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.

CovS inactivation reduces CovR promoter binding at diverse virulence factor encoding genes in group A Streptococcus.

The control of virulence gene regulator (CovR), also called caspsule synthesis regulator (CsrR), is critical to how the major human pathogen group A Streptococcus fine-tunes virulence factor production. CovR phosphorylation (CovR~P) levels are determined by its cognate sensor kinase CovS, and functional abrogating mutations in CovS can occur in invasive GAS isolates leading to hypervirulence. Presently, the mechanism of CovR-DNA binding specificity is unclear, and the impact of CovS inactivation on global CovR binding has not been assessed. Thus, we performed CovR chromatin immunoprecipitation sequencing (ChIP-seq) analysis in the emm1 strain MGAS2221 and its CovS kinase deficient derivative strain 2221-CovS-E281A. We identified that CovR bound in the promoter regions of nearly all virulence factor encoding genes in the CovR regulon. Additionally, direct CovR binding was observed for numerous genes encoding proteins involved in amino acid metabolism, but we found limited direct CovR binding to genes encoding other transcriptional regulators. The consensus sequence AATRANAAAARVABTAAA was present in the promoters of genes directly regulated by CovR, and mutations of highly conserved positions within this motif relieved CovR repression of the hasA and MGAS2221_0187 promoters. Analysis of strain 2221-CovS-E281A revealed that binding of CovR at repressed, but not activated, promoters is highly dependent on CovR~P state. CovR repressed virulence factor encoding genes could be grouped dependent on how CovR~P dependent variation in DNA binding correlated with gene transcript levels. Taken together, the data show that CovR repression of virulence factor encoding genes is primarily direct in nature, involves binding to a newly-identified DNA binding motif, and is relieved by CovS inactivation. These data provide new mechanistic insights into one of the most important bacterial virulence regulators and allow for subsequent focused investigations into how CovR-DNA interaction at directly controlled promoters impacts GAS pathogenesis.

Hyperphosphorylation of the Group A Streptococcal Control of Virulence Regulator Increases Promoter Occupancy Specifically at Virulence Factor-Encoding Genes.

The control of virulence two-component gene regulatory system (CovRS) is critical to the pathogenesis of many medically important streptococci. In emm1 group A streptococci (GAS), CovR directly binds the promoters of numerous GAS virulence factor-encoding genes. Elimination of CovS phosphatase activity increases CovR phosphorylation (CovR~P) levels and abrogates GAS virulence. Given the emm type-specific diversity of CovRS function, in this study we used chromatin immunoprecipitation sequencing (ChIP-seq) to define global CovR DNA occupancy in the wild-type emm3 strain MGAS10870 (medium CovR~P) and its CovS phosphatase-negative derivative 10870-CovS-T284A (high CovR~P). In the wild-type emm3 strain, 89% of the previously identified emm1 CovR binding sites present in the emm3 genome were also enriched; additionally, we ascertained unique CovR binding, primarily to genes in mobile genetic elements and other sites of interstrain chromosomal differences. Elimination of CovS phosphatase activity specifically increased CovR occupancy at the promoters of a broad array of CovR repressed virulence factor-encoding genes, including those encoding the key GAS regulator Mga and M protein. However, a limited number of promoters had augmented enrichment at low CovR~P levels. Differential motif searches using sequences enriched at high versus low CovR~P levels revealed two distinct binding patterns. At high CovR~P, a pseudopalindromic AT-rich consensus sequence (WTWTTATAAWAAAAWNATDA) consistent with CovR binding as a dimer was determined. Conversely, sequences specifically enriched at low CovR~P contained isolated ATTARA motifs suggesting an interaction with a monomer. These data extend understanding of global CovR DNA occupancy beyond emm1 GAS and provide a mechanism for previous observations regarding hypovirulence induced by CovS phosphatase abrogation. IMPORTANCE Given its key role in pathogenesis of Gram-positive bacteria, CovR is one of the most important members of the OmpR/PhoB family of transcriptional regulators. Herein we extend recent GAS CovR global binding analyses done in emm1 to a non-emm1 strain, which is important considering the known inter-emm-type heterogeneity in GAS CovRS function. Our data provide mechanistic understanding for variation in CovRS function between emm types and the profound hypovirulence of CovS phosphatase-negative strains in addition to indicating differential targeting by phosphorylated and nonphosphorylated CovR isoforms at specific CovR binding sites. These findings advance knowledge regarding how a key bacterial virulence regulator impacts pathogenesis and add to the growing appreciation of the function of nonphosphorylated OmpR/PhoB family members.

