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.

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.

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.

Regulation of PhoB on biofilm formation and hemolysin gene hlyA and ciaR of Streptococcus agalactiae.

PhoB is a response regulator protein that plays a key role in the PhoBR two-component signal transduction system. In this study, we used transcriptome and proteomics techniques to evaluate the detect the gene network regulated by PhoB of Streptococcus agalactiae. The results showed that expression of biofilm formation and virulence-related genes were changed after phoB deficiency. Crystal violet and CLSM assay confirmed that the deletion of the phoB increased the thickness of S. agalactiae biofilm. The results of lacZ reporter and the bacterial one-hybridization method showed that PhoB could directly bind to the promoter regions of hemolysin A and ciaR genes but not to the promoter regions of cylE and hemolysin III. Through the construction of an 18-base pair deoxyribose nucleic acid (DNA) random fragment library and the bacterial one-hybridization system, it was found that the conservative sequence of PhoB binding was TTGGAGAA(G/T). Our research has uncovered the virulence potential of the PhoBR two-component system of S. agalactiae. The findings of this study provide the theoretical foundation for in-depth research on the pathogenic mechanism of S. agalactiae.

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.

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.

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.

C9 regulates the complement-mediated cell lysis in association with CD59 to resist bacterial infection in a primary animal.

Complement component 9 (C9), as an essential component of terminal membrane attack complex of complement system, plays an important role in innate immune defense. However, the function and regulatory mechanism of C9 in the antimicrobial immune response of teleost fish remain unclear. In this study, the open reading frame of Nile tilapia (Oreochromis niloticus) C9 (OnC9) gene was amplified. The mRNA and protein expression of OnC9 were significantly changed upon infection with Streptococcus agalactiae and Aeromonas hydrophila in vivo and in vitro. Upon bacterial challenge, the OnC9 knockdown could lead to rapid proliferation of the pathogenic bacteria, ultimately resulting in tilapia death. However, the phenotype was rescued by re-injection of OnC9, which restored the healthy status of the knockdown tilapia. Further, the OnC9 was an essential component in complement-mediated cell lysis and associated with OnCD59 to regulate the efficiency of lysis. Overall, this study indicates that OnC9 is involved in host defense against bacterial infection, and provides a valuable reference for further exploration of the molecular regulatory mechanism of C9 in innate immune defense in a primary animal.

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.

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 c-di-AMP-binding protein CbpB modulates the level of ppGpp alarmone in Streptococcus agalactiae.

Cyclic di-AMP is an essential signalling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.

Antibacterial activity and metabolomic analysis of linalool against bovine mastitis pathogen Streptococcus agalactiae.

Streptococcus agalactiae is among the major causative pathogens of bovine mastitis, as well as crucial pathogen leading to human morbidity and mortality. Being a promising natural antibacterial agent, linalool has been broadly applied in medicine and food processing. However, its antibacterial effect against S. agalactiae has barely been elucidated. This study is the first to investigate the antibacterial activity and action mechanism of linalool against S. agalactiae causing bovine mastitis. Linalool exhibited significant antibacterial activity against S. agalactiae, with an inhibition zone diameter of 23 mm and a minimum inhibitory concentration of 1.875 µL/mL. In addition, linalool damaged cell structural integrity of S. agalactiae, leading to the leakage of intracellular components (alkaline phosphatase, nucleic acids and protein). Linalool also exhibited a scavenging effect on biofilm. Moreover, untargeted metabolomics analysis revealed that linalool stress substantially disrupted intracellular metabolism of S. agalactiae. Linalool caused energy metabolism disorder, and obstructed nucleic acid synthesis in S. agalactiae. Furthermore, downregulation of amino acids (e.g., proline, alanine) and upregulation of saturated fatty acids provide strong evidence for linalool induced cell wall and membrane damage. Overall, linalool exhibited strong antibacterial activity against S. agalactiae by destroying the cell structure and disrupting intracellular metabolism. This study provides a new insight and theoretical foundation for linalool application in preventing S. agalactiae infection.

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.

The Role of Regulator Catabolite Control Protein A (CcpA) in Streptococcus agalactiae Physiology and Stress Response.

Streptococcus agalactiae is a leading cause of infections in neonates. This opportunistic pathogen colonizes the vagina, where it has to cope with acidic pH and hydrogen peroxide produced by lactobacilli. Thus, in the host, this bacterium possesses numerous adaptation mechanisms in which the pleiotropic regulators play a major role. The transcriptional regulator CcpA (catabolite control protein A) has previously been shown to be the major regulator involved in carbon catabolite repression in Gram-positive bacteria but is also involved in other functions. By transcriptomic analysis, we characterized the CcpA-dependent gene regulation in S. agalactiae. Approximately 13.5% of the genome of S. agalactiae depends on CcpA for regulation and comprises genes involved in sugar uptake and fermentation, confirming the role of CcpA in carbon metabolism. We confirmed by electrophoretic mobility shift assays (EMSAs) that the DNA binding site called cis-acting catabolite responsive element (cre) determined for other streptococci was effective in S. agalactiae. We also showed that CcpA is of capital importance for survival under acidic and oxidative stresses and is implicated in macrophage survival by regulating several genes putatively or already described as involved in stress response. Among them, we focused our study on SAK_1689, which codes a putative UspA protein. We demonstrated that SAK_1689, highly downregulated by CcpA, is overexpressed under oxidative stress conditions, this overexpression being harmful for the bacterium in a ΔccpA mutant. IMPORTANCE Streptococcus agalactiae is a major cause of disease burden leading to morbidity and mortality in neonates worldwide. Deciphering its adaptation mechanisms is essential to understand how this bacterium manages to colonize its host. Here, we determined the regulon of the pleiotropic regulator CcpA in S. agalactiae. Our findings reveal that CcpA is not only involved in carbon catabolite repression, but is also important for acidic and oxidative stress resistance and survival in macrophages.

