Multiple spillovers from humans and onward transmission of SARS-CoV-2 in white-tailed deer.

Many animal species are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and could act as reservoirs; however, transmission in free-living animals has not been documented. White-tailed deer, the predominant cervid in North America, are susceptible to SARS-CoV-2 infection, and experimentally infected fawns can transmit the virus. To test the hypothesis that SARS-CoV-2 is circulating in deer, 283 retropharyngeal lymph node (RPLN) samples collected from 151 free-living and 132 captive deer in Iowa from April 2020 through January of 2021 were assayed for the presence of SARS-CoV-2 RNA. Ninety-four of the 283 (33.2%) deer samples were positive for SARS-CoV-2 RNA as assessed by RT-PCR. Notably, following the November 2020 peak of human cases in Iowa, and coinciding with the onset of winter and the peak deer hunting season, SARS-CoV-2 RNA was detected in 80 of 97 (82.5%) RPLN samples collected over a 7-wk period. Whole genome sequencing of all 94 positive RPLN samples identified 12 SARS-CoV-2 lineages, with B.1.2 (n = 51; 54.5%) and B.1.311 (n = 19; 20%) accounting for ∼75% of all samples. The geographic distribution and nesting of clusters of deer and human lineages strongly suggest multiple human-to-deer transmission events followed by subsequent deer-to-deer spread. These discoveries have important implications for the long-term persistence of the SARS-CoV-2 pandemic. Our findings highlight an urgent need for a robust and proactive "One Health" approach to obtain enhanced understanding of the ecology, molecular evolution, and dissemination of SARS-CoV-2.

Systems biology analysis of human genomes points to key pathways conferring spina bifida risk.

Spina bifida (SB) is a debilitating birth defect caused by multiple gene and environment interactions. Though SB shows non-Mendelian inheritance, genetic factors contribute to an estimated 70% of cases. Nevertheless, identifying human mutations conferring SB risk is challenging due to its relative rarity, genetic heterogeneity, incomplete penetrance, and environmental influences that hamper genome-wide association studies approaches to untargeted discovery. Thus, SB genetic studies may suffer from population substructure and/or selection bias introduced by typical candidate gene searches. We report a population based, ancestry-matched whole-genome sequence analysis of SB genetic predisposition using a systems biology strategy to interrogate 298 case-control subject genomes (149 pairs). Genes that were enriched in likely gene disrupting (LGD), rare protein-coding variants were subjected to machine learning analysis to identify genes in which LGD variants occur with a different frequency in cases versus controls and so discriminate between these groups. Those genes with high discriminatory potential for SB significantly enriched pathways pertaining to carbon metabolism, inflammation, innate immunity, cytoskeletal regulation, and essential transcriptional regulation consistent with their having impact on the pathogenesis of human SB. Additionally, an interrogation of conserved noncoding sequences identified robust variant enrichment in regulatory regions of several transcription factors critical to embryonic development. This genome-wide perspective offers an effective approach to the interrogation of coding and noncoding sequence variant contributions to rare complex genetic disorders.

Increase in invasive Streptococcus pyogenes M1 infections with close evolutionary genetic relationship, Iceland and Scotland, 2022 to 2023.

Group A Streptococcus isolates of the recently described M1UK clade have emerged to cause human infections in several European countries and elsewhere. Full-genome sequence analysis of M1 isolates discovered a close genomic relationship between some isolates from Scotland and the majority of isolates from Iceland causing serious infections in 2022 and 2023. Phylogenetic analysis strongly suggests that an isolate from or related to Scotland was the precursor to an M1UK variant responsible for almost all recent M1 infections in Iceland.

A Chimeric Penicillin Binding Protein 2X Significantly Decreases in Vitro Beta-Lactam Susceptibility and Increases in Vivo Fitness of Streptococcus pyogenes.

