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.

Group B Streptococcus transcriptome when interacting with brain endothelial cells.

Bacterial meningitis is a life-threatening infection of the central nervous system (CNS) that occurs when bacteria are able to cross the blood-brain barrier (BBB) or the meningeal-cerebrospinal fluid barrier (mBCSFB). The BBB and mBCSFB comprise highly specialized brain endothelial cells (BECs) that typically restrict pathogen entry. Group B Streptococcus (GBS or Streptococcus agalactiae) is the leading cause of neonatal meningitis. Until recently, identification of GBS virulence factors has relied on genetic screening approaches. Instead, we here conducted RNA-seq analysis on GBS when interacting with induced pluripotent stem cell-derived BECs (iBECs) to pinpoint virulence-associated genes. Of the 2,068 annotated protein-coding genes of GBS, 430 transcripts displayed significant changes in expression after interacting with BECs. Notably, we found that the majority of differentially expressed GBS transcripts were downregulated (360 genes) during infection of iBECs. Interestingly, codY, encoding a pleiotropic transcriptional repressor in low-G + C Gram-positive bacteria, was identified as being highly downregulated. We conducted qPCR to confirm the codY downregulation observed via RNA-seq during the GBS-iBEC interaction and obtained codY mutants in three different GBS background parental strains. As anticipated from the RNA-seq results, the [Formula: see text]codY strains were more adherent and invasive in two in vitro BEC models. Together, this demonstrates the utility of RNA-seq during the BEC interaction to identify GBS virulence modulators. IMPORTANCE: Group B Streptococcus (GBS) meningitis remains the leading cause of neonatal meningitis. Research work has identified surface factors and two-component systems that contribute to GBS disruption of the blood-brain barrier (BBB). These discoveries often relied on genetic screening approaches. Here, we provide transcriptomic data describing how GBS changes its transcriptome when interacting with brain endothelial cells. Additionally, we have phenotypically validated these data by obtaining mutants of a select regulator that is highly down-regulated during infection and testing on our BBB model. This work provides the research field with a validated data set that can provide an insight into potential pathways that GBS requires to interact with the BBB and open the door to new discoveries.

The 1.7 Å crystal structure of the C5a peptidase from Streptococcus agalactiae (ScpB) reveals an active site competent for catalysis.

A 1.7 Å structure is presented for an active form of the virulence factor ScpB, the C5a peptidase from Streptococcus agalactiae. The previously reported structure of the ScpB active site mutant exhibited a large separation (~20 Å) between the catalytic His and Ser residues. Significant differences are observed in the catalytic domain between the current and mutant ScpB structures resulting with a high RMSDCα (4.6 Å). The fold of the active form of ScpB is nearly identical to ScpA (RMSDCα 0.2 Å), the C5a-peptidase from Streptococcus pyogenes. Both ScpA and ScpB have comparable activity against human C5a, indicating neither enzyme require host proteins for C5a-ase activity. These studies are a first step in resolving reported differences in the specificities of these enzymes.

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.

Group B streptococcus induces cellular senescence in human amnion epithelial cells through a partial interleukin-1-mediated mechanism.

Group B streptococcus (GBS) infection is a significant public health concern associated with adverse pregnancy complications and increased neonatal mortality and morbidity. However, the mechanisms underlying the impact of GBS on the fetal membrane, the first line of defense against pathogens, are not fully understood. Here, we propose that GBS induces senescence and inflammatory factors (IL-6 and IL-8) in the fetal membrane through interleukin-1 (IL-1). Utilizing the existing transcriptomic data on GBS-exposed human fetal membrane, we showed that GBS affects senescence-related pathways and genes. Next, we treated primary amnion epithelial cells with conditioned medium from the choriodecidual layer of human fetal membrane exposed to GBS (GBS collected choriodecidual [CD] conditioned medium) in the absence or presence of an IL-1 receptor antagonist (IL-1Ra). GBS CD conditioned medium significantly increased ß-galactosidase activity, IL-6 and IL-8 release from the amnion epithelial cells. Cotreatment with IL1Ra reduced GBS-induced ß-galactosidase activity and IL-6 and IL-8 secretion. Direct treatment with IL-1α or IL-1ß confirmed the role of IL-1 signaling in the regulation of senescence in the fetal membrane. We further showed that GBS CD conditioned medium and IL-1 decreased cell proliferation in amnion epithelial cells. In summary, for the first time, we demonstrate GBS-induced senescence in the fetal membrane and present evidence of IL-1 pathway signaling between the choriodecidua and amnion layer of fetal membrane in a paracrine manner. Further studies will be warranted to understand the pathogenesis of adverse pregnancy outcomes associated with GBS infection and develop therapeutic interventions to mitigate these complications.