Increases in group A streptococcal infections in the pediatric population in Houston, TX, 2022.

Beginning in October 2022, we observed a substantial increase in the total number of cases of invasive GAS disease (iGAS) in the pediatric population in Houston, TX. Emm12 GAS strains were disproportionately represented but the overall proportion of iGAS infections observed during the current spike was similar to pre-pandemic years.

Evolving antibiotic resistance in Group B Streptococci causing invasive infant disease: 1970-2021.

BACKGROUND: We sought to define the frequency of antibiotic resistance over time in a collection of invasive GBS isolates derived from infant early-onset disease (EOD), late-onset disease (LOD), and late-late onset disease (LLOD). METHODS: A multicenter retrospective review of infants born from 1970 to 2021 with GBS isolated from blood, cerebrospinal fluid, synovial fluid, cellulitis, or bone. All isolates were serotyped and antimicrobial susceptibility testing performed using disk diffusion. RESULTS: The most common serotypes in our 2017 isolates were III (n = 1112, 55.1%), Ia (n = 445, 22%), Ib (n = 182, 9%) and II (n = 146, 7.2%). A total of 945 (46.8%) isolates were from infants with EOD, 976 (48.3%) from LOD, and 96 (4.75%) from LLOD. All isolates were penicillin-susceptible. Compared to strains isolated <2000, strains isolated ≥2000 showed significantly greater frequency of erythromycin (4.0% to 32.3%, P < 0.0001) and clindamycin (1.5% to 17.5%, P < 0.0001) resistance. Year of isolation (≥2000) and serotype V were significantly associated with erythromycin and/or clindamycin resistance. CONCLUSIONS: We document a rapid and significant increase in clindamycin and erythromycin resistance. As clindamycin may be considered in severely penicillin-allergic women needing GBS intrapartum prophylaxis, obstetricians, pediatricians, and neonatologist should be aware of this disturbing trend. IMPACT: Group B streptococcal strains isolated from infants with invasive infection have become more resistant to second-line antibiotics over time. In this epidemiologic study of 2017 group B streptococci isolated from 1970 to 2021, penicillin susceptibility remained uniform; however, resistance to erythromycin and clindamycin increased significantly over time across all capsular serotypes. Clindamycin resistance exceeded 20% by 2010 in most serotypes. While penicillin remains the treatment of choice for group B streptococcal infant disease, pediatricians and neonatologists should be aware of the high prevalence of resistance to clindamycin, a recommended alternative drug used for intrapartum-antibiotic prophylaxis in penicillin-allergic women.

The Integrative Conjugative Element ICESpyM92 Contributes to Pathogenicity of Emergent Antimicrobial-Resistant emm92 Group A Streptococcus.

Antimicrobial resistance-encoding mobile genetic elements (MGEs) may contribute to the disease potential of bacterial pathogens. We previously described the association of Group A Streptococcus (GAS) derived from invasive disease with increasingly frequent antimicrobial resistance (AMR). We hypothesized that a 65-kb AMR-encoding MGE (ICESpyM92), highly conserved among closely related emergent invasive emm92 GAS, contributes to GAS disease potential. Here, we provide evidence that a combination of ICESpyM92- and core genome-dependent differential gene expression (DGE) contributes to invasive disease phenotypes of emergent emm92 GAS. Using isogenic ICESpyM92 mutants generated in distinct emm92 genomic backgrounds, we determined the presence of ICESpyM92 enhances GAS virulence in a mouse subcutaneous infection model. Measurement of in vitro and ex vivo DGE indicates ICESpyM92 influences GAS global gene expression in a background-dependent manner. Our study links virulence and AMR on a unique MGE via MGE-related DGE and highlights the importance of investigating associations between AMR-encoding MGEs and pathogenicity.

Genome-wide analysis of in vivo CcpA binding with and without its key co-factor HPr in the major human pathogen group A Streptococcus.

Catabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram-positive bacteria. Most functional assessments of CcpA, including interaction with its key co-factor HPr, have been performed in nonpathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA-mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIP-seq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL-8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221-CcpA-V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIP-seq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild-type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr-dependent and independent activities of CcpA in a key pathogenic bacterium.

Reduced Ceftaroline Susceptibility among Invasive MRSA Infections in Children: a Clinical and Genomic Investigation.

