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Microorganisms colonizing the human vaginal mucosa are associated with healthy states, as well as conditions such as bacterial vaginosis and infection-associated preterm birth. Here, we report complete genome sequences of 37 bacterial isolates from the human vaginal tract.

Group B Streptococcus (GBS) is a pervasive neonatal pathogen accounting for a combined half a million deaths and stillbirths annually. The most common source of fetal or neonatal GBS exposure is the maternal microbiota.

SUMMARYType 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 BStreptococcus(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 EsxAin vitroand that in subtype I strain CJB111,esxA1appears 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.

Urinary tract infections (UTIs) are among the most common infections treated worldwide each year and are primarily caused by uropathogenic E. coli (UPEC). Rising rates of antibiotic resistance among uropathogens have spurred consideration of alternative strategies such as bacteriophage (phage) therapy; however, phage-bacterial interactions within the urinary environment are poorly defined. Here, we assess the activity of two phages, HP3 and ES17, against clinical UPEC isolates using in vitro and in vivo models of UTI. In both bacteriologic medium and pooled human urine, we identified phage resistance arising within the first 6-8 hours of coincubation. Whole genome sequencing revealed that UPEC resistant to HP3 and ES17 harbored mutations in genes involved in lipopolysaccharide (LPS) biosynthesis. These mutations coincided with several in vitro phenotypes, including alterations to adherence to and invasion of human bladder epithelial HTB-9 cells, and increased biofilm formation. Interestingly, these phage-resistant UPEC demonstrated reduced growth in pooled human urine, which could be partially rescued by nutrient supplementation, and were more sensitive to several outer membrane targeting antibiotics than parental strains. Additionally, these phage-resistant UPEC were attenuated in a murine UTI model. In total, our findings suggest that while resistance to phages, such as LPS-targeted HP3 and ES17, may readily arise in the urinary environment, phage resistance is accompanied by fitness costs rendering UPEC more susceptible to host immunity or antibiotics.IMPORTANCEUTIs are one of the most common causes of outpatient antibiotic use, and rising antibiotic resistance threatens the ability to control these infections unless alternative treatments are developed. Bacteriophage (phage) therapy is gaining renewed interest, however, much like antibiotics, bacteria can readily become resistant to phage. For successful UTI treatment, we must predict how bacteria will evade killing by phage and identify the downstream consequences of phage-resistant bacterial infections. In our current study, we found that while phage-resistant mutant bacteria quickly emerged, these mutations left bacteria less capable of growing in human urine and colonizing the murine bladder. These results suggest that phage therapy poses a viable UTI treatment if phage resistance confers fitness costs for the uropathogen. These results have implications for developing cocktails of phage with multiple different bacterial targets, each of which is only evaded at the cost of bacterial fitness.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of adult life-threatening sepsis and urinary tract infections (UTI). The emergence and spread of multidrug-resistant (MDR) ExPEC strains result in a considerable amount of treatment failure and hospitalization costs, and contribute to the spread of drug resistance amongst the human microbiome. Thus, an effective vaccine against ExPEC would reduce morbidity and mortality and possibly decrease carriage in healthy or diseased populations. A comparative genomic analysis demonstrated a gene encoding an invasin-like protein, termed sinH, annotated as an autotransporter protein, shows high prevalence in various invasive ExPEC phylogroups, especially those associated with systemic bacteremia and UTI. Here, we evaluated the protective efficacy and immunogenicity of a recombinant SinH-based vaccine consisting of either domain-3 or domains-1,2, and 3 of the putative extracellular region of surface-localized SinH. Immunization of a murine host with SinH-based antigens elicited significant protection against various strains of the pandemic ExPEC sequence type 131 (ST131) as well as multiple sequence types in two distinct models of infection (colonization and bacteremia). SinH immunization also provided significant protection against ExPEC colonization in the bladder in an acute UTI model. Immunized cohorts produced significantly higher levels of vaccine-specific serum IgG and urinary IgG and IgA, findings consistent with mucosal protection. Collectively, these results demonstrate that autotransporter antigens such as SinH may constitute promising ExPEC phylogroup-specific and sequence-type effective vaccine targets that reduce E. coli colonization and virulence.