Host inflammatory dynamics reveal placental immune modulation by Group B Streptococcus during pregnancy.

Group B Streptococcus (GBS) is a pathobiont that can ascend to the placenta and cause adverse pregnancy outcomes, in part through production of the toxin ß-hemolysin/cytolysin (ß-h/c). Innate immune cells have been implicated in the response to GBS infection, but the impact of ß-h/c on their response is poorly defined. We show that GBS modulates innate immune cell states by subversion of host inflammation through ß-h/c, allowing worse outcomes. We used an ascending mouse model of GBS infection to measure placental cell state changes over time following infection with a ß-h/c-deficient and isogenic wild type GBS strain. Transcriptomic analysis suggests that ß-h/c-producing GBS elicit a worse phenotype through suppression of host inflammatory signaling in placental macrophages and neutrophils, and comparison of human placental macrophages infected with the same strains recapitulates these results. Our findings have implications for identification of new targets in GBS disease to support host defense against pathogenic challenge.

The serine-rich repeat glycoprotein Srr2 mediates Streptococcus agalactiae interaction with host fibronectin.

BACKGROUND: Group B Streptococcus (GBS) is a commensal of healthy adults and an important pathogen in newborns, the elderly and immunocompromised individuals. GBS displays several virulence factors that promote colonisation and host infection, including the ST-17 strain-specific adhesin Srr2, previously characterised for its binding to fibrinogen. Another common target for bacterial adhesins and for host colonization is fibronectin, a multi-domain glycoprotein found ubiquitously in body fluids, in the extracellular matrix and on the surface of cells. RESULTS: In this study, fibronectin was identified as a novel ligand for the Srr2 adhesin of GBS. A derivative of the ST-17 strain BM110 overexpressing the srr2 gene showed an increased ability to bind fibrinogen and fibronectin, compared to the isogenic wild-type strain. Conversely, the deletion of srr2 impaired bacterial adhesion to both ligands. ELISA assays and surface plasmon resonance studies using the recombinant binding region (BR) form of Srr2 confirmed a direct interaction with fibronectin with an estimated Kd of 92 nM. Srr2-BR variants defective in fibrinogen binding also exhibited no interaction with fibronectin, suggesting that Srr2 binds this ligand through the dock-lock-latch mechanism, previously described for fibrinogen binding. The fibronectin site responsible for recombinant Srr2-BR binding was identified and localised in the central cell-binding domain of the protein. Finally, in the presence of fibronectin, the ability of a Δsrr2 mutant to adhere to human cervico-vaginal epithelial cells was significantly lower than that of the wild-type strain. CONCLUSION: By combining genetic and biochemical approaches, we demonstrate a new role for Srr2, namely interacting with fibronectin. We characterised the molecular mechanism of this interaction and demonstrated that it plays a role in promoting the adhesion of GBS to human cervico-vaginal epithelial cells, further substantiating the role of Srr2 as a factor responsible for the hypervirulence of GBS ST-17 strains. The discovery of the previously undescribed interaction between Srr2 and fibronectin establishes this adhesin as a key factor for GBS colonisation of host tissues.

Molecular characterization, expression and functional analysis of TAK1, TAB1 and TAB2 in Nile tilapia (Oreochromis niloticus).