Ceftaroline represents an attractive therapy option for methicillin-resistant Staphylococcus aureus (MRSA). Little data is available, however, regarding the frequency of reduced susceptibility (RS) to ceftaroline among pediatric MRSA infections. We screened invasive MRSA isolates at a tertiary children's hospital for ceftaroline RS. Ceftaroline RS occurred in 2.9% of isolates and only among health care associated infections. Ceftaroline RS isolates were more often clindamycin-resistant. Sequencing data indicated the predominance of the CC5 lineage among ceftaroline RS isolates.

Population Genomics Reveals Distinct Temporal Association with the Emergence of ST1 Serotype V Group B Streptococcus and Macrolide Resistance in North America.

Identified in the 1970s as the leading cause of invasive bacterial disease in neonates and young infants, group B Streptococcus (GBS) is now also recognized as a significant cause of morbidity and mortality among adults with underlying medical conditions and the elderly. Concomitant with the increasing incidence of GBS invasive disease in adults is the rise of resistance among GBS isolates to second line antibiotics. Previous research shows that among serotype V GBS, one of the most common capsular types causing adult invasive disease, sequence type 1 (ST1), accounts for an overwhelming majority of adult invasive disease isolates and frequently harbors macrolide resistance. In this study, using whole-genome sequencing data from strains isolated in the United States and Canada over a 45-year period, we examined the association of antimicrobial resistance with the emergence of invasive serotype V ST1 GBS. Our findings show a strong temporal association between increased macrolide resistance and the emergence of serotype V ST1 GBS subpopulations that currently co-circulate to cause invasive disease in adults and young infants. ST1 GBS subpopulations are defined, in part, by the presence of macrolide resistance genes in mobile genetic elements. Increased frequency of macrolide resistance-encoding mobile genetic elements among invasive GBS ST1 strains suggests the presence of such elements contributes to GBS virulence. Our work provides a foundation for the investigation of genetic features contributing to the increasing prevalence and pathogenesis of serotype V GBS in adult invasive disease.

Staphylococcus aureus Colonization in Healthy Children during the First Year of the Severe Acute Respiratory Syndrome Coronavirus 2 Pandemic.

The early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic was temporally associated with a reduction in many childhood infections, although the impact on bacterial colonization is unknown. We longitudinally assessed Staphylococcusaureus colonization prior to and through the first year of the pandemic. We observed a decline in methicillin-resistant Staphylococcus aureus colonization associated with SARS-CoV-2 prevention mandates.

The LiaFSR Transcriptome Reveals an Interconnected Regulatory Network in Group A Streptococcus.

The mechanisms by which bacteria sense the host environment and alter gene expression are poorly understood. LiaFSR is a gene regulatory system unique to Gram-positive bacteria, including group A Streptococcus (GAS), and responds to cell envelope stress. We previously showed that LiaF acts as an inhibitor to LiaFSR activation in GAS. To better understand gene regulation associated with LiaFSR activation, we performed RNA sequencing on isogenic deletion mutants fixed in a LiaFSR "always on" (ΔliaF) or "always off" (ΔliaR) state. Transcriptome analyses of ΔliaF and ΔliaR in GAS showed near perfect inverse correlation, including the gene encoding the global transcriptional regulator SpxA2. In addition, mutant transcriptomes included genes encoding multiple virulence factors and showed substantial overlap with the CovRS regulon. Chromatin immunoprecipitation quantitative PCR demonstrated direct spxA2 gene regulation following activation of the response regulator, LiaR. High SpxA2 levels as a result of LiaFSR activation were directly correlated with increased CovR-regulated virulence gene transcription. Furthermore, consistent with known virulence gene repression by phosphorylated CovR, elevated SpxA2 levels were inversely correlated with CovR phosphorylation. Despite increased transcription of several virulence factors, ΔliaF (high SpxA2) exhibited a paradoxical virulence phenotype in both in vivo mouse and ex vivo human blood models of disease. Likewise, despite decreased virulence factor transcription with ΔliaR (low SpxA2), increased virulence was observed in an in vivo mouse model of disease-a phenotype attributable, in part, to known SpxA2-associated speB transcription. Our findings provide evidence of a critical role of LiaFSR in sensing the host environment and suggest a potential mechanism for gene regulatory system cross talk shared by many Gram-positive pathogens.

Strain-Dependent Effect of Capsule on Transmission and Persistence in an Infant Mouse Model of Group A Streptococcus Infection.

