Group B Streptococcus in the Diabetic Wound.

microbiology↗

bioRxiv2021

A type VII secretion system in Group B Streptococcus mediates cytotoxicity and virulence

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, or interbacterial killing in a few genera. Bioinformatic analysis indicates that Group B streptococcal (GBS) isolates encode 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 not known. Here we show that the most abundant GBS T7SS subtype is important for virulence and cytotoxicity in brain endothelium and that these phenotypes are dependent on the WXG100 effector EsxA. We further show that the WXG motif is required for cytotoxicity in brain endothelium and that EsxA is a pore-forming protein. This work reveals the importance of a T7SS in host-GBS interactions and has implications for the functions of T7SS effectors in other Gram-positive bacteria.

microbiology↗

bioRxiv2020

Streptococcus agalactiae MprF synthesizes a novel cationic glycolipid that promotes brain entry and meningitis

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) that 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 two positive net charges per molecule, distinct from the widely described lysylated phospholipid Lysyl-phosphatidylglycerol (Lys-PG) which 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 GBS{Delta}mprF mutant developed bacteremia comparably to Wild-Type 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.

microbiology↗

bioRxiv2020

Bacterial protein domains with a novel Ig-like fold bind human CEACAM receptors

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 at mucosal surfaces. Though several GBS adhesins have been identified, the host receptor targets of these adhesins remain unknown. We report here that surface-expressed {beta} protein from GBS binds to human CEACAM1 and CEACAM5 receptors. A crystal structure of the complex showed that the IgSF domain in {beta} represents a novel Ig-fold subtype called IgI3, in which unique features allow binding to CEACAM1. Bioinformatic assessments revealed that this newly identified IgI3 fold is not exclusively present in GBS. Instead, the IgI3 fold is predicted to be present in adhesins from other clinically important human pathogens. We confirmed the interaction between CEACAM1 and the predicted IgI3-containing adhesin in two different streptococcal pathogens. Overall, our results indicate that the IgI3 fold could provide a broadly applied mechanism for bacteria to target CEACAMs.

microbiology↗

bioRxiv2020

Genome-wide mutagenesis identifies factors involved in Enterococcus faecalis vaginal adherence and persistence

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 post-inoculation 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. IMPORTANCEDespite increasing prevalence and association of E. faecalis with aerobic vaginitis, essentially nothing is known about the bacterial factors that influence E. faecalis vaginal colonization. We have adapted an animal model of vaginal colonization that supports colonization of multiple E. faecalis strains. Additionally, we determined that ethanolamine utilization and type VII secretion system genes contribute to vaginal colonization and persistence. Identification of factors important for vaginal colonization and persistence provides potential targets for the development of therapeutics. This study is the first to identify key determinants that promote vaginal colonization by E. faecalis, which may represent an important reservoir for antibiotic resistant enterococci.

microbiology↗

bioRxiv2019

The Group B Streptococcal surface antigen I/II protein, BspC, interacts with host vimentin to promote adherence to brain endothelium and inflammation during the pathogenesis of meningitis

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 {Delta}bspC mutants exhibited lower mortality as well as decreased brain bacterial counts and inflammatory infiltrate compared with 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, 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. AUTHOR SUMMARYGroup B Streptococcus (GBS) typically colonizes healthy adults but can cause severe disease in immune compromised individuals, including newborns. Despite wide-spread intrapartum antibiotic prophylaxis given to pregnant women, GBS remains a leading cause of neonatal meningitis. To cause meningitis, GBS must interact with and penetrate the blood-brain barrier (BBB), which separates bacteria and immune cells in the blood from the brain. In order to develop targeted therapies to treat GBS meningitis, it is important to understand the mechanisms of BBB crossing. Here, we describe the role of the GBS surface factor, BspC, in promoting meningitis and discover the host ligand for BspC, vimentin, which is an intermediate filament protein that is constitutively expressed by endothelial cells. We determined that BspC interacts with the C-terminal domain of cell-surface vimentin to promote bacterial attachment to brain endothelial cells and that purified BspC protein can induce immune signaling pathways. In a mouse model of hematogenous meningitis, we observed that a GBS mutant lacking BspC was less virulent compared to WT GBS and resulted in less inflammatory disease. We also observed that mice lacking vimentin were protected from GBS infection. These results reveal the importance of the BspC-vimentin interaction in the progression of GBS meningitis disease.

microbiology↗