An extracellular bacterial pathogen modulates host metabolism to regulate its own sensing and proliferation.

Successful infection depends on the ability of the pathogen to gain nutrients from the host. The extracellular pathogenic bacterium group A Streptococcus (GAS) causes a vast array of human diseases. By using the quorum-sensing sil system as a reporter, we found that, during adherence to host cells, GAS delivers streptolysin toxins, creating endoplasmic reticulum stress. This, in turn, increases asparagine (ASN) synthetase expression and the production of ASN. The released ASN is sensed by the bacteria, altering the expression of ∼17% of GAS genes of which about one-third are dependent on the two-component system TrxSR. The expression of the streptolysin toxins is strongly upregulated, whereas genes linked to proliferation are downregulated in ASN absence. Asparaginase, a widely used chemotherapeutic agent, arrests GAS growth in human blood and blocks GAS proliferation in a mouse model of human bacteremia. These results delineate a pathogenic pathway and propose a therapeutic strategy against GAS infections.

Unmutated but T cell dependent IgM antibodies targeting Streptococcus suis play an essential role in bacterial clearance.

Streptococcus suis serotype 2 is an important encapsulated bacterial swine pathogen and zoonotic agent for which no effective vaccine exists. The interaction with B cells and the humoral response against S. suis are poorly understood despite their likely relevance for a potential vaccine. We evaluated germinal center (GC) B cell kinetics, as well as the production and role of S. suis-specific antibodies following infections in a mouse model. We found that mice infected with S. suis developed GC that peaked 13-21 days post-infection. GC further increased and persisted upon periodic reinfection that mimics real life conditions in swine farms. Anti-S. suis IgM and several IgG subclasses were produced, but antibodies against the S. suis capsular polysaccharide (CPS) were largely IgM. Interestingly, depletion of total IgG from the wild-type mice sera had no effect on bacterial killing by opsonophagocytosis in vitro. Somatic hypermutation and isotype switching were dispensable for controlling the infection or anti-CPS IgM production. However, T cell-deficient (Tcrb-/-) mice were unable to control bacteremia, produce optimal anti-CPS IgM titers, or elicit antibodies with opsonophagocytic activity. SAP deficiency, which prevents GC formation but not extrafollicular B cell responses, ablated anti S. suis-IgG production but maintained IgM production and eliminated the infection. In contrast, B cell deficient mice were unable to control bacteremia. Collectively, our results indicate that the antibody response plays a large role in immunity against S. suis, with GC-independent but T cell-dependent germline IgM being the major effective antibody specificities. Our results further highlight the importance IgM, and potentially anti-CPS antibodies, in clearing S. suis infections and provide insight for future development of S. suis vaccines.

Genomic and functional determinants of host spectrum in Group B Streptococcus.

Group B Streptococcus (GBS) is a major human and animal pathogen that threatens public health and food security. Spill-over and spill-back between host species is possible due to adaptation and amplification of GBS in new niches but the evolutionary and functional mechanisms underpinning those phenomena are poorly known. Based on analysis of 1,254 curated genomes from all major GBS host species and six continents, we found that the global GBS population comprises host-generalist, host-adapted and host-restricted sublineages, which are found across host groups, preferentially within one host group, or exclusively within one host group, respectively, and show distinct levels of recombination. Strikingly, the association of GBS genomes with the three major host groups (humans, cattle, fish) is driven by a single accessory gene cluster per host, regardless of sublineage or the breadth of host spectrum. Moreover, those gene clusters are shared with other streptococcal species occupying the same niche and are functionally relevant for host tropism. Our findings demonstrate (1) the heterogeneity of genome plasticity within a bacterial species of public health importance, enabling the identification of high-risk clones; (2) the contribution of inter-species gene transmission to the evolution of GBS; and (3) the importance of considering the role of animal hosts, and the accessory gene pool associated with their microbiota, in the evolution of multi-host bacterial pathogens. Collectively, these phenomena may explain the adaptation and clonal expansion of GBS in animal reservoirs and the risk of spill-over and spill-back between animals and humans.