All tested strains of Streptococcus pyogenes (group A streptococcus, GAS) remain susceptible to penicillin. However, GAS strains with amino acid substitutions in penicillin-binding proteins that confer decreased susceptibility to beta-lactam antibiotics have been identified recently. This discovery raises concerns about emergence of beta-lactam antibiotic resistance in GAS. Whole genome sequencing recently identified GAS strains with a chimeric penicillin-binding protein 2X (PBP2X) containing a recombinant segment from Streptococcus dysgalactiae subspecies equisimilis (SDSE). To directly test the hypothesis that the chimeric SDSE-like PBP2X alters beta-lactam susceptibility in vitro and fitness in vivo, an isogenic mutant strain was generated and virulence assessed in a mouse model of necrotizing myositis. Compared with naturally occurring and isogenic strains with a wild-type GAS-like PBP2X, strains with the chimeric SDSE-like PBP2X had reduced susceptibility in vitro to nine beta-lactam antibiotics. In a mouse model of necrotizing myositis, the strains had identical fitness in the absence of benzylpenicillin treatment. However, mice treated intermittently with a subtherapeutic dose of benzylpenicillin had significantly more colony-forming units recovered from limbs infected with strains with the chimeric SDSE-like PBP2X. These results show that mutations such as the PBP2X chimera may result in significantly decreased beta-lactam susceptibility and increased fitness and virulence. Expanded diagnostic laboratory surveillance, genome sequencing, and molecular pathogenesis study of potentially emergent beta-lactam antibiotic resistance among GAS are needed.

Signals of Significantly Increased Vaccine Breakthrough, Decreased Hospitalization Rates, and Less Severe Disease in Patients with Coronavirus Disease 2019 Caused by the Omicron Variant of Severe Acute Respiratory Syndrome Coronavirus 2 in Houston, Texas.

Genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to dramatically alter the landscape of the coronavirus disease 2019 (COVID-19) pandemic. The recently described variant of concern designated Omicron (B.1.1.529) has rapidly spread worldwide and is now responsible for the majority of COVID-19 cases in many countries. Because Omicron was recognized recently, many knowledge gaps exist about its epidemiology, clinical severity, and disease course. A genome sequencing study of SARS-CoV-2 in the Houston Methodist health care system identified 4468 symptomatic patients with infections caused by Omicron from late November 2021 through January 5, 2022. Omicron rapidly increased in only 3 weeks to cause 90% of all new COVID-19 cases, and at the end of the study period caused 98% of new cases. Compared with patients infected with either Alpha or Delta variants in our health care system, Omicron patients were significantly younger, had significantly increased vaccine breakthrough rates, and were significantly less likely to be hospitalized. Omicron patients required less intense respiratory support and had a shorter length of hospital stay, consistent with on average decreased disease severity. Two patients with Omicron stealth sublineage BA.2 also were identified. The data document the unusually rapid spread and increased occurrence of COVID-19 caused by the Omicron variant in metropolitan Houston, Texas, and address the lack of information about disease character among US patients.

Delta Variants of SARS-CoV-2 Cause Significantly Increased Vaccine Breakthrough COVID-19 Cases in Houston, Texas.

Genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have repeatedly altered the course of the coronavirus disease 2019 (COVID-19) pandemic. Delta variants are now the focus of intense international attention because they are causing widespread COVID-19 globally and are associated with vaccine breakthrough cases. We sequenced 16,965 SARS-CoV-2 genomes from samples acquired March 15, 2021, through September 20, 2021, in the Houston Methodist hospital system. This sample represents 91% of all Methodist system COVID-19 patients during the study period. Delta variants increased rapidly from late April onward to cause 99.9% of all COVID-19 cases and spread throughout the Houston metroplex. Compared with all other variants combined, Delta caused a significantly higher rate of vaccine breakthrough cases (23.7% for Delta compared with 6.6% for all other variants combined). Importantly, significantly fewer fully vaccinated individuals required hospitalization. Vaccine breakthrough cases caused by Delta had a low median PCR cycle threshold value (a proxy for high virus load). This value was similar to the median cycle threshold value for unvaccinated patients with COVID-19 caused by Delta variants, suggesting that fully vaccinated individuals can transmit SARS-CoV-2 to others. Patients infected with Alpha and Delta variants had several significant differences. The integrated analysis indicates that vaccines used in the United States are highly effective in decreasing severe COVID-19, hospitalizations, and deaths.