Group B Streptococcus (GBS) colonizes the vaginal mucosa of a significant percentage of healthy women and is a leading cause of neonatal bacterial infections. Currently, pregnant women are screened in the last month of pregnancy and GBS-positive women are given antibiotics during parturition to prevent bacterial transmission to the neonate. Recently, human milk oligosaccharides (HMOs) isolated from breastmilk were found to inhibit GBS growth and biofilm formation in vitro, and women that make certain HMOs are less likely to be vaginally colonized with GBS. Using in vitro human vaginal epithelial cells and a murine vaginal colonization model, we tested the impact of HMO treatment on GBS burdens and the composition of the endogenous microbiota by 16S rRNA amplicon sequencing. HMO treatment reduced GBS vaginal burdens in vivo with minimal alterations to the vaginal microbiota. HMOs displayed potent inhibitory activity against GBS in vitro, but HMO pretreatment did not alter adherence of GBS or the probiotic Lactobacillus rhamnosus to human vaginal epithelial cells. Additionally, disruption of a putative GBS glycosyltransferase (Δsan_0913) rendered the bacterium largely resistant to HMO inhibition in vitro and in vivo but did not compromise its adherence, colonization, or biofilm formation in the absence of HMOs. We conclude that HMOs are a promising therapeutic bioactive to limit GBS vaginal colonization with minimal impacts on the vaginal microenvironment.IMPORTANCEDuring pregnancy, GBS ascension into the uterus can cause fetal infection or preterm birth. Additionally, GBS exposure during labor creates a risk of serious disease in the vulnerable newborn and mother postpartum. Current recommended prophylaxis consists of administering broad-spectrum antibiotics to GBS-positive mothers during labor. Although antibiotics have significantly reduced GBS neonatal disease, there are several unintended consequences including altered neonatal gut bacteria and increased risk for other types of infection. Innovative preventions displaying more targeted antimicrobial activity, while leaving the maternal microbiota intact, are thus appealing. Using a mouse model, we found that human milk oligosaccharides (HMOs) reduce GBS burdens without perturbing the vaginal microbiota. We conclude that HMOs are a promising alternative to antibiotics to reduce GBS neonatal disease.

Multidrug-resistant (MDR) Enterococcus faecalis are major causes of hospital-acquired infections. Numerous clinical strains of E. faecalis harbor a large pathogenicity island that encodes enterococcal surface protein (Esp), which is suggested to promote biofilm production and virulence, but this remains controversial. To resolve this issue, we characterized the Esp N-terminal region, the portion implicated in biofilm production. Small angle X-ray scattering indicated that the N-terminal region had a globular head, which consisted of two DEv-Ig domains as visualized by X-ray crystallography, followed by an extended tail. The N-terminal region was not required for biofilm production but instead significantly strengthened biofilms against mechanical or degradative disruption, greatly increasing retention of Enterococcus within biofilms. Biofilm strengthening required low pH, which resulted in Esp unfolding, aggregating, and forming amyloid-like structures. The pH threshold for biofilm strengthening depended on protein stability. A truncated fragment of the first DEv-Ig domain, plausibly generated by a host protease, was the least stable and sufficient to strengthen biofilms at pH ≤ 5.0, while the entire N-terminal region and intact Esp on the enterococcal surface was more stable and required a pH ≤ 4.3. These results suggested a virulence role of Esp in strengthening enterococcal biofilms in acidic abiotic or host environments.

Streptococcus canis is a common colonizing bacterium of the urogenital tract of cats and dogs that can also cause invasive disease in these animal populations and in humans. Although the virulence mechanisms of S. canis are not well-characterized, an M-like protein, SCM, has recently identified been as a potential virulence factor. SCM is a surface-associated protein that binds to host plasminogen and IgGs suggesting its possible importance in host-pathogen interactions. In this study, we developed in vitro and ex vivo blood component models and murine models of S. canis vaginal colonization, systemic infection, and dermal infection to compare the virulence potential of the zoonotic S. canis vaginal isolate G361 and its isogenic SCM-deficient mutant (G361∆scm). We found that while S. canis establishes vaginal colonization and causes invasive disease in vivo, the contribution of the SCM protein to virulence phenotypes in these models is modest. We conclude that SCM is dispensable for invasive disease in murine models and for resistance to human blood components ex vivo, but may contribute to mucosal persistence, highlighting a potential contribution to the recently appreciated genetic diversity of SCM across strains and hosts.