A novel phage-encoded endolysin EN534-C active against clinical strain Streptococcus agalactiae GBS.

Streptococcus agalactiae (Group B Streptococcus, GBS) is primarily known as a major neonatal pathogen. In adults, these bacteria often colonize the gastrointestinal and urogenital tracts. Treatment of infections using antibiotics is often complicated by recurrences caused by multi-resistant streptococci. Endolysin EN534 from prophage A2 of human isolate Streptococcus agalactiae KMB-534 has a modular structure consisting of two terminal catalytic domains, amidase_3 and CHAP, and one central binding domain, LysM. The EN534 gene was cloned into an expression vector, and the corresponding recombinant protein EN534-C was expressed in Escherichia coli in a soluble form and isolated by affinity chromatography. The lytic activity of this endolysin was tested on cell wall substrates from different GBS serotypes, B. subtilis, L. jensenii, and E. coli. The enzyme lysed streptococci, but not beneficial vaginal lactobacilli. The isolated protein is stable in a temperature range of 20-37 °C. Calcium ions enhanced the activity of the enzyme in the pH range from 5.0 to 8.0. The exolytic activity of EN534-C was observed by time-lapse fluorescence microscopy on a S. agalactiae CCM 6187 substrate. Recombinant endolysin EN534-C may have the potential to become an antimicrobial agent for the treatment of S. agalactiae infections.

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.

Mast cell-derived factor XIIIA contributes to sexual dimorphic defense against group B streptococcal infections.

Invasive bacterial infections remain a major cause of human morbidity. Group B streptococcus (GBS) are Gram-positive bacteria that cause invasive infections in humans. Here, we show that factor XIIIA-deficient (FXIIIA-deficient) female mice exhibited significantly increased susceptibility to GBS infections. Additionally, female WT mice had increased levels of FXIIIA and were more resistant to GBS infection compared with isogenic male mice. We observed that administration of exogenous FXIIIA to male mice increased host resistance to GBS infection. Conversely, administration of a FXIIIA transglutaminase inhibitor to female mice decreased host resistance to GBS infection. Interestingly, male gonadectomized mice exhibited decreased sensitivity to GBS infection, suggesting a role for gonadal androgens in host susceptibility. FXIIIA promoted GBS entrapment within fibrin clots by crosslinking fibronectin with ScpB, a fibronectin-binding GBS surface protein. Thus, ScpB-deficient GBS exhibited decreased entrapment within fibrin clots in vitro and increased dissemination during systemic infections. Finally, using mice in which FXIIIA expression was depleted in mast cells, we observed that mast cell-derived FXIIIA contributes to host defense against GBS infection. Our studies provide insights into the effects of sexual dimorphism and mast cells on FXIIIA expression and its interactions with GBS adhesins that mediate bacterial dissemination and pathogenesis.

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.

NADPH oxidase-derived reactive oxygen species production activates the ERK1/2 pathway in neutrophil extracellular traps formation by Streptococcus agalactiae isolated from clinical mastitis bovine.

Streptococcus agalactiae (S. agalactiae) continues to be challenging for milk quality in some countries and leads to huge economic losses. A large number of neutrophils are recruited into inflammatory foci when S. agalactiae infection occurs, and most studies have focused on the interaction between neutrophil extracellular traps (NETs) and this bacterium in the context of human pathogenicity. However, there is little information on the NET formation mechanism induced by S. agalactiae in the context of bovine mastitis. Here, neutrophils isolated from BALB/c mice were infected with S. agalactiae SAG-FX17, and NET formation was evaluated. SAG-FX17 could induce NADPH oxidase-derived reactive oxygen species (NOX-ROS)-dependent NET formation, and 21.8% of bacteria could be eliminated by NETs via NET DNA and associated proteins. SAG-FX17 could induce the phosphorylation of p38 MAPK, ERK1/2 MAPK, and JNK/SAPK in neutrophils. However, only ERK1/2 MAPK was shown to play an important role in SAG-FX17-induced NET formation. Importantly, NOX-ROS production occurs upstream of ERK1/2 MAPK activation and then induces NET release. ERK1/2 MAPK phosphorylation can, in turn, enhance NOX-ROS generation, which further contributes to NET release and bacterial elimination. This study provides evidence of the molecular mechanism underlying serotype Ia S. agalactiae SAG-FX17-induced NET formation and the interaction between bacteria and NETs, and these findings will increase our knowledge about bacterial mastitis in dairy cattle and contribute to the prevention and clinical treatment of bovine mastitis.

Role of MUC5B during Group B Streptococcal Vaginal Colonization.

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