Characterization of M-Type-Specific Pilus Expression in Group A Streptococcus.

In addition to providing a typing mechanism for group A Streptococcus (GAS) isolates (T typing), cell surface pilus production impacts GAS virulence characteristics, including adherence and immune evasion. The pilus biosynthesis genes are located in the fibronectin- and collagen-binding T-antigen (FCT) region of the genome, and nine different FCT types, encoding more than 20 different T types, have been described. GAS isolates are not uniform in their degree or pattern of pilus expression, as highlighted by pilus production being thermoregulated in isolates that harbor the FCT-type FCT-3 (e.g., M-types M3 and M49) but not in isolates that harbor FCT-2 (e.g., M-type M1). Here, we investigated the molecular basis underlying our previous finding that M3 GAS isolates produce pili in lower abundance than M1 or M49 isolates do. We discovered that, at least in part, the low pilus expression observed for M3 isolates is a consequence of the repression of pilus gene expression by the CovR/CovS two-component regulatory system and of an M3-specific mutation in the nra gene, encoding a positive regulator of pilus gene expression. We also discovered that the orthologous transcriptional regulators RofA and Nra, whose encoding genes are located within FCT-2 and FCT-3, respectively, are not functionally identical. Finally, we sequenced the genome of an M3 isolate that had naturally undergone recombinational replacement of the FCT region, changing the FCT and T types of this strain from FCT-3/T3 to FCT-2/T1. Our study furthers the understanding of strain- and type-specific variation in virulence factor production by an important human pathogen. IMPORTANCE Our ability to characterize how a pathogen infects and causes disease, and consequently our ability to devise approaches to prevent or attenuate such infections, is inhibited by the finding that isolates of a given pathogen often show phenotypic variability, for example, in their ability to adhere to host cells through modulation of cell surface adhesins. Such variability is observed between isolates of group A Streptococcus (GAS), and this study investigates the molecular basis for why some GAS isolates produce pili, cell wall-anchored adhesins, in lower abundance than other isolates do. Given that pili are being considered as potential antigens in formulations of future GAS vaccines, this study may inform vaccine design.

Genome-Wide Transposon Mutagenesis Screens Identify Group A Streptococcus Genes Affecting Susceptibility to ß-Lactam Antibiotics.

Group A streptococcus (GAS) is a Gram-positive human bacterial pathogen responsible for more than 700 million infections annually worldwide. Beta-lactam antibiotics are the primary agents used to treat GAS infections. Naturally occurring GAS clinical isolates with decreased susceptibility to beta-lactam antibiotics attributed to mutations in PBP2X have recently been documented. This prompted us to perform a genome-wide screen to identify GAS genes that alter beta-lactam susceptibility in vitro. Using saturated transposon mutagenesis, we screened for GAS gene mutations conferring altered in vitro susceptibility to penicillin G and/or ceftriaxone, two beta-lactam antibiotics commonly used to treat GAS infections. In the aggregate, we found that inactivating mutations in 150 GAS genes are associated with altered susceptibility to penicillin G and/or ceftriaxone. Many of the genes identified were previously not known to alter beta-lactam susceptibility or affect cell wall biosynthesis. Using isogenic mutant strains, we confirmed that inactivation of clpX (Clp protease ATP-binding subunit) or cppA (CppA proteinase) resulted in decreased in vitro susceptibility to penicillin G and ceftriaxone. Deletion of murA1 (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) conferred increased susceptibility to ceftriaxone. Our results provide new information about the GAS genes affecting susceptibility to beta-lactam antibiotics. IMPORTANCE Beta-lactam antibiotics are the primary drugs prescribed to treat infections caused by group A streptococcus (GAS), an important human pathogen. However, the molecular mechanisms of GAS interactions with beta-lactam antibiotics are not fully understood. In this study, we performed a genome-wide mutagenesis screen to identify GAS mutations conferring altered susceptibility to beta-lactam antibiotics. In the aggregate, we discovered that mutations in 150 GAS genes were associated with altered beta-lactam susceptibility. Many identified genes were previously not known to alter beta-lactam susceptibility or affect cell wall biosynthesis. Our results provide new information about the molecular mechanisms of GAS interaction with beta-lactam antibiotics.