The MAPK pathway is the common intersection of signal transduction pathways such as inflammation, differentiation and proliferation and plays an important role in the process of antiviral immunity. Streptococcus agalactiae will have a great impact on tilapia aquaculture, so it is necessary to study the immune response mechanism of tilapia to S. agalactiae. In this study, we isolated the cDNA sequences of TAK1, TAB1 and TAB2 from Nile tilapia (Oreochromis niloticus). The TAK1 gene was 3492 bp in length, contained an open reading frame (ORF) of 1809 bp and encoded a polypeptide of 602 amino acids. The cDNA sequence of the TAB1 gene was 4001 bp, and its ORF was 1491 bp, which encoded 497 amino acids. The cDNA sequence of the TAB2 gene was 4792 bp, and its ORF was 2217 bp, encoding 738 amino acids. TAK1 has an S_TKc domain and a coiled coil structure; the TAB1 protein structure contains a PP2C_SIG domain and a conserved PYVDXA/TXF sequence model; and TAB2 contains a CUE domain, a coiled coil domain and a Znf_RBZ domain. Homology analysis showed that TAK1 and TAB1 had the highest homology with Neolamprologus brichardi, and TAB2 had the highest homology with Simochromis diagramma (98.28 %). In the phylogenetic tree, TAK1, TAB1 and TAB2 formed a large branch with other scleractinian fishes. The tissue expression analysis showed that the expression of TAK1, TAB1 and TAB2 was highest in the muscle. The expression of TAK1, TAB1 and TAB2 was significantly induced in most of the tested tissues after stimulation with LPS, Poly I:C and S. agalactiae. The subcellular localization results showed that TAK1 was located in the cytoplasm, and TAB1 and TAB2 had certain distributions in the cytoplasm and nucleus. Coimmunoprecipitation (Co-IP) results showed that TRAF6 did not interact with the TAK1 protein but interacted with TAB2, while TAB1 did not interact with P38γ but interacted with TAK1. There was also an interaction between TAK1 and TAB2.

The Streptococcus agalactiae Exonuclease ExoVII Is Required for Resistance to Exogenous DNA-Damaging Agents.

Streptococcus agalactiae is a human pathogen responsible for severe invasive infections in newborns. In this bacterium, XseB, a part of the ExoVII exonuclease, was shown to be specifically more abundant in the hypervirulent ST-17 strains. In Escherichia coli, ExoVII is associated either with mismatch repair or with recombinational DNA repair and is redundant with other exonucleases. In this study, the biological role of S. agalactiae ExoVII was examined. The ΔexoVII mutant strain was subjected to different DNA-damaging agents, as well as a large set of mutants impaired either in the mismatch repair pathway or in processes of recombinational DNA repair. Our results clarified the role of this protein in Gram-positive bacteria as we showed that ExoVII is not significantly involved in mismatch repair but is involved in bacterial recovery after exposure to exogenous DNA-damaging agents such as ciprofloxacin, UV irradiation, or hydrogen peroxide. We found that ExoVII is more particularly important for resistance to ciprofloxacin, likely as part of the RecF DNA repair pathway. Depending on the tested agent, ExoVII appeared to be fully redundant or nonredundant with another exonuclease, RecJ. The importance of each exonuclease, ExoVII or RecJ, in the process of DNA repair is thus dependent on the considered DNA lesion. IMPORTANCE This study examined the role of the ExoVII exonuclease of Streptococcus agalactiae within the different DNA repair processes. Our results concluded that ExoVII is involved in bacterial recovery after exposure to different exogenous DNA-damaging agents but not in the mismatch repair pathway. We found that ExoVII is particularly important for resistance to ciprofloxacin, likely as part of the RecF DNA repair pathway.

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.

High Glucose Promotes Inflammation and Weakens Placental Defenses against E. coli and S. agalactiae Infection: Protective Role of Insulin and Metformin.