Type II and IV toxin-antitoxin systems coordinately stabilize the integrative and conjugative element of the ICESa2603 family conferring multiple drug resistance in Streptococcus suis.

Integrative and conjugative elements (ICEs) play a vital role in bacterial evolution by carrying essential genes that confer adaptive functions to the host. Despite their importance, the mechanism underlying the stable inheritance of ICEs, which is necessary for the acquisition of new traits in bacteria, remains poorly understood. Here, we identified SezAT, a type II toxin-antitoxin (TA) system, and AbiE, a type IV TA system encoded within the ICESsuHN105, coordinately promote ICE stabilization and mediate multidrug resistance in Streptococcus suis. Deletion of SezAT or AbiE did not affect the strain's antibiotic susceptibility, but their duple deletion increased susceptibility, mainly mediated by the antitoxins SezA and AbiEi. Further studies have revealed that SezA and AbiEi affect the genetic stability of ICESsuHN105 by moderating the excision and extrachromosomal copy number, consequently affecting the antibiotic resistance conferred by ICE. The DNA-binding proteins AbiEi and SezA, which bind palindromic sequences in the promoter, coordinately modulate ICE excision and extracellular copy number by binding to sequences in the origin-of-transfer (oriT) and the attL sites, respectively. Furthermore, AbiEi negatively regulates the transcription of SezAT by binding directly to its promoter, optimizing the coordinate network of SezAT and AbiE in maintaining ICESsuHN105 stability. Importantly, SezAT and AbiE are widespread and conserved in ICEs harbouring diverse drug-resistance genes, and their coordinated effects in promoting ICE stability and mediating drug resistance may be broadly applicable to other ICEs. Altogether, our study uncovers the TA system's role in maintaining the genetic stability of ICE and offers potential targets for overcoming the dissemination and evolution of drug resistance.

Streptococcus dysgalactiae subsp. equisimilis infection and its intersection with Streptococcus pyogenes.

SUMMARYStreptococcus dysgalactiae subsp. equisimilis (SDSE) is an increasingly recognized cause of disease in humans. Disease manifestations range from non-invasive superficial skin and soft tissue infections to life-threatening streptococcal toxic shock syndrome and necrotizing fasciitis. Invasive disease is usually associated with co-morbidities, immunosuppression, and advancing age. The crude incidence of invasive disease approaches that of the closely related pathogen, Streptococcus pyogenes. Genomic epidemiology using whole-genome sequencing has revealed important insights into global SDSE population dynamics including emerging lineages and spread of anti-microbial resistance. It has also complemented observations of overlapping pathobiology between SDSE and S. pyogenes, including shared virulence factors and mobile gene content, potentially underlying shared pathogen phenotypes. This review provides an overview of the clinical and genomic epidemiology, disease manifestations, treatment, and virulence determinants of human infections with SDSE with a particular focus on its overlap with S. pyogenes. In doing so, we highlight the importance of understanding the overlap of SDSE and S. pyogenes to inform surveillance and disease control strategies.

Phosphorylation of GntR reduces Streptococcus suis oxidative stress resistance and virulence by inhibiting NADH oxidase transcription.

GntR transcription factor of Streptococcus suis serotype 2 (SS2) is a potential substrate protein of STK, but the regulation mechanisms of GntR phosphorylation are still unclear. This study confirmed that STK phosphorylated GntR in vivo, and in vitro phosphorylation experiments showed that STK phosphorylated GntR at Ser-41. The phosphomimetic strain (GntR-S41E) had significantly reduced lethality in mice and reduced bacterial load in the blood, lung, liver, spleen, and brain of infected mice compared to wild-type (WT) SS2. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments demonstrated that the promoter of nox was bound by GntR. The phosphomimetic protein GntR-S41E cannot bind to the promoter of nox, and the nox transcription levels were significantly reduced in the GntR-S41E mutant compared to WT SS2. The virulence in mice and the ability to resist oxidative stress of the GntR-S41E strain were restored by complementing transcript levels of nox. NOX is an NADH oxidase that catalyzes the oxidation of NADH to NAD+ with the reduction of oxygen to water. We found that NADH is likely accumulated under oxidative stress in the GntR-S41E strain, and higher NADH levels resulted in increased amplified ROS killing. In total, we report GntR phosphorylation could inhibit the transcription of nox, which impaired the ability of SS2 to resist oxidative stress and virulence.