Trajectory of Growth of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants in Houston, Texas, January through May 2021, Based on 12,476 Genome Sequences.

Certain genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of substantial concern because they may be more transmissible or detrimentally alter the pandemic course and disease features in individual patients. SARS-CoV-2 genome sequences from 12,476 patients in the Houston Methodist health care system diagnosed from January 1 through May 31, 2021 are reported here. Prevalence of the B.1.1.7 (Alpha) variant increased rapidly and caused 63% to 90% of new cases in the latter half of May. Eleven B.1.1.7 genomes had an E484K replacement in spike protein, a change also identified in other SARS-CoV-2 lineages. Compared with non-B.1.1.7-infected patients, individuals with B.1.1.7 had a significantly lower cycle threshold (a proxy for higher virus load) and significantly higher hospitalization rate. Other variants [eg, B.1.429 and B.1.427 (Epsilon), P.1 (Gamma), P.2 (Zeta), and R.1] also increased rapidly, although the magnitude was less than that in B.1.1.7. Twenty-two patients infected with B.1.617.1 (Kappa) or B.1.617.2 (Delta) variants had a high rate of hospitalization. Breakthrough cases (n = 207) in fully vaccinated patients were caused by a heterogeneous array of virus genotypes, including many not currently designated variants of interest or concern. In the aggregate, this study delineates the trajectory of SARS-CoV-2 variants circulating in a major metropolitan area, documents B.1.1.7 as the major cause of new cases in Houston, TX, and heralds the arrival of B.1.617 variants in the metroplex.

Sequence Analysis of 20,453 Severe Acute Respiratory Syndrome Coronavirus 2 Genomes from the Houston Metropolitan Area Identifies the Emergence and Widespread Distribution of Multiple Isolates of All Major Variants of Concern.

Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there has been international concern about the emergence of virus variants with mutations that increase transmissibility, enhance escape from the human immune response, or otherwise alter biologically important phenotypes. In late 2020, several variants of concern emerged globally, including the UK variant (B.1.1.7), the South Africa variant (B.1.351), Brazil variants (P.1 and P.2), and two related California variants of interest (B.1.429 and B.1.427). These variants are believed to have enhanced transmissibility. For the South Africa and Brazil variants, there is evidence that mutations in spike protein permit it to escape from some vaccines and therapeutic monoclonal antibodies. On the basis of our extensive genome sequencing program involving 20,453 coronavirus disease 2019 patient samples collected from March 2020 to February 2021, we report identification of all six of these SARS-CoV-2 variants among Houston Methodist Hospital (Houston, TX) patients residing in the greater metropolitan area. Although these variants are currently at relatively low frequency (aggregate of 1.1%) in the population, they are geographically widespread. Houston is the first city in the United States in which active circulation of all six current variants of concern has been documented by genome sequencing. As vaccine deployment accelerates, increased genomic surveillance of SARS-CoV-2 is essential to understanding the presence, frequency, and medical impact of consequential variants and their patterns and trajectory of dissemination.

Significantly Decreased Mortality in a Large Cohort of Coronavirus Disease 2019 (COVID-19) Patients Transfused Early with Convalescent Plasma Containing High-Titer Anti-Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike Protein IgG.