Summary Streptococcus agalactiae (Group B Streptococcus, GBS) is the most common neonatal pathogen. However, the cellular and molecular mechanisms for neonatal susceptibility to GBS pneumonia and sepsis are incompletely understood. Here we optimized a mouse model of GBS pneumonia to test the role of alveolar macrophage (ΑΜΦ) maturation in host vulnerability to disease. Compared with juvenile and adult mice, neonatal mice infected with GBS had increased mortality and persistence of lung injury. In addition, neonatal mice were defective in GBS phagocytosis and killing. ΑΜΦ depletion and disruption of ΑΜΦ differentiation in Csf2−/− mice both impaired GBS clearance. AMΦ engage the heavily sialylated GBS capsule via the cell surface Siglec receptors Sn and Siglec-E. Although both newborn and adult ΑΜΦ expressed Siglec-E, newborn ΑΜΦ expressed significantly lower levels of Sn. We propose that a developmental delay in Sn expression on ΑΜΦ may prevent effective killing and clearing of GBS from the newborn lung., Graphical Abstract, Highlights • Newborn mice fail to kill GBS, developing persistent lung injury • Mature AMΦ detect the Sialic acid capsule on GBS to mediate bacterial clearance • Immature newborn AMΦ lack mature Siglec expression required for killing GBS • GBS engages the inhibitory Siglec-E on newborn AMΦ to suppress innate immunity, Medical Microbiology; Reproductive Medicine; Microbiome

Urinary tract infection (UTI) is a prominent global health care burden. Although UTI is readily treated with antibiotics in healthy adults, complicated cases in immune-compromised individuals and the emerging antibiotic resistance of several uropathogens have accelerated the need for new treatment strategies. Here, we surveyed the composition of urinary exosomes in a mouse model of uropathgenic Escherichia coli (UPEC) UTI to identify specific urinary tract defense constituents for therapeutic development. We found an enrichment of the iron-binding glycoprotein lactoferrin in the urinary exosomes of infected mice. In subsequent in vitro studies, we identified human bladder epithelial cells as a source of lactoferrin during UPEC infection. We further established that exogenous treatment with human lactoferrin (hLf) reduces UPEC epithelial adherence and enhances neutrophil antimicrobial functions including bacterial killing and extracellular trap production. Notably, a single intravesicular dose of hLf drastically reduced bladder bacterial burden and neutrophil infiltration in our murine UTI model. We propose that lactoferrin is an important modulator of innate immune responses in the urinary tract and has potential application in novel therapeutic design for UTI.

Certain populations such as diabetic individuals are at increased risk for developing urinary tract infections (UTI), although the underlying reasons for this susceptibility are not fully known. Additionally, diabetics are more likely to become infected with certain types of bacteria, such as group B Streptococcus (GBS). In this study, we find that an antimicrobial peptide called cathelicidin, which is thought to protect the bladder from infection, is ineffective in controlling GBS and alters the type of immune cells that migrate to the bladder during infection. Using a mouse model of diabetes, we observe that diabetic mice are more susceptible to GBS infection even though they also have more infiltrating immune cells and increased production of cathelicidin. Taken together, our findings identify this antimicrobial peptide as a potential contributor to increased susceptibility of diabetic individuals to GBS UTI., Group B Streptococcus (GBS) causes frequent urinary tract infection (UTI) in susceptible populations, including individuals with type 2 diabetes and pregnant women; however, specific host factors responsible for increased GBS susceptibility in these populations are not well characterized. Here, we investigate cathelicidin, a cationic antimicrobial peptide, known to be critical for defense during UTI with uropathogenic Escherichia coli (UPEC). We observed a loss of antimicrobial activity of human and mouse cathelicidins against GBS and UPEC in synthetic urine and no evidence for increased cathelicidin resistance in GBS urinary isolates. Furthermore, we found that GBS degrades cathelicidin in a protease-dependent manner. Surprisingly, in a UTI model, cathelicidin-deficient (Camp−/−) mice showed decreased GBS burdens and mast cell recruitment in the bladder compared to levels in wild-type (WT) mice. Pharmacologic inhibition of mast cells reduced GBS burdens and histamine release in WT but not Camp−/− mice. Streptozotocin-induced diabetic mice had increased bladder cathelicidin production and mast cell recruitment at 24 h postinfection with GBS compared to levels in nondiabetic controls. We propose that cathelicidin is an important immune regulator but ineffective antimicrobial peptide against GBS in urine. Combined, our findings may in part explain the increased frequency of GBS UTI in diabetic and pregnant individuals. IMPORTANCE Certain populations such as diabetic individuals are at increased risk for developing urinary tract infections (UTI), although the underlying reasons for this susceptibility are not fully known. Additionally, diabetics are more likely to become infected with certain types of bacteria, such as group B Streptococcus (GBS). In this study, we find that an antimicrobial peptide called cathelicidin, which is thought to protect the bladder from infection, is ineffective in controlling GBS and alters the type of immune cells that migrate to the bladder during infection. Using a mouse model of diabetes, we observe that diabetic mice are more susceptible to GBS infection even though they also have more infiltrating immune cells and increased production of cathelicidin. Taken together, our findings identify this antimicrobial peptide as a potential contributor to increased susceptibility of diabetic individuals to GBS UTI.