Placentas from gestational diabetes mellitus (GDM) patients undergo significant metabolic and immunologic adaptations due to hyperglycemia, which results in an exacerbated synthesis of proinflammatory cytokines and an increased risk for infections. Insulin or metformin are clinically indicated for the treatment of GDM; however, there is limited information about the immunomodulatory activity of these drugs in the human placenta, especially in the context of maternal infections. Our objective was to study the role of insulin and metformin in the placental inflammatory response and innate defense against common etiopathological agents of pregnancy bacterial infections, such as E. coli and S. agalactiae, in a hyperglycemic environment. Term placental explants were cultivated with glucose (10 and 50 mM), insulin (50-500 nM) or metformin (125-500 µM) for 48 h, and then they were challenged with live bacteria (1 × 105 CFU/mL). We evaluated the inflammatory cytokine secretion, beta defensins production, bacterial count and bacterial tissue invasiveness after 4-8 h of infection. Our results showed that a GDM-associated hyperglycemic environment induced an inflammatory response and a decreased beta defensins synthesis unable to restrain bacterial infection. Notably, both insulin and metformin exerted anti-inflammatory effects under hyperglycemic infectious and non-infectious scenarios. Moreover, both drugs fortified placental barrier defenses, resulting in reduced E. coli counts, as well as decreased S. agalactiae and E. coli invasiveness of placental villous trees. Remarkably, the double challenge of high glucose and infection provoked a pathogen-specific attenuated placental inflammatory response in the hyperglycemic condition, mainly denoted by reduced TNF-α and IL-6 secretion after S. agalactiae infection and by IL-1ß after E. coli infection. Altogether, these results suggest that metabolically uncontrolled GDM mothers develop diverse immune placental alterations, which may help to explain their increased vulnerability to bacterial pathogens.

Using Azadirachta indica protein hydrolysate as a plant protein in Nile tilapia (Oreochromis niloticus) diet: Effects on the growth, economic efficiency, antioxidant-immune response and resistance to Streptococcus agalactiae.

A feeding trial for 90 days was conducted on Nile tilapia (Oreochromis niloticus) (average weight: 25.50 ± 0.05 g) to evaluate the effect of dietary inclusion of Azadirachta indica seed protein hydrolysate (AIPH). The evaluation included the impact on the growth metrics, economic efficiency, antioxidant potential, hemato-biochemical indices, immune response, and histological architectures. A total of 250 fish were randomly distributed in five treatments (n = 50) and received diets included with five levels of AIPH (%): 0 (control diet, AIPH0), 2 (AIPH2), 4 (AIPH4), 6 (AIPH6) or 8 (AIPH8), where AIPH partially replace fish meal by 0, 8.7%, 17.4%, 26.1%, and 34.8%, respectively. After the feeding trial, a pathogenic bacterium (Streptococcus agalactiae, 1.5 × 108 CFU/mL) was intraperitoneally injected into the fish and the survival rate was recorded. The results elucidated that AIPH-included diets significantly (p < 0.05) enhanced the growth indices (final body weight, total feed intake, total body weight gain, and specific growth rate) and intestinal morpho-metrics (villous width, length, muscular coat thickness, and goblet cells count) in comparison to the control diet, with the AIPH8 diet recording the highest values. Dietary AIPH inclusion significantly improved (p < 0.05) the economic efficacy indicated by reduced feed cost/kg gain and increased performance index. The fish fed on the AIPH diets had noticeably significantly higher (p < 0.05) protein profile variables (total proteins and globulin) and antioxidant capabilities (superoxide dismutase and total antioxidant capacity) than the AIPH0 group. The dietary inclusion of AIPH significantly (p < 0.05) boosted the haematological parameters (haemoglobin, packed cell volume %, and counts of red blood cells and white blood cells) and immune indices (serum bactericidal activity %, antiprotease activity, and immunoglobulin M level) in a concentration-dependent manner. The blood glucose and malondialdehyde levels were significantly (p < 0.05) lowered by dietary AIPH (2%-8%). The albumin level and hepatorenal functioning parameters (aspartate aminotransferase, alanine aminotransferase, and creatinine) were not significantly (p > 0.05) altered by AIPH diets. Additionally, AIPH diets did not adversely alter the histology of the hepatic, renal or splenic tissues with moderately activated melano-macrophage centres. The mortality rate among S. agalactiae-infected fish declined as dietary AIPH levels rose, where the highest survival rate (86.67%) was found in the AIPH8 group (p < 0.05). Based on the broken line regression model, our study suggests using dietary AIPH at the optimal level of 6%. Overall, dietary AIPH inclusion enhanced the growth rate, economic efficiency, health status, and resistance of Nile tilapia to the S. agalactiae challenge. These beneficial impacts can help the aquaculture sector to be more sustainable.

