Bacteria hijack a meningeal neuroimmune axis to facilitate brain invasion.

The meninges are densely innervated by nociceptive sensory neurons that mediate pain and headache1,2. Bacterial meningitis causes life-threatening infections of the meninges and central nervous system, affecting more than 2.5 million people a year3-5. 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.

Bacterial protein domains with a novel Ig-like fold target 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 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 ß protein of GBS binds to human CEACAM1 and CEACAM5 receptors. A crystal structure of the complex showed that an IgSF domain in ß 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.

Identification of a novel cationic glycolipid in Streptococcus agalactiae that contributes to 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), 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.

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 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.

Targeting the BspC-vimentin interaction to develop anti-virulence therapies during Group B streptococcal meningitis.

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.

Staphylococcus aureus Fibronectin-Binding Proteins Contribute to Colonization of the Female Reproductive Tract.

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 α5ß1 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 ΔfnbAB 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.

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, 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.

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 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.

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 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.

Mast cell chymase decreases the severity of group B Streptococcus infections.

BACKGROUND: Group B Streptococcus (GBS) or Streptococcus agalactiae are ß-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.

Human milk oligosaccharides inhibit growth of group B Streptococcus.

Streptococcus agalactiae (group B Streptococcus, GBS) is a leading cause of invasive bacterial infections in newborns, typically acquired vertically during childbirth secondary to maternal vaginal colonization. Human milk oligosaccharides (HMOs) have important nutritional and biological activities that guide the development of the immune system of the infant and shape the composition of normal gut microbiota. In this manner, HMOs help protect against pathogen colonization and reduce the risk of infection. In the course of our studies of HMO-microbial interactions, we unexpectedly uncovered a novel HMO property to directly inhibit the growth of GBS independent of host immunity. By separating different HMO fractions through multidimensional chromatography, we found the bacteriostatic activity to be confined to specific non-sialylated HMOs and synergistic with a number of conventional antibiotic agents. Phenotypic screening of a GBS transposon insertion library identified a mutation within a GBS-specific gene encoding a putative glycosyltransferase that confers resistance to HMOs, suggesting that HMOs may function as an alternative substrate to modify a GBS component in a manner that impairs growth kinetics. Our study uncovers a unique antibacterial role for HMOs against a leading neonatal pathogen and expands the potential therapeutic utility of these versatile molecules.

Characterization of a Two-Component System Transcriptional Regulator, LtdR, That Impacts Group B Streptococcal Colonization and Disease.

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 ΔltdR 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 ΔltdR mutant was cleared more readily from the vaginal tract and also that infection with the ΔltdR mutant resulted in increased cytokine production from human vaginal epithelial cells. RNA sequencing revealed global transcriptional differences between the ΔltdR 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.

Contribution of pilus type 2b to invasive disease caused by a Streptococcus agalactiae ST-17 strain.

BACKGROUND: Group B Streptococcus (GBS) is a major cause of invasive disease especially in neonates. In GBS three structurally distinct pilus polymers have been identified as important virulence factors and promising vaccine candidates. The vast majority of Group B Streptococci belonging to the hypervirulent serotype III ST-17 lineage bear pilus types 1 and 2b. The purpose of this study was to investigate the relative contribution of these two pilus types to the pathogenesis of a ST-17 strain. RESULTS: We performed in vivo and in vitro analysis of isogenic knockout mutants derived from the GBS COH1 ST-17 strain deprived of either pilus type 1 or 2b. We compared the two pilus mutants with the wild type strain in a mouse model of invasive disease, in vitro survival in macrophages, and adherence/invasion assays using human brain endothelial and lung epithelial cell lines. Significantly less of the pilus 2b mutant was recovered from the blood, lungs and brain tissue of infected mice compared to the wild-type and pilus 1 mutant strains. Further, while the pilus 2b mutant survived similarly in murine macrophages, it exhibited a lower capacity to adhere and invade human brain epithelial and lung endothelial cell lines. CONCLUSIONS: The data suggest an important role of pilus 2b in mediating GBS infection and host cell interaction of strains belonging to the hypervirulent GBS ST-17 lineage.