A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease1–9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11–13 to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry14. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis. Metabolomics data from germ-free and specific-pathogen-free mice reveal effects of the microbiome on host chemistry, identifying conjugations of bile acids that are also enriched in patients with inflammatory bowel disease or cystic fibrosis.

Trichomonas vaginalis is a sexually transmitted, eukaryotic parasite that causes trichomoniasis, the most common nonviral, sexually transmitted disease in the USA and worldwide. Little is known about the molecular mechanisms involved in the host immune response to this widespread parasite. Here we report that T. vaginalis induces NLRP3 inflammasome activation in human macrophages, leading to caspase-1 activation and the processing of pro-IL-1β to the mature and bioactive form of the cytokine. Using inhibitor-based approaches, we show that NLRP3 activation by T. vaginalis involves host cell detection of extracellular ATP via P2X7 receptors and potassium efflux. In addition, our data reveal that T. vaginalis inflammasome activation induces macrophage inflammatory cell death by pyroptosis, known to occur via caspase-1 cleavage of the gasdermin D protein, which assembles to form pores in the host cell membrane. We found that T. vaginalis-induced cytolysis of macrophages is attenuated in gasdermin D knockout cells. Lastly, in a murine challenge model, we detected IL-1β production in vaginal fluids in response to T. vaginalis infection in vivo. Together, our findings mechanistically dissect how T. vaginalis contributes to the production of the proinflammatory IL-1β cytokine and uncover pyroptosis as a mechanism by which the parasite can trigger host macrophage cell death.

Group B Streptococcus (GBS) is a common cause of bacterial urinary tract infections (UTI) in susceptible populations, including pregnant women and the elderly. However, the factors that govern GBS persistence and disease severity in this niche are not fully understood. Here, we report that the presence of the fungus Candida albicans, a common urogenital colonizer, can promote GBS UTI. Co-inoculation of GBS with C. albicans increased bacterial adherence to bladder epithelium and promoted GBS colonization in vivo in a C. albicans adhesin-dependent manner. This study demonstrates that fungal colonization of the urogenital tract may be an important determinant of bacterial pathogenesis during UTI.

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 Δcas9 mutants. Compared to parental strains, Δcas9 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 Δcas9 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 Δcas9 mutant strain. RNA-Seq analysis of the WT and ΔciaR 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.

A mosaic of cross-phyla chemical interactions occurs between all metazoans and their microbiomes. In humans, the gut harbors the heaviest microbial load, but many organs, particularly those with a mucosal surface, associate with highly adapted and evolved microbial consortia1. The microbial residents within these organ systems are increasingly well characterized, yielding a good understanding of human microbiome composition, but we have yet to elucidate the full chemical impact the microbiome exerts on an animal and the breadth of the chemical diversity it contributes2. A number of molecular families are known to be shaped by the microbiome including short-chain fatty acids, indoles, aromatic amino acid metabolites, complex polysaccharides, and host lipids; such as sphingolipids and bile acids3–11. These metabolites profoundly affect host physiology and are being explored for their roles in both health and disease. Considering the diversity of the human microbiome, numbering over 40,000 operational taxonomic units12, a plethora of molecular diversity remains to be discovered. Here, we use unique mass spectrometry informatics approaches and data mapping onto a murine 3D-model13–15to provide an untargeted assessment of the chemical diversity between germ-free (GF) and colonized mice (specific-pathogen free, SPF), and report the finding of novel bile acids produced by the microbiome in both mice and humans that have evaded characterization despite 170 years of research on bile acid chemistry16.

Uncontrolled bacteremia is a common and life threatening condition that can lead to sepsis and septic shock with significant morbidity and mortality. Neutrophil granulocytes, the most abundant phagocytic leukocyte of the innate immune system, play an essential role in capturing and killing invading pathogens. Their antimicrobial repertoire includes the formation of Neutrophil Extracellular Traps (NETs), chromatin-based, web-like structures of DNA that facilitate the capture and killing of bacteria. In sepsis, however, it has been suggested that the uncontrolled release of NETs worsens disseminated coagulation and promotes venous thrombosis. Here, we describe how clinically relevant concentrations of the commonly used sedative propofol as well as a lipid composition similar to the propofol carrier impair NET production by human neutrophils. Drugs commonly administered in the Intensive Care Unit (ICU) may impact the inflammatory response to either worsen or improve clinical outcomes and may therefore be considered for additional therapeutic effects if clinical studies confirm such findings.