Coronavirus disease 2019 (COVID-19) convalescent plasma has emerged as a promising therapy and has been granted Emergency Use Authorization by the US Food and Drug Administration for hospitalized COVID-19 patients. We recently reported results from interim analysis of a propensity score-matched study suggesting that early treatment of COVID-19 patients with convalescent plasma containing high-titer anti-spike protein receptor binding domain (RBD) IgG significantly decreases mortality. We herein present results from a 60-day follow-up of a cohort of 351 transfused hospitalized patients. Prospective determination of enzyme-linked immunosorbent assay anti-RBD IgG titer facilitated selection and transfusion of the highest titer units available. Retrospective analysis by the Ortho VITROS IgG assay revealed a median signal/cutoff ratio of 24.0 for transfused units, a value far exceeding the recent US Food and Drug Administration-required cutoff of 12.0 for designation of high-titer convalescent plasma. With respect to altering mortality, our analysis identified an optimal window of 44 hours after hospitalization for transfusing COVID-19 patients with high-titer convalescent plasma. In the aggregate, the analysis confirms and extends our previous preliminary finding that transfusion of COVID-19 patients soon after hospitalization with high-titer anti-spike protein RBD IgG present in convalescent plasma significantly reduces mortality.

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

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

A Single Amino Acid Replacement in Penicillin-Binding Protein 2X in Streptococcus pyogenes Significantly Increases Fitness on Subtherapeutic Benzylpenicillin Treatment in a Mouse Model of Necrotizing Myositis.

Invasive strains of Streptococcus pyogenes with significantly reduced susceptibility to ß-lactam antibiotics have been recently described. These reports have caused considerable concern in the international infectious disease, medical microbiology, and public health communities because S. pyogenes has remained universally susceptible to ß-lactam antibiotics for 70 years. Virtually all analyzed strains had single amino acid replacements in penicillin-binding protein 2X (PBP2X), a major target of ß-lactam antibiotics in pathogenic bacteria. We used isogenic strains to test the hypothesis that a single amino acid replacement in PBP2X conferred a fitness advantage in a mouse model of necrotizing myositis. We determined that when mice were administered intermittent subtherapeutic dosing of benzylpenicillin, the strain with a Pro601Leu amino acid replacement in PBP2X that confers reduced ß-lactam susceptibility in vitro was more fit, as assessed by the magnitude of colony-forming units recovered from disease tissue. These data provide important pathogenesis information that bears on this emerging global infectious disease problem.

Genome-Wide Screens Identify Group A Streptococcus Surface Proteins Promoting Female Genital Tract Colonization and Virulence.

Group A streptococcus (GAS) is a major pathogen that impacts health and economic affairs worldwide. Although the oropharynx is the primary site of infection, GAS can colonize the female genital tract and cause severe diseases, such as puerperal sepsis, neonatal infections, and necrotizing myometritis. Our understanding of how GAS genes contribute to interaction with the primate female genital tract is limited by the lack of relevant animal models. Using two genome-wide transposon mutagenesis screens, we identified 69 GAS genes required for colonization of the primate vaginal mucosa in vivo and 96 genes required for infection of the uterine wall ex vivo. We discovered a common set of 39 genes important for GAS fitness in both environments. They include genes encoding transporters, surface proteins, transcriptional regulators, and metabolic pathways. Notably, the genes that encode the surface-exclusion protein (SpyAD) and the immunogenic secreted protein 2 (Isp2) were found to be crucial for GAS fitness in the female primate genital tract. Targeted gene deletion confirmed that isogenic mutant strains ΔspyAD and Δisp2 are significantly impaired in ability to colonize the primate genital tract and cause uterine wall pathologic findings. Our studies identified novel GAS genes that contribute to female reproductive tract interaction that warrant translational research investigation.

