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.

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.

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.

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.

Genome-Wide Assessment of Streptococcus agalactiae Genes Required for Survival in Human Whole Blood and Plasma.

Streptococcus agalactiae (group B streptococcus, or GBS) is a common cause of bacteremia and sepsis in newborns, pregnant women, and immunocompromised patients. The molecular mechanisms used by GBS to survive and proliferate in blood are not well understood. Here, using a highly virulent GBS strain and transposon-directed insertion site sequencing (TraDIS), we performed genome-wide screens to discover novel GBS genes required for bacterial survival in human whole blood and plasma. The screen identified 85 and 41 genes that are required for GBS growth in whole blood and plasma, respectively. A common set of 29 genes was required in both whole blood and plasma. Targeted gene deletion confirmed that (i) genes encoding methionine transporter (metP) and manganese transporter (mtsA) are crucial for GBS survival in whole blood and plasma, (ii) gene W903_1820, encoding a small multidrug export family protein, contributes significantly to GBS survival in whole blood, (iii) the shikimate pathway gene aroA is essential for GBS growth in whole blood and plasma, and (iv) deletion of srr1, encoding a fibrinogen-binding adhesin, increases GBS survival in whole blood. Our findings provide new insight into the GBS-host interactions in human blood.

Population Genomic Molecular Epidemiological Study of Macrolide-Resistant Streptococcus pyogenes in Iceland, 1995 to 2016: Identification of a Large Clonal Population with a pbp2x Mutation Conferring Reduced In Vitro ß-Lactam Susceptibility.

Resistance to macrolide antibiotics is a global concern in the treatment of Streptococcus pyogenes (group A Streptococcus [GAS]) infections. In Iceland, since the detection of the first macrolide-resistant isolate in 1998, three epidemic waves of macrolide-resistant GAS infections have occurred, with peaks in 1999, 2004, and 2008. We conducted whole-genome sequencing of all 1,575 available GAS macrolide-resistant clinical isolates of all infection types collected at the national reference laboratory in Reykjavik, Iceland, from 1998 to 2016. Among 1,515 erythromycin-resistant isolates, 90.3% were of only three emm types, emm4 (n = 713), emm6 (n = 324), and emm12 (n = 332), with each being predominant in a distinct epidemic peak. The antibiotic efflux pump genes, mef(A) and msr(D), were present on chimeric mobile genetic elements in 99.3% of the macrolide-resistant isolates of these emm types. Of note, in addition to macrolide resistance, virtually all emm12 isolates had a single amino acid substitution in penicillin-binding protein PBP2X that conferred a 2-fold increased penicillin G and ampicillin MIC among the isolates tested. We conclude that each of the three large epidemic peaks of macrolide-resistant GAS infections occurring in Iceland since 1998 was caused by the emergence and clonal expansion of progenitor strains, with macrolide resistance being conferred predominantly by inducible Mef(A) and Msr(D) drug efflux pumps. The occurrence of emm12 strains with macrolide resistance and decreased beta-lactam susceptibility was unexpected and is of public health concern.

Reduced In Vitro Susceptibility of Streptococcus pyogenes to ß-Lactam Antibiotics Associated with Mutations in the pbp2x Gene Is Geographically Widespread.

Recently, two related Streptococcus pyogenes strains with reduced susceptibility to ampicillin, amoxicillin, and cefotaxime, antibiotics commonly used to treat S. pyogenes infections, were reported. The two strains had the same nonsynonymous (amino acid-substituting) mutation in the pbp2x gene, encoding penicillin-binding protein 2X (PBP2X). This concerning report led us to investigate our library of 7,025 genome sequences of type emm1, emm28, and emm89S. pyogenes clinical strains recovered from intercontinental sources for mutations in pbp2x We identified 137 strains that, combined, had 37 nonsynonymous mutations in 36 codons in pbp2x Although to a lesser magnitude than the two previously published isolates, many of our strains had decreased susceptibility in vitro to multiple beta-lactam antibiotics. Many pbp2x mutations were found only in single strains, but 16 groups of two or more isolates of the same emm type had an identical amino acid replacement. Phylogenetic analysis showed that, with one exception, strains of the same emm type with the same amino acid replacement were clonally related by descent. This finding indicates that strains with some amino acid changes in PBP2X can successfully spread to new human hosts and cause invasive infections. Mapping of the amino acid changes onto a three-dimensional structure of the related Streptococcus pneumoniae PBP2X suggests that some substitutions are located in regions functionally important in related pathogenic bacterial species. Decreased beta-lactam susceptibility is geographically widespread in strains of numerically common emm gene subtypes. Enhanced surveillance and further epidemiological and molecular genetic study of this potential emergent antimicrobial problem are warranted.

Polymorphisms in Regulator of Cov Contribute to the Molecular Pathogenesis of Serotype M28 Group A Streptococcus.

Two-component systems (TCSs) are signal transduction proteins that enable bacteria to respond to external stimuli by altering the global transcriptome. Accessory proteins interact with TCSs to fine-tune their activity. In group A Streptococcus (GAS), regulator of Cov (RocA) is an accessory protein that functions with the control of virulence regulator/sensor TCS, which regulates approximately 15% of the GAS transcriptome. Whole-genome sequencing analysis of serotype M28 GAS strains collected from invasive infections in humans identified a higher number of missense (amino acid-altering) and nonsense (protein-truncating) polymorphisms in rocA than expected. We hypothesized that polymorphisms in RocA alter the global transcriptome and virulence of serotype M28 GAS. We used naturally occurring clinical isolates with rocA polymorphisms (n = 48), an isogenic rocA deletion mutant strain, and five isogenic rocA polymorphism mutant strains to perform genome-wide transcript analysis (RNA sequencing), in vitro virulence factor assays, and mouse and nonhuman primate pathogenesis studies to test this hypothesis. Results demonstrated that polymorphisms in rocA result in either a subtle transcriptome change, causing a wild-type-like virulence phenotype, or a substantial transcriptome change, leading to a significantly increased virulence phenotype. Each polymorphism had a unique effect on the global GAS transcriptome. Taken together, our data show that naturally occurring polymorphisms in one gene encoding an accessory protein can significantly alter the global transcriptome and virulence phenotype of GAS, an important human pathogen.