Streptococcus pyogenes (group A Streptococcus [GAS]) is a human pathogen responsible for a wide range of diseases. Asymptomatic carriage of GAS in the human pharynx is commonplace and a potential reservoir for GAS transmission. Early studies showed that GAS transmission correlated with high bacterial burdens during the acute symptomatic phase of the disease. Human studies and the nonhuman primate model are generally impractical for investigation of the bacterial mechanisms contributing to GAS transmission and persistence. To address this gap, we adapted an infant mouse model of pneumococcal colonization and transmission to investigate factors that influence GAS transmission and persistence. The model recapitulated the direct correlation between GAS burden and transmission during the acute phase of infection observed in humans and nonhuman primates. Furthermore, our results indicate that the ratio of colonized to uncolonized hosts influences the rates of GAS transmission and persistence. We used the model to test the hypothesis that capsule production influences GAS transmission and persistence in a strain-dependent manner. We detected significant differences in rates of transmission and persistence between capsule-positive (emm3) and capsule-negative (emm87) GAS strains. Capsule was associated with higher levels of GAS shedding, independent of the strain background. In contrast to the capsule-positive emm3 strain, restoring capsule production in emm87 GAS did not increase transmissibility, and the absence of capsule enhanced persistence only in the capsule-negative (emm87) strain background. These data suggest that strain background (capsule positive versus capsule negative) influences the effect of capsule in GAS transmission and persistence and that as-yet-undefined factors are required for the transmission of capsule-negative emm types.

Cefazolin Inoculum Effect and Methicillin-Susceptible Staphylococcus aureus Osteoarticular Infections in Children.

Select methicillin-susceptible Staphylococcus aureus (MSSA) strains may produce ß-lactamases with affinity for first-generation cephalosporins (1GCs). In the setting of a high inoculum, these ß-lactamases may promote the cleavage of 1GCs, a phenomenon known as the cefazolin inoculum effect (CzIE). We evaluated the prevalence and impact of CzIE on clinical outcomes among MSSA acute hematogenous osteomyelitis (AHO) cases. MSSA AHO isolates obtained from two children's hospitals between January 2011 and December 2018 were procured through ongoing surveillance studies. Isolates were tested for CzIE via a broth macrodilution assay using an inoculum of 107 CFU/ml; CzIE was defined as a cefazolin MIC of ≥16 µg/ml. Isolates were characterized by accessory gene regulator group (agr). The progression from acute to chronic osteomyelitis was considered an important outcome. A total of 250 cases with viable isolates were included. Notably, 14.4% of isolates exhibited CzIE with no observed temporal trend; and 4% and 76% of patients received a 1GC as an empirical and definitive therapy, respectively. CzIE isolates were more often resistant to clindamycin, belonged to agrIII, and associated with the development of chronic osteomyelitis. In multivariable analyses, agrIII, multiple surgical debridements, delayed source control, and CzIE were independently associated with progression to chronic osteomyelitis. A higher rate of chronic osteomyelitis was observed with CzIE isolates regardless of definitive antibiotic choice. CzIE is exhibited by 14.4% of MSSA AHO isolates in children. CzIE is independently associated with progression to chronic osteomyelitis in cases of AHO irrespective of final antibiotic choice. These data suggest that negative outcomes reported with CzIE may more accurately reflect strain-dependent virulence factors rather than true antibiotic failure.

Sequence type 1 group B Streptococcus, an emerging cause of invasive disease in adults, evolves by small genetic changes.

The molecular mechanisms underlying pathogen emergence in humans is a critical but poorly understood area of microbiologic investigation. Serotype V group B Streptococcus (GBS) was first isolated from humans in 1975, and rates of invasive serotype V GBS disease significantly increased starting in the early 1990s. We found that 210 of 229 serotype V GBS strains (92%) isolated from the bloodstream of nonpregnant adults in the United States and Canada between 1992 and 2013 were multilocus sequence type (ST) 1. Elucidation of the complete genome of a 1992 ST-1 strain revealed that this strain had the highest homology with a GBS strain causing cow mastitis and that the 1992 ST-1 strain differed from serotype V strains isolated in the late 1970s by acquisition of cell surface proteins and antimicrobial resistance determinants. Whole-genome comparison of 202 invasive ST-1 strains detected significant recombination in only eight strains. The remaining 194 strains differed by an average of 97 SNPs. Phylogenetic analysis revealed a temporally dependent mode of genetic diversification consistent with the emergence in the 1990s of ST-1 GBS as major agents of human disease. Thirty-one loci were identified as being under positive selective pressure, and mutations at loci encoding polysaccharide capsule production proteins, regulators of pilus expression, and two-component gene regulatory systems were shown to affect the bacterial phenotype. These data reveal that phenotypic diversity among ST-1 GBS is mainly driven by small genetic changes rather than extensive recombination, thereby extending knowledge into how pathogens adapt to humans.