Group B Streptococcus (GBS) is a major neonatal pathogen but rarely causes disease in adults. We previously showed in mice that GBS escapes killing in the neonatal lung via its heavily sialylated capsule. Immune cells detect sialic acid moieties via expression of a repertoire of Siglec receptors. Combinatory expression of proinflammatory and anti-inflammatory Siglec receptors allows differentiation between host and pathogenic microbial sialic acid modifications. We test here the hypothesis that neonatal alveolar macrophages (AMs) fail to detect and kill GBS due to developmental immaturity of Siglec receptor expression. Adult AMs express the proinflammatory sialic acid receptor Sialoadhesin (Sn, Siglec-1) and the anti-inflammatory receptors Siglec-E and Siglec-F. However, real time PCR, immunofluorescence, and FACS detected only Siglec-E in neonatal lung macrophage populations. Sn expression increased soon after birth and was restricted to AMs. The timing of increased Sn expression in newborn mice correlated with susceptibility to GBS. Mice infected with GBS on day 1 suffered early onset mortality, while mice infected on day 2 displayed late onset disease. Mice infected on day 3 survived GBS infection. Further implicating AM immaturity, Csf2−/− mice, which have defects in AM differentiation, lacked Sn expression and had reduced GBS clearance following infection. The presence of Siglec-E but absence of Sn in newborn AMs appeared to promote tolerance to GBS. Newborn SigE−/− mice had increased GBS phagocytosis and killing compared to WT controls. We therefore conclude that GBS exploits developmental immaturity of Siglec expression in AMs via its sialic acid capsule in causing neonatal disease.

Urinary tract infection (UTI) is a prominent global health care burden. Although UTI is readily treated with antibiotics in healthy adults, complicated cases in immune-compromised individuals and the emerging antibiotic resistance of several uropathogens have accelerated the need for new treatment strategies. Here, we surveyed the composition of urinary exosomes in a mouse model of uropathgenic Escherichia coli (UPEC) UTI to identify specific urinary tract defense constituents for therapeutic development. We found an enrichment of the iron-binding glycoprotein lactoferrin in the urinary exosomes of infected mice. In subsequent in vitro studies, we identified human bladder epithelial cells as a source of lactoferrin during UPEC infection. We further established that exogenous treatment with human lactoferrin (hLf) reduces UPEC epithelial adherence and enhances neutrophil antimicrobial functions including bacterial killing and extracellular trap production. Notably, a single intravesicular dose of hLf drastically reduced bladder bacterial burden and neutrophil infiltration in our murine UTI model. We propose that lactoferrin is an important modulator of innate immune responses in the urinary tract and has potential application in novel therapeutic design for UTI.

Urinary tract infections (UTIs) caused by the human fungal pathogen Candida albicans and related species are prevalent in hospitalized patients, especially those on antibiotic therapy, with indwelling catheters, or with predisposing conditions such as diabetes or immunodeficiency. Understanding of key host defenses against Candida UTI is critical for developing effective treatment strategies. Tamm-Horsfall glycoprotein (THP) is the most abundant urine protein, with multiple roles in renal physiology and bladder protection. THP protects against bacterial UTI by blocking bacterial adherence to the bladder epithelium, but its role in defense against fungal pathogens is not yet described. Here we demonstrate that THP restricts colonization of the urinary tract by C. albicans. THP binds to C. albicans hyphae, but not the yeast form, in a manner dependent on fungal expression of the Als3 adhesion glycoprotein. THP directly blocks C. albicans adherence to bladder epithelial cells in vitro, and THP-deficient mice display increased fungal burden in a C. albicans UTI model. This work outlines a previously unknown role for THP as an essential component for host immune defense against fungal urinary tract infection.