The Copper Resistome of Group B Streptococcus Reveals Insight into the Genetic Basis of Cellular Survival during Metal Ion Stress.

In bacteria, copper (Cu) can support metabolic processes as an enzymatic cofactor but can also cause cell damage if present in excess, leading to intoxication. In group B Streptococcus (GBS), a system for control of Cu efflux based on the prototypical cop operon supports survival during Cu stress. In some other bacteria, genetic systems additional to the cop operon are engaged during Cu stress and also contribute to the management of cellular Cu homeostasis. Here, we examined genetic systems beyond the cop operon in GBS for regions that contribute to survival of GBS in Cu stress using a forward genetic screen and probe of the entire bacterial genome. A high-density mutant library, generated using pGh9-ISS1, was used to expose GBS to Cu stress and compare it to nonexposed controls en masse. Eight genes were identified as essential for GBS survival in Cu stress, whereas five genes constrained GBS growth in Cu stress. The genes encode varied factors including enzymes for metabolism, cell wall synthesis, transporters, and cell signaling factors. Targeted mutation of the genes validated their roles in GBS resistance to Cu stress. Excepting copA, the genes identified are new to the area of bacterial metal ion intoxication. We conclude that a discrete and limited suite of genes beyond the cop operon in GBS contributes to a repertoire of mechanisms used to survive Cu stress in vitro and achieve cellular homeostasis. IMPORTANCE Genetic systems for copper (Cu) homeostasis in bacteria, including streptococci, are vital to survive metal ion stress. Genetic systems that underpin survival of GBS during Cu stress, beyond the archetypal cop operon for Cu management, are undefined. We show that Streptococcus resists Cu intoxication by utilizing a discrete and limited suite of genes beyond the cop operon, including several genes that are new to the area of bacterial cell metal ion homeostasis. The Cu resistome of GBS defined here enhances our understanding of metal ion homeostasis in GBS.

Streptococcus agalactiae imports spermidine by a member of the amino acid/polyamine antiporter family to endure citric acid stress at the vaginal pH.

Polyamines bind to various cellular components, such as nucleic acids, phospholipids, proteins and nucleotides. They are involved in the virulence and protection against physiological stresses of several bacterial species. Streptococcus agalactiae is able to colonize the vaginal tract of asymptomatic pregnant women and to resist, by an as yet poorly characterized mechanism, pH 4.0, the low physiological pH of this environment. We identified a transporter of the amino acid/polyamine antiporter family (SAK_1604 in strain A909) that shares 39.8 % similar amino acids with CadB and 34.7 % with PotE, two transporters implicated in acid resistance in Escherichia coli. We found that sak_1604 is overexpressed in the presence of spermidine and during citric acid stress at the vaginal pH, but not during lactic acid or HCl stresses at the same pH or during a sodium citrate stress at pH 7.4. Dihydrogen citrate is the predominant form of citric acid at pH 4.0. Using a deletion mutant, we proved that SAK_1604 is involved in the survival of S. agalactiae during citric acid stress at pH 4.0 in the presence of spermidine, and we showed by TLC analysis that it is involved in spermidine transport in these conditions. Our data open new perspectives on the comprehension of the molecular mechanisms allowing S. agalactiae to survive at the physiological pH of the vagina and on the unsuspected role of an ionic form of citric acid.