Cluster of Fatal Group A Streptococcal emm87 Infections in a Single Family: Molecular Basis for Invasion and Transmission.

Hypervirulent disease due to group A Streptococcus (GAS) can result from strains with mutations that enhance virulence gene expression but reduce subsequent transmission. We used whole-genome sequencing to investigate intrafamilial spread among 4 siblings of infection due to a hypervirulent GAS strain that resulted in a fatality. All invasive and pharyngeal GAS isolates had an identical mutation in a gene encoding a key regulatory protein that yielded a hyperinvasive phenotype. These data challenge the prevailing theory of reduced transmission induced by mutations that lead to hypervirulent GAS by showing that spread of hypervirulent GAS may lead to clusters of invasive disease.

Increased Pilus Production Conferred by a Naturally Occurring Mutation Alters Host-Pathogen Interaction in Favor of Carriage in Streptococcus pyogenes.

Studies of the human pathogen group A Streptococcus (GAS) define the carrier phenotype to be an increased ability to adhere to and persist on epithelial surfaces and a decreased ability to cause disease. We tested the hypothesis that a single amino acid change (Arg135Gly) in a highly conserved sensor kinase (LiaS) of a poorly defined GAS regulatory system contributes to a carrier phenotype through increased pilus production. When introduced into an emm serotype-matched invasive strain, the carrier allele (the gene encoding the LiaS protein with an arginine-to-glycine change at position 135 [liaSR135G]) recapitulated a carrier phenotype defined by an increased ability to adhere to mucosal surfaces and a decreased ability to cause disease. Gene transcript analyses revealed that the liaS mutation significantly altered transcription of the genes encoding pilus in the presence of bacitracin. Elimination of pilus production in the isogenic carrier mutant decreased its ability to colonize the mouse nasopharynx and to adhere to and be internalized by cultured human epithelial cells and restored the virulence phenotype in a mouse model of necrotizing fasciitis. We also observed significantly reduced survival of the isogenic carrier mutant compared to that of the parental invasive strain after exposure to human neutrophils. Elimination of pilus in the isogenic carrier mutant increased the level of survival after exposure to human neutrophils to that for the parental invasive strain. Together, our data demonstrate that the carrier mutation (liaSR135G) affects pilus expression. Our data suggest new mechanisms of pilus gene regulation in GAS and that the invasiveness associated with pilus gene regulation in GAS differs from the enhanced invasiveness associated with increased pilus production in other bacterial pathogens.

Adhesin competence repressor (AdcR) from Streptococcus pyogenes controls adaptive responses to zinc limitation and contributes to virulence.

Altering zinc bioavailability to bacterial pathogens is a key component of host innate immunity. Thus, the ability to sense and adapt to the alterations in zinc concentrations is critical for bacterial survival and pathogenesis. To understand the adaptive responses of group A Streptococcus (GAS) to zinc limitation and its regulation by AdcR, we characterized gene regulation by AdcR. AdcR regulates the expression of 70 genes involved in zinc acquisition and virulence. Zinc-bound AdcR interacts with operator sequences in the negatively regulated promoters and mediates differential regulation of target genes in response to zinc deficiency. Genes involved in zinc mobilization and conservation are derepressed during mild zinc deficiency, whereas the energy-dependent zinc importers are upregulated during severe zinc deficiency. Further, we demonstrated that transcription activation by AdcR occurs by direct binding to the promoter. However, the repression and activation by AdcR is mediated by its interactions with two distinct operator sequences. Finally, mutational analysis of the metal ligands of AdcR caused impaired DNA binding and attenuated virulence, indicating that zinc sensing by AdcR is critical for GAS pathogenesis. Together, we demonstrate that AdcR regulates GAS adaptive responses to zinc limitation and identify molecular components required for GAS survival during zinc deficiency.

A naturally occurring single amino acid replacement in multiple gene regulator of group A Streptococcus significantly increases virulence.

Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation within a species; however, few investigations demonstrate how naturally occurring SNPs may increase strain virulence. We recently used group A Streptococcus as a model pathogen to study bacteria strain genotype-patient disease phenotype relationships. Whole-genome sequencing of approximately 800 serotype M59 group A Streptococcus strains, recovered during an outbreak of severe invasive infections across North America, identified a disproportionate number of SNPs in the gene encoding multiple gene regulator of group A Streptococcus (mga). Herein, we report results of studies designed to test the hypothesis that the most commonly occurring SNP, encoding a replacement of arginine for histidine at codon 201 of Mga (H201R), significantly increases virulence. Whole transcriptome analysis revealed that the H201R replacement significantly increased expression of mga and 54 other genes, including many proven virulence factors. Compared to the wild-type strain, a H201R isogenic mutant strain caused significantly larger skin lesions in mice. Serial quantitative bacterial culture and noninvasive magnetic resonance imaging also demonstrated that the isogenic H201R strain was significantly more virulent in a nonhuman primate model of joint infection. These findings show that the H201R replacement in Mga increases the virulence of M59 group A Streptococcus and provide new insight to how a naturally occurring SNP in bacteria contributes to human disease phenotypes.

A Single Amino Acid Replacement in the Sensor Kinase LiaS Contributes to a Carrier Phenotype in Group A Streptococcus.

Despite the high frequency of asymptomatic carriage of bacterial pathogens, we understand little about the bacterial molecular genetic underpinnings of this phenomenon. To obtain new information about the molecular genetic mechanisms underlying carriage of group A Streptococcus (GAS), we performed whole-genome sequencing of GAS strains recovered from a single individual during acute pharyngitis and subsequent asymptomatic carriage. We discovered that compared to the initial infection isolate, the strain recovered during asymptomatic carriage contained three single nucleotide polymorphisms, one of which was in a highly conserved region of a gene encoding a sensor kinase, liaS, resulting in an arginine-to-glycine amino acid replacement at position 135 of LiaS (LiaS(R135G)). Using gene replacement, we demonstrate that introduction of the carrier allele (liaS(R135G)) into a serotype-matched invasive strain increased mouse nasopharyngeal colonization and adherence to cultured human epithelial cells. The carrier mutation also resulted in a reduced ability to grow in human blood and reduced virulence in a mouse model of necrotizing fasciitis. Repair of the mutation in the GAS carrier strain restored virulence and decreased adherence to cultured human epithelial cells. We also provide evidence that the carrier mutation alters the GAS transcriptome, including altered transcription of GAS virulence genes, providing a potential mechanism for the pleiotropic phenotypic effects. Our data obtained using isogenic strains suggest that the liaS(R135G) mutation in the carrier strain contributes to the transition from disease to asymptomatic carriage and provides new information about this poorly described regulatory system in GAS.

Characterization of the effect of the histidine kinase CovS on response regulator phosphorylation in group A Streptococcus.

Two-component gene regulatory systems (TCSs) are a major mechanism by which bacteria respond to environmental stimuli and thus are critical to infectivity. For example, the control of virulence regulator/sensor kinase (CovRS) TCS is central to the virulence of the major human pathogen group A Streptococcus (GAS). Here, we used a combination of quantitative in vivo phosphorylation assays, isoallelic strains that varied by only a single amino acid in CovS, and transcriptome analyses to characterize the impact of CovS on CovR phosphorylation and GAS global gene expression. We discovered that CovS primarily serves to phosphorylate CovR, thereby resulting in the repression of virulence factor-encoding genes. However, a GAS strain selectively deficient in CovS phosphatase activity had a distinct transcriptome relative to that of its parental strain, indicating that both CovS kinase and phosphatase activities influence the CovR phosphorylation status. Surprisingly, compared to a serotype M3 strain, serotype M1 GAS strains had high levels of phosphorylated CovR, low transcript levels of CovR-repressed genes, and strikingly different responses to environmental cues. Moreover, the inactivation of CovS in the serotype M1 background resulted in a greater decrease in phosphorylated CovR levels and a greater increase in the transcript levels of CovR-repressed genes than did CovS inactivation in a serotype M3 strain. These data clarify the influence of CovS on the CovR phosphorylation status and provide insight into why serotype M1 GAS strains have high rates of spontaneous mutations in covS during invasive GAS infection, thus providing a link between TCS molecular function and the epidemiology of deadly bacterial infections.