M proteins of group A Streptococcus bind hyaluronic acid via arginine-arginine/serine-arginine motifs.

Tissue injury, including extracellular matrix (ECM) degradation, is a hallmark of group A Streptococcus (GAS) skin infection and is partially mediated by M proteins which possess lectin-like properties. Hyaluronic acid is a glycosaminoglycan enriched in the cutaneous ECM, yet an interaction with M proteins has yet to be explored. This study revealed that hyaluronic acid binding was conserved across phylogenetically diverse M proteins, mediated by RR/SR motifs predominantly localized in the C repeat region. Keratinocyte wound healing was decreased through the recruitment of hyaluronic acid by M proteins in an M type-specific manner. GAS strains 5448 (M1 serotype) and ALAB49 (M53 serotype) also bound hyaluronic acid via M proteins, but hyaluronic acid could increase bacterial adherence independently of M proteins. The identification of host-pathogen mechanisms that affect ECM composition and cell repair responses may facilitate the development of nonantibiotic therapeutics that arrest GAS disease progression in the skin.

Enolase of Streptococcus suis serotype 2 promotes biomolecular condensation of ribosomal protein SA for HBMECs apoptosis.

BACKGROUND: Ribosomal protein SA (RPSA) of human brain microvascular endothelial cells (HBMECs) can transfer from the cytosol to the cell surface and act as a receptor for some pathogens, including Streptococcus suis serotype 2 (SS2), a zoonotic pathogen causing meningitis in pigs and humans. We previously reported that SS2 virulence factor enolase (ENO) binds to RPSA on the cell surface of HBMECs and induces apoptosis. However, the mechanism that activates RPSA translocation to the cell surface and induces ENO-mediated HBMEC apoptosis is unclear. RESULTS: Here, we show that RPSA localization and condensation on the host cell surface depend on its internally disordered region (IDR). ENO binds to the IDR of RPSA and promotes its interaction with RPSA and vimentin (VIM), which is significantly suppressed after 1,6-Hexanediol (1,6-Hex, a widely used tool to disrupt phase separation) treatment, indicating that ENO incorporation and thus the concentration of RPSA/VIM complexes via co-condensation. Furthermore, increasing intracellular calcium ions (Ca2+) in response to SS2 infection further facilitates the liquid-like condensation of RPSA and aggravates ENO-induced HBMEC cell apoptosis. CONCLUSIONS: Together, our study provides a previously underappreciated molecular mechanism illuminating that ENO-induced RPSA condensation activates the migration of RPSA to the bacterial cell surface and stimulates SS2-infected HBMEC death and, potentially, disease progression. This study offers a fresh avenue for investigation into the mechanism by which other harmful bacteria infect hosts via cell surfaces' RPSA.

Large Clusters of Invasive emm49 Group A Streptococcus Identified Within Arizona Health Care Facilities Through Statewide Genomic Surveillance System, 2019-2021.

A statewide genomic surveillance system for invasive Group A Streptococcus was implemented in Arizona in June 2019, resulting in 1046 isolates being submitted for genomic analysis to characterize emm types and identify transmission clusters. Eleven of the 32 identified distinct emm types comprised >80% of samples, with 29.7% of all isolates being typed as emm49 (and its genetic derivative emm151). Phylogenetic analysis initially identified an emm49 genomic cluster of 4 isolates that rapidly expanded over subsequent months (June 2019 to February 2020). Public health investigations identified epidemiologic links with 3 different long-term care facilities, resulting in specific interventions. Unbiased genomic surveillance allowed for identification and response to clusters that would have otherwise remained undetected.