Galacto-Oligosaccharide Supplementation Modulates Pathogen-Commensal Competition between Streptococcus agalactiae and Streptococcus salivarius.

The members of the infant microbiome are governed by feeding method (breastmilk vs. formula). Regardless of the source of nutrition, a competitive growth advantage can be provided to commensals through prebiotics - either human milk oligosaccharides (HMOs) or plant oligosaccharides that are supplemented into formula. To characterize how prebiotics modulate commensal - pathogen interactions, we have designed and studied a minimal microbiome where a pathogen, Streptococcus agalactiae engages with a commensal, Streptococcus salivarius. We discovered that while S. agalactiae suppresses the growth of S. salivarius via increased lactic acid production, galacto-oligosaccharides (GOS) supplementation reverses the effect. This result has major implications in characterizing how single species survive in the gut, what niche they occupy, and how they engage with other community members.

Global gene expression analysis of Streptococcus agalactiae at exponential growth phase.

A comparative overview of the global gene expression levels of S. agalactiae reference strain NEM316 at the exponential growth phase was done through RNA-sequencing. The expression levels of 47 genes potentially linked to virulence evidenced that: i) the major nuclease, GBS_RS03720/gbs0661, presented higher mean expression values than the remainder of DNase genes; ii) the genetic pilus island PI-2a genes presented higher mean expression values than PI-1 coding genes; and, iii) three virulence-associated genes ranked among the top-100 most expressed genes (GBS_RS07760, GBS_RS09445 and GBS_RS03485). Among this top-100, genes encoding proteins involved in "Translation, ribosomal structure and biogenesis" represented 46%. Curiously, genes with no assigned function were grouped in the category of highly expressed genes. As very little is known about the molecular mechanisms behind the release of DNases, preliminary assays were developed to understand whether direct DNA exposure would affect gene expression at the exponential growth phase. No differentially expressed genes were detected, indicating that follow-up studies are needed to disclose the complex molecular pathways (and stimuli) triggering the release of DNases. In general, our insights on the global expression levels of NEM316 at exponential growth phase with and without DNA exposure should open novel research lines to decipher S. agalactiae puzzling adaptation and virulence mechanisms, such as DNase production.

Dual actions of group B Streptococcus capsular sialic acid provide resistance to platelet-mediated antimicrobial killing.

Circulating platelets have important functions in thrombosis and in modulating immune and inflammatory responses. However, the role of platelets in innate immunity to bacterial infection is largely unexplored. While human platelets rapidly kill Staphylococcus aureus, we found the neonatal pathogen group B Streptococcus (GBS) to be remarkably resistant to platelet killing. GBS possesses a capsule polysaccharide (CPS) with terminal α2,3-linked sialic acid (Sia) residues that mimic a common epitope present on the human cell surface glycocalyx. A GBS mutant deficient in CPS Sia was more efficiently killed by human platelets, thrombin-activated platelet releasate, and synthetic platelet-associated antimicrobial peptides. GBS Sia is known to bind inhibitory Sia-recognizing Ig superfamily lectins (Siglecs) to block neutrophil and macrophage activation. We show that human platelets also express high levels of inhibitory Siglec-9 on their surface, and that GBS can engage this receptor in a Sia-dependent manner to suppress platelet activation. In a mouse i.v. infection model, antibody-mediated platelet depletion increased susceptibility to platelet-sensitive S. aureus but did not alter susceptibility to platelet-resistant GBS. Elimination of murine inhibitory Siglec-E partially reversed platelet suppression in response to GBS infection. We conclude that GBS Sia has dual roles in counteracting platelet antimicrobial immunity: conferring intrinsic resistance to platelet-derived antimicrobial components and inhibiting platelet activation through engagement of inhibitory Siglecs. We report a bacterial virulence factor for evasion of platelet-mediated innate immunity.

Functional Insights into the High-Molecular-Mass Penicillin-Binding Proteins of Streptococcus agalactiae Revealed by Gene Deletion and Transposon Mutagenesis Analysis.