Trichomonas vaginalis is a sexually transmitted, eukaryotic parasite that causes trichomoniasis, the most common nonviral, sexually transmitted disease in the USA and worldwide. Little is known about the molecular mechanisms involved in the host immune response to this widespread parasite. Here we report that T. vaginalis induces NLRP3 inflammasome activation in human macrophages, leading to caspase-1 activation and the processing of pro-IL-1β to the mature and bioactive form of the cytokine. Using inhibitor-based approaches, we show that NLRP3 activation by T. vaginalis involves host cell detection of extracellular ATP via P2X7 receptors and potassium efflux. In addition, our data reveal that T. vaginalis inflammasome activation induces macrophage inflammatory cell death by pyroptosis, known to occur via caspase-1 cleavage of the gasdermin D protein, which assembles to form pores in the host cell membrane. We found that T. vaginalis-induced cytolysis of macrophages is attenuated in gasdermin D knockout cells. Lastly, in a murine challenge model, we detected IL-1β production in vaginal fluids in response to T. vaginalis infection in vivo. Together, our findings mechanistically dissect how T. vaginalis contributes to the production of the proinflammatory IL-1β cytokine and uncover pyroptosis as a mechanism by which the parasite can trigger host macrophage cell death.

Background Streptococcus agalactiae (group B Streptococcus [GBS]) asymptomatically colonizes approximately 20% of adults; however, GBS causes severe disease in susceptible populations, including newborns, pregnant women, and elderly individuals. In shifting between commensal and pathogenic states, GBS reveals multiple mechanisms of virulence factor control. Here we describe a GBS protein that we named "biofilm regulatory protein A" (BrpA) on the basis of its homology with BrpA from Streptococcus mutans. Methods We coupled phenotypic assays, RNA sequencing, human neutrophil and whole-blood killing assays, and murine infection models to investigate the contribution of BrpA to GBS physiology and virulence. Results Sequence analysis identified BrpA as a LytR-CpsA-Psr enzyme. Targeted mutagenesis yielded a GBS mutant (ΔbrpA) with normal ultrastructural morphology but a 6-fold increase in chain length, a biofilm defect, and decreased acid tolerance. GBS ΔbrpA stimulated increased neutrophil reactive oxygen species and proved more susceptible to human and murine blood and neutrophil killing. Notably, the wild-type parent outcompeted ΔbrpA GBS in murine sepsis and vaginal colonization models. RNA sequencing of ΔbrpA uncovered multiple differences from the wild-type parent, including pathways of cell wall synthesis and cellular metabolism. Conclusions We propose that BrpA is an important virulence regulator and potential target for design of novel antibacterial therapeutics against GBS.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive bacterium, which colonizes the vaginal tract in 10-30% of women. Colonization is transient in nature, and little is known about the host and bacterial factors controlling GBS persistence. Gaining insight into these factors is essential for developing therapeutics to limit maternal GBS carriage and prevent transmission to the susceptible newborn. In this work, we have used human cervical and vaginal epithelial cells, and our established mouse model of GBS vaginal colonization, to characterize key host factors that respond during GBS colonization. We identify a GBS strain that persists beyond a month in the murine vagina, whereas other strains are more readily cleared. Correspondingly, we have detected differential cytokine production in human cell lines after challenge with the persistent strain vs. other GBS strains. We also demonstrate that the persistent strain more readily invades cervical cells compared with vaginal cells, suggesting that GBS may potentially use the cervix as a reservoir to establish long-term colonization. Furthermore, we have identified interleukin-17 production in response to long-term colonization, which is associated with eventual clearance of GBS. We conclude that both GBS strain differences and concurrent host immune responses are crucial in modulating vaginal colonization.

Group B Streptococcus (GBS) colonizes the gastrointestinal and vaginal epithelium of a significant percentage of healthy women, with potential for ascending intrauterine infection or transmission during parturition, creating a risk of serious disease in the vulnerable newborn. This review highlights new insights on the bacterial virulence determinants, host immune responses, and microbiome interactions that underpin GBS vaginal colonization, the proximal step in newborn infectious disease pathogenesis. From the pathogen perspective, the function GBS adhesins and biofilms, β-hemolysin/cytolysin toxin, immune resistance factors, sialic acid mimicry, and two-component transcriptional regulatory systems are reviewed. From the host standpoint, pathogen recognition, cytokine responses, and the vaginal mucosal and placental immunity to the pathogen are detailed. Finally, the rationale, efficacy, and potential unintended consequences of current universal recommended intrapartum antibiotic prophylaxis are considered, with updates on new developments toward a GBS vaccine or alternative approaches to reducing vaginal colonization.