Streptococcus suis Serotype 2 Type IV Secretion Effector SspA-1 Induces Proinflammatory Cytokine Production via TLR2 Endosomal and Type I Interferon Signaling.

The subtilisin-like protease-1 (SspA-1) plays an important role in the pathogenesis of a highly virulent strain of Streptococcus suis 2. However, the mechanism of SspA-1-triggered excessive inflammatory response is still unknown. In this study, we demonstrated that activation of type I IFN signaling is required for SspA-1-induced excessive proinflammatory cytokine production. Further experiments showed that the TLR2 endosomal pathway mediates SspA-1-induced type I IFN signaling and the inflammatory response. Finally, we mapped the major signaling components of the related pathway and found that the TIR adaptor proteins Mal, TRAM, and MyD88 and the downstream activation of IRF1 and IRF7 were involved in this pathway. These results explain the molecular mechanism by which SspA-1 triggers an excessive inflammatory response and reveal a novel effect of type I IFN in S. suis 2 infection, possibly providing further insights into the pathogenesis of this highly virulent S. suis 2 strain.

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.

Dpr-mediated H2O2 resistance contributes to streptococcus survival in a cystic fibrosis airway model system.

The cystic fibrosis (CF) lung environment is conducive to the colonization of bacteria as polymicrobial biofilms, which are associated with poor clinical outcomes for persons with CF (pwCF). Streptococcus spp. are highly prevalent in the CF airway, but its role in the CF lung microbiome is poorly understood. Some studies have shown Streptococcus spp. to be associated with better clinical outcomes for pwCF, while others show that high abundance of Streptococcus spp. is correlated with exacerbations. Our lab previously reported a polymicrobial culture system consisting of four CF-relevant pathogens that can be used to study microbial behavior in a more clinically relevant setting. Here, we use this model system to identify genetic pathways that are important for Streptococcus sanguinis survival in the context of the polymicrobial community. We identified genes related to reactive oxygen species as differentially expressed in S. sanguinis monoculture versus growth of this microbe in the mixed community. Genetic studies identified Dpr as important for S. sanguinis survival in the community. We show that Dpr, a DNA-binding ferritin-like protein, and PerR, a peroxide-responsive transcriptional regulator of Dpr, are important for protecting S. sanguinis from phenazine-mediated toxicity in co-culture with Pseudomonas aeruginosa and when exposed to hydrogen peroxide, both of which mimic the CF lung environment. Characterizing such interactions in a clinically relevant model system contributes to our understanding of microbial behavior in the context of polymicrobial biofilm infections. IMPORTANCE: Streptococcus spp. are recognized as a highly prevalent pathogen in cystic fibrosis (CF) airway infections. However, the role of this microbe in clinical outcomes for persons with CF is poorly understood. Here, we leverage a polymicrobial community system previously developed by our group to model CF airway infections as a tool to investigate a Pseudomonas-Streptococcus interaction involving reactive oxygen species (ROS). We show that protection against ROS is required for Streptococcus sanguinis survival in a clinically relevant polymicrobial system. Using this model system to study interspecies interactions contributes to our broader understanding of the complex role of Streptococcus spp. in the CF lung.

Specific interaction between Group B Streptococcus CC17 hypervirulent clone and phagocytes.