High-molecular-mass penicillin-binding proteins (PBPs) are enzymes that catalyze the biosynthesis of bacterial cell wall peptidoglycan. The Gram-positive bacterial pathogen Streptococcus agalactiae (group B streptococcus [GBS]) produces five high-molecular-mass PBPs, namely, PBP1A, PBP1B, PBP2A, PBP2B, and PBP2X. Among these, only PBP2X is essential for cell viability, whereas the other four PBPs are individually dispensable. The biological function of the four nonessential PBPs is poorly characterized in GBS. We deleted the pbp1a, pbp1b, pbp2a, and pbp2b genes individually from a genetically well-characterized serotype V GBS strain and studied the phenotypes of the four isogenic mutant strains. Compared to the wild-type parental strain, (i) none of the pbp isogenic mutant strains had a significant growth defect in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY) rich medium, (ii) isogenic mutant Δpbp1a and Δpbp1b strains had significantly increased susceptibility to penicillin and ampicillin, and (iii) isogenic mutant Δpbp1a and Δpbp2b strains had significantly longer chain lengths. Using saturated transposon mutagenesis and transposon insertion site sequencing, we determined the genes essential for the viability of the wild-type GBS strain and each of the four isogenic pbp deletion mutant strains in THY rich medium. The pbp1a gene is essential for cell viability in the pbp2b deletion background. Reciprocally, pbp2b is essential in the pbp1a deletion background. Moreover, the gene encoding RodA, a peptidoglycan polymerase that works in conjunction with PBP2B, is also essential in the pbp1a deletion background. Together, our results suggest functional overlap between PBP1A and the PBP2B-RodA complex in GBS cell wall peptidoglycan biosynthesis. IMPORTANCE High-molecular-mass penicillin-binding proteins (HMM PBPs) are enzymes required for bacterial cell wall biosynthesis. Bacterial pathogen group B streptococcus (GBS) produces five distinct HMM PBPs. The biological functions of these proteins are not well characterized in GBS. In this study, we performed a comprehensive deletion analysis of genes encoding HMM PBPs in GBS. We found that deleting certain PBP-encoding genes altered bacterial susceptibility to beta-lactam antibiotics, cell morphology, and the essentiality of other enzymes involved in cell wall peptidoglycan synthesis. The results of our study shed new light on the biological functions of PBPs in GBS.

Copper Intoxication in Group B Streptococcus Triggers Transcriptional Activation of the cop Operon That Contributes to Enhanced Virulence during Acute Infection.

Bacteria can utilize copper (Cu) as a trace element to support cellular processes; however, excess Cu can intoxicate bacteria. Here, we characterize the cop operon in group B streptococcus (GBS) and establish its role in evasion of Cu intoxication and the response to Cu stress on virulence. Growth of a GBS mutant deficient in the copA Cu exporter was severely compromised under Cu stress conditions. GBS survival of Cu stress reflected a mechanism of CopY derepression of the CopA efflux system. However, neither mutant was attenuated for intracellular survival in macrophages. Analysis of global transcriptional responses to Cu by RNA sequencing (RNA-seq) revealed a stress signature encompassing homeostasis of multiple metals. Genes induced by Cu stress included putative metal transporters for manganese import, whereas a system for iron export was repressed. In addition, copA promoted the ability of GBS to colonize the blood, liver, and spleen of mice following disseminated infection. Together, these findings show that GBS copA mediates resistance to Cu intoxication via regulation by the Cu-sensing transcriptional repressor copY. Cu stress responses in GBS reflect a transcriptional signature that heightens virulence and represents an important part of the bacterium's ability to survive in different environments. IMPORTANCE Understanding how bacteria manage cellular levels of metal ions, such as copper, helps to explain how microbial cells can survive in different stressful environments. We show the opportunistic pathogen group B streptococcus (GBS) achieve homeostasis of intracellular copper through the activities of the genes that comprise the cop operon, and we describe how this helps GBS survive in stressful environments, including in the mammalian host during systemic disseminated infection.