Effect of myeloid differentiation primary response gene 88 on expression profiles of genes during the development and progression of Helicobacter-induced gastric cancer.

BACKGROUND: Gastric cancer is one of the most common and lethal type of cancer worldwide. Infection with Helicobacter pylori (H. pylori) is recognized as the major cause of gastric cancer. However, it remains unclear the mechanism by which Helicobacter infection leads to gastric cancer. Furthermore, the underlying molecular events involved during the progression of Helicobacter infection to gastric malignancy are not well understood. In previous studies, we demonstrated that that H. felis-infected Myd88 -/- mice exhibited dramatic pathology and an accelerated progression to gastric dysplasia; however, the MyD88 downstream gene targets responsible for this pathology have not been described. This study was designed to identify MyD88-dependent genes involved in the progression towards gastric cancer during the course of Helicobacter infection. METHODS: Wild type (WT) and Myd88 deficient mice (Myd88 -/-) were infected with H. felis for 25 and 47 weeks and global transcriptome analysis performed on gastric tissue using MouseWG-6 v2 expression BeadChips microarrays. Function and pathway enrichment analyses of statistically significant, differential expressed genes (p < 0.05) were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tools. RESULTS: Helicobacter infection affected the transcriptional profile of more genes in Myd88 -/- mice compared to WT mice. Infection of Myd88 -/- mice resulted in the differential expression of 1,989 genes at 25 weeks (1031 up and 958 downregulated). At 47 weeks post-H.felis infection, 2,162 (1140 up and 1022 downregulated) were differentially expressed. The most significant differentially upregulated gene during Helicobacter infection in Myd88 -/- mice was chitinase-like 4 (chil4), which is involved in tissue remodeling and wound healing. Other highly upregulated genes in H. felis-infected Myd88 -/- mice included, Indoleamine 2,3-Dioxygenase 1 (Ido1), Guanylate binding protein 2 (Gbp2), ubiquitin D (Ubd), ß 2 -Microglobulin (B2m), CD74 antigen (Cd74), which have been reported to promote cancer progression by enhancing angiogenesis, proliferation, migration, metastasis, invasion, and tumorigenecity. For downregulated genes, the highly expressed genes included, ATPase H+/K+ transporting, alpha subunit (Atp4a), Atp4b, Mucin 5 AC (Muc5ac), Apolipoprotein A-1 (Apoa1), and gastric intrinsic factor (Gif), whose optimal function is important in maintaining gastric hemostasis and lower expression has been associated with increased risk of gastric carcinogenesis. CONCLUSIONS: These results provide a global transcriptional gene profile during the development and progression of Helicobacter-induced gastric cancer. The data show that our mouse model system is useful for identifying genes involved in gastric cancer progression.

Coxsackievirus B exits the host cell in shed microvesicles displaying autophagosomal markers.

Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, "fluorescent timer" protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. "Fluorescent timer" protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of "fluorescent timer" protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. "Fluorescent timer" protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured "fluorescent timer" protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.

Host-pathogen interactions in bacterial meningitis.

Bacterial meningitis is a devastating disease occurring worldwide with up to half of the survivors left with permanent neurological sequelae. Due to intrinsic properties of the meningeal pathogens and the host responses they induce, infection can cause relatively specific lesions and clinical syndromes that result from interference with the function of the affected nervous system tissue. Pathogenesis is based on complex host-pathogen interactions, some of which are specific for certain bacteria, whereas others are shared among different pathogens. In this review, we summarize the recent progress made in understanding the molecular and cellular events involved in these interactions. We focus on selected major pathogens, Streptococcus pneumonia, S. agalactiae (Group B Streptococcus), Neisseria meningitidis, and Escherichia coli K1, and also include a neglected zoonotic pathogen, Streptococcus suis. These neuroinvasive pathogens represent common themes of host-pathogen interactions, such as colonization and invasion of mucosal barriers, survival in the blood stream, entry into the central nervous system by translocation of the blood-brain and blood-cerebrospinal fluid barrier, and induction of meningeal inflammation, affecting pia mater, the arachnoid and subarachnoid spaces.