Treatment of Coronavirus Disease 2019 Patients with Convalescent Plasma Reveals a Signal of Significantly Decreased Mortality.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and proven treatments are limited. Transfusion of convalescent plasma collected from donors who have recovered from COVID-19 is among many approaches being studied as potentially efficacious therapy. We are conducting a prospective, propensity score-matched study assessing the efficacy of COVID-19 convalescent plasma transfusion versus standard of care as treatment for severe and/or critical COVID-19. We present herein the results of an interim analysis of 316 patients enrolled at Houston Methodist hospitals from March 28 to July 6, 2020. Of the 316 transfused patients, 136 met a 28-day outcome and were matched to 251 non-transfused control COVID-19 patients. Matching criteria included age, sex, body mass index, comorbidities, and baseline ventilation requirement 48 hours from admission, and in a second matching analysis, ventilation status at day 0. Variability in the timing of transfusion relative to admission and titer of antibodies of plasma transfused allowed for analysis in specific matched cohorts. The analysis showed a significant reduction (P = 0.047) in mortality within 28 days, specifically in patients transfused within 72 hours of admission with plasma with an anti-spike protein receptor binding domain titer of ≥1:1350. These data suggest that treatment of COVID-19 with high anti-receptor binding domain IgG titer convalescent plasma is efficacious in early-disease patients.

Treatment of Coronavirus Disease 2019 (COVID-19) Patients with Convalescent Plasma.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for >100 years. Patients (n = 25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28, 2020, to April 14, 2020. Patients were transfused with convalescent plasma, obtained from donors with confirmed severe acute respiratory syndrome coronavirus 2 infection who had recovered. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 after transfusion. Clinical improvement was assessed on the basis of a modified World Health Organization six-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. At day 7 after transfusion with convalescent plasma, nine patients had at least a one-point improvement in clinical scale, and seven of those were discharged. By day 14 after transfusion, 19 (76%) patients had at least a one-point improvement in clinical status, and 11 were discharged. No adverse events as a result of plasma transfusion were observed. Whole genome sequencing data did not identify a strain genotype-disease severity correlation. The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease.

Metal sensing and regulation of adaptive responses to manganese limitation by MtsR is critical for group A streptococcus virulence.

Pathogenic bacteria encounter host-imposed manganese (Mn) limitation during infection. Herein we report that in the human pathogen Streptococcus pyogenes, the adaptive response to Mn limitation is controlled by a DtxR family metalloregulator, MtsR. Genes upregulated by MtsR during Mn limitation include Mn (mtsABC) and Fe acquisition systems (sia operon), and a metal-independent DNA synthesis enzyme (nrdFEI.2). To elucidate the mechanism of metal sensing and gene regulation by MtsR, we determined the crystal structure of MtsR. MtsR employs two Mn-sensing sites to monitor metal availability, and metal occupancy at each site influences MtsR regulatory activity. The site 1 acts as the primary Mn sensing site, and loss of metal at site 1 causes robust upregulation of mtsABC. The vacant site 2 causes partial induction of mtsABC, indicating that site 2 functions as secondary Mn sensing site. Furthermore, we show that the C-terminal FeoA domains of adjacent dimers participate in the oligomerization of MtsR on DNA, and multimerization is critical for MtsR regulatory activity. Finally, the mtsR mutant strains defective in metal sensing and oligomerization are attenuated for virulence in a mouse model of invasive infection, indicating that Mn sensing and gene regulation by MtsR are critical processes during S. pyogenes infection.

Genetic Basis Underlying the Hyperhemolytic Phenotype of Streptococcus agalactiae Strain CNCTC10/84.