Urinary tract infections are a major problem in human medicine for which better understanding of native immune defenses may reveal new pathways for therapeutic intervention. Tamm-Horsfall glycoprotein (THP), the most abundant urinary protein, interacts with bacteria including uropathogenic Escherichia coli (UPEC) as well host immune cells. In addition to its well-studied functions to antagonize bacterial colonization, we hypothesize that THP serves a critical host defense function through innate immune modulation. Using isolated human neutrophils, we found that THP binds neutrophils and that this interaction reduces reactive oxygen species generation, chemotaxis and killing of UPEC. We discovered that THP engages the inhibitory neutrophil receptor sialic acid-binding Ig-like lectin-9 (Siglec-9), and mouse functional ortholog Siglec-E, in a manner dependent on sialic acid on its N-glycan moieties. THP-null mice have significantly more neutrophils present in the urine compared with wild-type mice, both with and without the presence of inflammatory stimuli. These data support THP as an important negative regulator of neutrophil activation in the urinary tract, with dual functions to counteract bacterial colonization and suppress excessive inflammation within the urinary tract.

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.

Group B Streptococcus (GBS) is the most common pathogen in neonates but rarely causes disease in adults. GBS causes pneumonia in newborns, suggesting a defect in lung innate immunity at birth. Using a novel neonatal mouse model of GBS pneumonia, we showed that newborn mice have reduced killing of GBS in the lung and more severe and persistent lung injury. Here we test the hypothesis that defects in alveolar macrophage (AM) maturation in newborns leads to GBS susceptibility. Clodronate depletion of AM from adult mice prevented GBS killing, supporting the requirement of AM for GBS protection. AM maturation and function requires GM-CSF, which is abundantly expressed in the lung. While AM maturation occurred on day 4 in WT mice, GM-CSF knockout mice (Csf2−/−) failed to develop AM. Consistent with the role of AM in GBS immunity, neonatal Csf2−/− mice had reduced GBS killing and lung macrophages failed to phagocytose GBS. Csf2−/− macrophages also had reduced expression of multiple Siglecs, cell surface receptors implicated in protection against encapsulated bacteria. Newborn lung macrophages expressed primarily Siglec-E (SigE), whereas expression of other Siglecs were only detected in more mature AM. We used SigE−/− mice to test its role in neonatal GBS pneumonia. GBS killing in SigE−/− neonates was similar to WT. However SigE−/− macrophages appeared to phagocytose GBS more efficiently in vivo, suggesting GBS may employ SigE to avoid detection by neonatal macrophages. Our data show that mature alveolar macrophages are required for protection against pulmonary GBS infection and potentially implicate the developmental regulation of macrophage Siglec expression in neonatal lung immunity.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a frequent commensal organism of the vaginal tract of healthy women. However, GBS can transition to a pathogen in susceptible hosts, but host and microbial factors that contribute to this conversion are not well understood. GBS CovR/S (CsrR/S) is a two component regulatory system that regulates key virulence elements including adherence and toxin production. We performed global transcription profiling of human vaginal epithelial cells exposed to WT, CovR deficient, and toxin deficient strains, and observed that insufficient regulation by CovR and subsequent increased toxin production results in a drastic increase in host inflammatory responses, particularly in cytokine signaling pathways promoted by IL-8 and CXCL2. Additionally, we observed that CovR regulation impacts epithelial cell attachment and intracellular invasion. In our mouse model of GBS vaginal colonization, we further demonstrated that CovR regulation promotes vaginal persistence, as infection with a CovR deficient strain resulted in a heightened host immune response as measured by cytokine production and neutrophil activation. Using CXCr2 KO mice, we determined that this immune alteration occurs, at least in part, via signaling through the CXCL2 receptor. Taken together, we conclude that CovR is an important regulator of GBS vaginal colonization and loss of this regulatory function may contribute to the inflammatory havoc seen during the course of infection.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single β-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

Group B Streptococcus (Streptococcus agalactiae, GBS) causes severe infections in neonates. While not a common adult pathogen, GBS is the leading cause of congenital pneumonia. To determine the molecular mechanisms of neonatal susceptibility, we developed a murine GBS pneumonia model. Neonatal, juvenile, and adult C57BL/6 mice were inoculated intranasally with 2800 CFU/g of GBS (COH1). After 7 d, survival was 100 % in adults and juveniles, with 10 % of neonates dying within 3 d after infection. Histopathological assessments showed severe lung injury scores 24 h after infection, with adult and juvenile mice demonstrating resolution of injury at 3 d post infection and normal histology after 7 d. In contrast, neonatal lungs had persistent lung injury up to 7 d post infection. By FACS, GBS induced rapid neutrophil recruitment into adult lungs and increased CD11b expression on adult lung macrophages. In neonatal lungs, GBS neither increased neutrophil numbers nor altered macrophage marker expression. Confocal imaging showed GBS in adult lungs almost entirely localized to alveolar macrophage phagolysosomes. However in neonatal lungs, GBS was only rarely phagocytosed by macrophages. These data were consistent with GBS killing data, which showed complete GBS killing within 24 h in adult and juvenile mice, but persistent GBS viability within the lung for up to 3 d in neonates. Defective GBS killing in neonates appeared to require the GBS capsule. Neonatal mice were much more efficient in killing the ΔcpsD capsule mutant compared to wild type GBS. Adult mice showed equal killing of ΔcpsD or wild type GBS. Our data suggest that the GBS capsule prevents killing by the newborn lung innate immune system, leading to neonatal disease susceptibility.