Streptococcus agalactiae also named Group B Streptococcus (GBS) is the most significant pathogen causing invasive infections, such as bacteremia and meningitis, in neonates. Worldwide epidemiological studies have shown that a particular clonal complex (CC) of capsular serotype III, the CC17, is strongly associated with meningitis in neonates and is therefore, designated as the hypervirulent clone. Macrophages are a permissive niche for intracellular bacteria of all GBS clones. In this study, we deciphered the specific interaction of GBS CC17 strains with macrophages. Our study revealed that CC17 strains are phagocytosed at a higher rate than GBS non-CC17 strains by human monocytes and macrophages both in cellular models and in primary cells. CC17-enhanced phagocytosis is due to an initial enhanced-attachment step to macrophages mediated by the CC17-specific surface protein HvgA and the PI-2b pilus (Spb1). We showed that two different inhibitors of scavenger receptors (fucoidan and poly(I)) specifically inhibited CC17 adhesion and phagocytosis while not affecting those of non-CC17 strains. Once phagocytosed, both CC17 and non-CC17 strains remained in a LAMP-1 positive vacuole that ultimately fuses with lysosomes where they can survive at similar rates. Finally, both strains displayed a basal egress which occurs independently from actin and microtubule networks. Our findings provide new insights into the interplay between the hypervirulent GBS CC17 and major players of the host's innate immune response. This enhanced adhesion, leading to increased phagocytosis, could reflect a peculiar capacity of the CC17 lineage to subvert the host immune defenses, establish a niche for persistence or disseminate.

Re-framing the importance of Group B Streptococcus as a gut-resident pathobiont.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive bacterial species that causes disease in humans across the lifespan. While antibiotics are used to mitigate GBS infections, it is evident that antibiotics disrupt human microbiomes (which can predispose people to other diseases later in life), and antibiotic resistance in GBS is on the rise. Taken together, these unintended negative impacts of antibiotics highlight the need for precision approaches for minimizing GBS disease. One possible approach involves selectively depleting GBS in its commensal niches before it can cause disease at other body sites or be transmitted to at-risk individuals. One understudied commensal niche of GBS is the adult gastrointestinal (GI) tract, which may predispose colonization at other body sites in individuals at risk for GBS disease. However, a better understanding of the host-, microbiome-, and GBS-determined variables that dictate GBS GI carriage is needed before precise GI decolonization approaches can be developed. In this review, we synthesize current knowledge of the diverse body sites occupied by GBS as a pathogen and as a commensal. We summarize key molecular factors GBS utilizes to colonize different host-associated niches to inform future efforts to study GBS in the GI tract. We also discuss other GI commensals that are pathogenic in other body sites to emphasize the broader utility of precise de-colonization approaches for mitigating infections by GBS and other bacterial pathogens. Finally, we highlight how GBS treatments could be improved with a more holistic understanding of GBS enabled by continued GI-focused study.

The Streptococcus pyogenes stand-alone regulator RofA exhibits characteristics of a PRD-containing virulence regulator.

Streptococcus pyogenes [group A streptococcus (GAS)] is a human pathogen capable of infecting diverse tissues. To successfully infect these sites, GAS must detect available nutrients and adapt accordingly. The phosphoenolpyruvate transferase system (PTS) mediates carbohydrate uptake and metabolic gene regulation to adapt to the nutritional environment. Regulation by the PTS can occur through phosphorylation of transcriptional regulators at conserved PTS-regulatory domains (PRDs). GAS has several PRD-containing stand-alone regulators with regulons encoding both metabolic genes and virulence factors [PRD-containing virulence regulators (PCVRs)]. One is RofA, which regulates the expression of virulence genes in multiple GAS serotypes. It was hypothesized that RofA is phosphorylated by the PTS in response to carbohydrate levels to coordinate virulence gene expression. In this study, the RofA regulon of M1T1 strain 5448 was determined using RNA sequencing. Two operons were consistently differentially expressed across growth in the absence of RofA; the pilus operon was downregulated, and the capsule operon was upregulated. This correlated with increased capsule production and decreased adherence to keratinocytes. Purified RofA-His was phosphorylated in vitro by PTS proteins EI and HPr, and phosphorylated RofA-FLAG was detected in vivo when GAS was grown in low-glucose C medium. Phosphorylated RofA was not observed when C medium was supplemented 10-fold with glucose. Mutations of select histidine residues within the putative PRDs contributed to the in vivo phosphorylation of RofA, although phosphorylation of RofA was still observed, suggesting other phosphorylation sites exist in the protein. Together, these findings support the hypothesis that RofA is a PCVR that may couple sugar metabolism with virulence regulation.