Streptococcus agalactiae (group B streptococcus [GBS]) is a major cause of infections in newborns, pregnant women, and immunocompromised patients. GBS strain CNCTC10/84 is a clinical isolate that has high virulence in animal models of infection and has been used extensively to study GBS pathogenesis. Two unusual features of this strain are hyperhemolytic activity and hypo-CAMP factor activity. These two phenotypes are typical of GBS strains that are functionally deficient in the CovR-CovS two-component regulatory system. A previous whole-genome sequencing study found that strain CNCTC10/84 has intact covR and covS regulatory genes. We investigated CovR-CovS regulation in CNCTC10/84 and discovered that a single-nucleotide insertion in a homopolymeric tract in the covR promoter region underlies the strong hemolytic activity and weak CAMP activity of this strain. Using isogenic mutant strains, we demonstrate that this single-nucleotide insertion confers significantly decreased expression of covR and covS and altered expression of CovR-CovS-regulated genes, including that of genes encoding ß-hemolysin and CAMP factor. This single-nucleotide insertion also confers significantly increased GBS survival in human whole blood ex vivoIMPORTANCE Group B streptococcus (GBS) is the leading cause of neonatal sepsis, pneumonia, and meningitis. GBS strain CNCTC10/84 is a highly virulent blood isolate that has been used extensively to study GBS pathogenesis for over 20 years. Strain CNCTC10/84 has an unusually strong hemolytic activity, but the genetic basis is unknown. In this study, we discovered that a single-nucleotide insertion in an intergenic homopolymeric tract is responsible for the elevated hemolytic activity of CNCTC10/84.

Single Amino Acid Replacements in RocA Disrupt Protein-Protein Interactions To Alter the Molecular Pathogenesis of Group A Streptococcus.

Group A Streptococcus (GAS) is a human-specific pathogen and major cause of disease worldwide. The molecular pathogenesis of GAS, like many pathogens, is dependent on the coordinated expression of genes encoding different virulence factors. The control of virulence regulator/sensor (CovRS) two-component system is a major virulence regulator of GAS that has been extensively studied. More recent investigations have also involved regulator of Cov (RocA), a regulatory accessory protein to CovRS. RocA interacts, in some manner, with CovRS; however, the precise molecular mechanism is unknown. Here, we demonstrate that RocA is a membrane protein containing seven transmembrane helices with an extracytoplasmically located N terminus and cytoplasmically located C terminus. For the first time, we demonstrate that RocA directly interacts with itself (RocA) and CovS, but not CovR, in intact cells. Single amino acid replacements along the entire length of RocA disrupt RocA-RocA and RocA-CovS interactions to significantly alter the GAS virulence phenotype as defined by secreted virulence factor activity in vitro and tissue destruction and mortality in vivo In summary, we show that single amino acid replacements in a regulatory accessory protein can affect protein-protein interactions to significantly alter the virulence of a major human pathogen.

Group A Streptococcus AdcR Regulon Participates in Bacterial Defense against Host-Mediated Zinc Sequestration and Contributes to Virulence.

Colonization by pathogenic bacteria depends on their ability to overcome host nutritional defenses and acquire nutrients. The human pathogen group A streptococcus (GAS) encounters the host defense factor calprotectin (CP) during infection. CP inhibits GAS growth in vitro by imposing zinc (Zn) limitation. However, GAS counterstrategies to combat CP-mediated Zn limitation and the in vivo relevance of CP-GAS interactions to bacterial pathogenesis remain unknown. Here, we report that GAS upregulates the AdcR regulon in response to CP-mediated Zn limitation. The AdcR regulon includes genes encoding Zn import (adcABC), Zn sparing (rpsN.2), and Zn scavenging systems (adcAII, phtD, and phtY). Each gene in the AdcR regulon contributes to GAS Zn acquisition and CP resistance. The ΔadcC and ΔrpsN.2 mutant strains were the most susceptible to CP, whereas the ΔadcA, ΔadcAII, and ΔphtD mutant strains displayed less CP sensitivity during growth in vitro However, the ΔphtY mutant strain did not display an increased CP sensitivity. The varied sensitivity of the mutant strains to CP-mediated Zn limitation suggests distinct roles for individual AdcR regulon genes in GAS Zn acquisition. GAS upregulates the AdcR regulon during necrotizing fasciitis infection in WT mice but not in S100a9-/- mice lacking CP. This suggests that CP induces Zn deficiency in the host. Finally, consistent with the in vitro results, several of the AdcR regulon genes are critical for GAS virulence in WT mice, whereas they are dispensable for virulence in S100a9-/- mice, indicating the direct competition for Zn between CP and proteins encoded by the GAS AdcR regulon during infection.