Group B Streptococcus (GBS) is a major causative agent of neonatal meningitis due to its ability to efficiently cross the blood-brain barrier (BBB) and enter the central nervous system (CNS). It has been demonstrated that GBS can invade human brain microvascular endothelial cells (hBMEC), a primary component of the BBB; however, the mechanism of intracellular survival and trafficking is unclear. We previously identified a two component regulatory system, CiaR/H, which promotes GBS intracellular survival in hBMEC. Here we show that a GBS strain deficient in the response regulator, CiaR, localized more frequently with Rab5, Rab7 and LAMP1 positive vesicles. Further, lysosomes isolated from hBMEC contained fewer viable bacteria following initial infection with the ΔciaR mutant compared to the WT strain. To characterize the contribution of CiaR-regulated genes, we constructed isogenic mutant strains lacking the two most down-regulated genes in the CiaR-deficient mutant, SAN_2180 and SAN_0039. These genes contributed to bacterial uptake and intracellular survival. Furthermore, competition experiments in mice showed that WT GBS had a significant survival advantage over the Δ2180 and Δ0039 mutants in the bloodstream and brain.

Streptococcus agalactiae (group B streptococcus [GBS]) colonizes the rectovaginal tract in 20% to 30% of women and during pregnancy can be transmitted to the newborn, causing severe invasive disease. Current routine screening and antibiotic prophylaxis have fallen short of complete prevention of GBS transmission, and GBS remains a leading cause of neonatal infection. We have investigated the ability of Streptococcus salivarius , a predominant member of the native human oral microbiota, to control GBS colonization. Comparison of the antibacterial activities of multiple S. salivarius strains by use of a deferred-antagonism test showed that S. salivarius strain K12 exhibited the broadest spectrum of activity against GBS. K12 effectively inhibited all GBS strains tested, including disease-implicated isolates from newborns and colonizing isolates from the vaginal tract of pregnant women. Inhibition was dependent on the presence of megaplasmid pSsal-K12, which encodes the bacteriocins salivaricin A and salivaricin B; however, in coculture experiments, GBS growth was impeded by K12 independently of the megaplasmid. We also demonstrated that K12 adheres to and invades human vaginal epithelial cells at levels comparable to GBS. Inhibitory activity of K12 was examined in vivo using a mouse model of GBS vaginal colonization. Mice colonized with GBS were treated vaginally with K12. K12 administration significantly reduced GBS vaginal colonization in comparison to nontreated controls, and this effect was partially dependent on the K12 megaplasmid. Our results suggest that K12 may have potential as a preventative therapy to control GBS vaginal colonization and thereby prevent its transmission to the neonate during pregnancy.

Group B Streptococcus (GBS), in the transition from commensal organisms to pathogens, will encounter diverse host environments and, thus, require coordinated control of the transcriptional responses to these changes. This work was aimed at better understanding the role of two-component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knockout strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1% to 3% of the genome. Interestingly, two sugar phosphotransferase systems appeared to be differentially regulated in the TCS-16 knockout strain (TCS loci were numbered in order of their appearance on the chromosome), suggesting an involvement in monitoring carbon source availability. High-throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for the growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16, with concomitant dramatic upregulation of the adjacent operon, which encodes a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and the data also provide experimental evidence for TCS-17/RgfAC involvement in virulence., IMPORTANCE Two-component systems have been evolved by bacteria to detect environmental changes, and they play key roles in pathogenicity. A comprehensive analysis of TCS in GBS has not been performed previously. In this work, we classify 21 TCS and present evidence for the involvement of two specific TCS in GBS virulence and colonization in vivo. Although pinpointing specific TCS stimuli is notoriously difficult, we used a combination of techniques to identify two systems with different effects on GBS pathogenesis. For one of the systems, we propose that fructose-6-phosphate, a metabolite in glycolysis, is sufficient to induce a regulatory response involving a sugar transport system. Our catalogue and classification of TCS may guide further studies into the role of TCS in GBS pathogenicity and biology.