Manganese uptake by MtsABC contributes to the pathogenesis of human pathogen group A streptococcus by resisting host nutritional immune defenses.

The interplay between host nutritional immune mechanisms and bacterial nutrient uptake systems has a major impact on the disease outcome. The host immune factor calprotectin (CP) limits the availability of essential transition metals, such as manganese (Mn) and zinc (Zn), to control the growth of invading pathogens. We previously demonstrated that the competition between CP and the human pathogen group A streptococcus (GAS) for Zn impacts GAS pathogenesis. However, the contribution of Mn sequestration by CP in GAS infection control and the role of GAS Mn acquisition systems in overcoming host-imposed Mn limitation remain unknown. Using a combination of in vitro and in vivo studies, we show that GAS-encoded mtsABC is a Mn uptake system that aids bacterial evasion of CP-imposed Mn scarcity and promotes GAS virulence. Mn deficiency caused by either the inactivation of mtsC or CP also impaired the protective function of GAS-encoded Mn-dependent superoxide dismutase. Our ex vivo studies using human saliva show that saliva is a Mn-scant body fluid, and Mn acquisition by MtsABC is critical for GAS survival in human saliva. Finally, animal infection studies using wild-type (WT) and CP-/- mice showed that MtsABC is critical for GAS virulence in WT mice but dispensable in mice lacking CP, indicating the direct interplay between MtsABC and CP in vivo. Together, our studies elucidate the role of the Mn import system in GAS evasion of host-imposed metal sequestration and underscore the translational potential of MtsABC as a therapeutic or prophylactic target.

Group A Streptococcus induces lysosomal dysfunction in THP-1 macrophages.

The human-specific bacterial pathogen group A Streptococcus (GAS) is a significant cause of morbidity and mortality. Macrophages are important to control GAS infection, but previous data indicate that GAS can persist in macrophages. In this study, we detail the molecular mechanisms by which GAS survives in THP-1 macrophages. Our fluorescence microscopy studies demonstrate that GAS is readily phagocytosed by macrophages, but persists within phagolysosomes. These phagolysosomes are not acidified, which is in agreement with our findings that GAS cannot survive in low pH environments. We find that the secreted pore-forming toxin Streptolysin O (SLO) perforates the phagolysosomal membrane, allowing leakage of not only protons but also large proteins including the lysosomal protease cathepsin B. Additionally, GAS recruits CD63/LAMP-3, which may contribute to lysosomal permeabilization, especially in the absence of SLO. Thus, although GAS does not inhibit fusion of the lysosome with the phagosome, it has multiple mechanisms to prevent proper phagolysosome function, allowing for persistence of the bacteria within the macrophage. This has important implications for not only the initial response but also the overall functionality of the macrophages, which may lead to the resulting pathologies in GAS infection. Our data suggest that therapies aimed at improving macrophage function may positively impact patient outcomes in GAS infection.

Emergent emm4 group A Streptococcus evidences a survival strategy during interaction with immune effector cells.

The major gram-positive pathogen group A Streptococcus (GAS) is a model organism for studying microbial epidemics as it causes waves of infections. Since 1980, several GAS epidemics have been ascribed to the emergence of clones producing increased amounts of key virulence factors such as streptolysin O (SLO). Herein, we sought to identify mechanisms underlying our recently identified temporal clonal emergence among emm4 GAS, given that emergent strains did not produce augmented levels of virulence factors relative to historic isolates. By creating and analyzing isoallelic strains, we determined that a conserved mutation in a previously undescribed gene encoding a putative carbonic anhydrase was responsible for the defective in vitro growth observed in the emergent strains. We also identified that the emergent strains survived better inside macrophages and killed macrophages at lower rates than the historic strains. Via the creation of isogenic mutant strains, we linked the emergent strain "survival" phenotype to the downregulation of the SLO encoding gene and upregulation of the msrAB operon which encodes proteins involved in defense against extracellular oxidative stress. Our findings are in accord with recent surveillance studies which found a high ratio of mucosal (i.e., pharyngeal) relative to invasive infections among emm4 GAS. Since ever-increasing virulence is unlikely to be evolutionarily advantageous for a microbial pathogen, our data further understanding of the well-described oscillating patterns of virulent GAS infections by demonstrating mechanisms by which emergent strains adapt a "survival" strategy to outcompete previously circulating isolates.

The impact of butyrate on group B Streptococcus-induced intestinal barrier disruption.

Group B Streptococcus (Streptococcus agalactiae; GBS) is a leading cause of neonatal sepsis worldwide. As a pathobiont of the intestinal tract, it is capable of translocating across barriers leading to invasive disease. Neonatal susceptibility to invasive disease stems from immature intestinal barriers. GBS intestinal colonization induces major transcriptomic changes in the intestinal epithelium related to barrier function. Butyrate, a microbial metabolite produced by fermentation of dietary fiber, bolsters intestinal barrier function against enteric pathogens, and these effects can be transferred in utero via the placenta to the developing fetus. Our aim was to determine if butyrate mitigates GBS disruption of intestinal barriers. We used human intestinal epithelial cell (IEC) lines to evaluate the impact of butyrate on GBS-induced cell death and GBS adhesion and invasion. IECs and human fetal tissue-derived enteroids were used to evaluate monolayer permeability. We evaluated the impact of maternal butyrate treatment (mButyrate) using our established mouse model of neonatal GBS intestinal colonization and late-onset sepsis. We found that butyrate reduces GBS-induced cell death, GBS invasion, monolayer permeability, and translocation in vitro. In mice, mButyrate decreases GBS intestinal burden in offspring. Our results demonstrate the importance of bacterial metabolites, such as butyrate, in their potential to bolster epithelial barrier function and mitigate neonatal sepsis risk.IMPORTANCEGroup B Streptococcus (GBS) is a leading cause of neonatal morbidity and mortality. It is a commensal of the intestines that can translocate across barriers leading to sepsis in vulnerable newborns. With the rise in antibiotic-resistant strains and no licensed vaccine, there is an urgent need for preventative strategies. Butyrate, a short-chain fatty acid metabolized in the gut, enhances barrier function against pathogens. Importantly, butyrate is transferred in utero, conferring these benefits to infants. Here, we demonstrate that butyrate reduces GBS colonization and epithelial invasion. These effects were not microbiome-driven, suggesting butyrate directly impacts epithelial barrier function. Our results highlight the potential impact of maternal dietary metabolites, like butyrate, as a strategy to mitigate neonatal sepsis risk.

Spatiotemporal Trends in Group A Streptococcal Pharyngitis in the United States.

BACKGROUND: Group A Streptococcus (GAS) causes an estimated 5.2 million outpatient visits for pharyngitis annually in the United States, with incidence peaking in winter, but the annual spatiotemporal pattern of GAS pharyngitis across the United States is poorly characterized. METHODS: We used outpatient claims data from individuals with private medical insurance between 2010 and 2018 to quantify GAS pharyngitis visit rates across U.S. census regions, subregions, and states. We evaluated seasonal and age-based patterns of geographic spread and the association between school start dates and the summertime upward inflection in GAS visits. RESULTS: The South had the most visits per person (yearly average, 39.11 visits per 1000 people; 95% confidence interval, 36.21-42.01) and the West had the fewest (yearly average, 17.63 visits per 1000 people; 95% confidence interval, 16.76-18.49). Visits increased earliest in the South and in school-age children. Differences in visits between the South and other regions were most pronounced in the late summer through early winter. Visits peaked earliest in central southern states, in December to January, and latest on the coasts, in March. The onset of the rise in GAS pharyngitis visits correlated with, but preceded, average school start times. CONCLUSIONS: The burden and timing of GAS pharyngitis varied across the continental United States, with the South experiencing the highest overall rates and earliest onset and peak in outpatient visits. Understanding the drivers of these regional differences in GAS pharyngitis will help in identifying and targeting prevention measures.