RocA Has Serotype-Specific Gene Regulatory and Pathogenesis Activities in Serotype M28 Group A Streptococcus.

Serotype M28 group A streptococcus (GAS) is a common cause of infections such as pharyngitis ("strep throat") and necrotizing fasciitis ("flesh-eating" disease). Relatively little is known about the molecular mechanisms underpinning M28 GAS pathogenesis. Whole-genome sequencing studies of M28 GAS strains recovered from patients with invasive infections found an unexpectedly high number of missense (amino acid-changing) and nonsense (protein-truncating) polymorphisms in rocA (regulator of Cov), leading us to hypothesize that altered RocA activity contributes to M28 GAS molecular pathogenesis. To test this hypothesis, an isogenic rocA deletion mutant strain was created. Transcriptome sequencing (RNA-seq) analysis revealed that RocA inactivation significantly alters the level of transcripts for 427 and 323 genes at mid-exponential and early stationary growth phases, respectively, including genes for 41 transcription regulators and 21 virulence factors. In contrast, RocA transcriptomes from other GAS M protein serotypes are much smaller and include fewer transcription regulators. The rocA mutant strain had significantly increased secreted activity of multiple virulence factors and grew to significantly higher colony counts under acid stress in vitro RocA inactivation also significantly increased GAS virulence in a mouse model of necrotizing myositis. Our results demonstrate that RocA is an important regulator of transcription regulators and virulence factors in M28 GAS and raise the possibility that naturally occurring polymorphisms in rocA in some fashion contribute to human invasive infections caused by M28 GAS strains.

Contribution of Secreted NADase and Streptolysin O to the Pathogenesis of Epidemic Serotype M1 Streptococcus pyogenes Infections.

Streptococcus pyogenes secretes many toxins that facilitate human colonization, invasion, and dissemination. NADase (SPN) and streptolysin O (SLO) are two toxins that play important roles in pathogenesis. We previously showed that increased production of SPN and SLO in epidemic serotype M1 and M89 S. pyogenes strains is associated with rapid intercontinental spread and enhanced virulence. The biological functions of SPN and SLO have been extensively studied using eukaryotic cell lines, but the relative contribution of each of these two toxins to pathogenesis of epidemic M1 or M89 strains remains unexplored. Herein, using a genetically representative epidemic M1 strain and a panel of isogenic mutant derivative strains, we evaluated the relative contributions of SPN and SLO toxins to virulence in mouse models of necrotizing myositis, bacteremia, and skin and soft tissue infection. We found that isogenic mutants lacking SPN, SLO, and both toxins are equally impaired in ability to cause necrotizing myositis. In addition, mutants lacking either SPN or SLO are significantly attenuated in the bacteremia and soft tissue infection models, and the mutant strain lacking production of both toxins is further attenuated. The mutant strain lacking both SPN and SLO production is severely attenuated in ability to resist killing by human polymorphonuclear leukocytes. We conclude that both SPN and SLO contribute significantly to S. pyogenes pathogenesis in these virulence assays.

Intergenic Variable-Number Tandem-Repeat Polymorphism Upstream of rocA Alters Toxin Production and Enhances Virulence in Streptococcus pyogenes.

Variable-number tandem-repeat (VNTR) polymorphisms are ubiquitous in bacteria. However, only a small fraction of them has been functionally studied. Here, we report an intergenic VNTR polymorphism that confers an altered level of toxin production and increased virulence in Streptococcus pyogenes The nature of the polymorphism is a one-unit deletion in a three-tandem-repeat locus upstream of the rocA gene encoding a sensor kinase. S. pyogenes strains with this type of polymorphism cause human infection and produce significantly larger amounts of the secreted cytotoxins S. pyogenes NADase (SPN) and streptolysin O (SLO). Using isogenic mutant strains, we demonstrate that deleting one or more units of the tandem repeats abolished RocA production, reduced CovR phosphorylation, derepressed multiple CovR-regulated virulence factors (such as SPN and SLO), and increased virulence in a mouse model of necrotizing fasciitis. The phenotypic effect of the VNTR polymorphism was nearly the same as that of inactivating the rocA gene. In summary, we identified and characterized an intergenic VNTR polymorphism in S. pyogenes that affects toxin production and virulence. These new findings enhance understanding of rocA biology and the function of VNTR polymorphisms in S. pyogenes.

Deletion analysis of Streptococcus pneumoniae late competence genes distinguishes virulence determinants that are dependent or independent of competence induction.

The competence regulon of Streptococcus pneumoniae (pneumococcus) is crucial for genetic transformation. During competence development, the alternative sigma factor ComX is activated, which in turn, initiates transcription of 80 'late' competence genes. Interestingly, only 16 late genes are essential for genetic transformation. We hypothesized that these late genes that are dispensable for competence are beneficial to pneumococcal fitness during infection. These late genes were systematically deleted, and the resulting mutants were examined for their fitness during mouse models of bacteremia and acute pneumonia. Among these, 14 late genes were important for fitness in mice. Significantly, deletion of some late genes attenuated pneumococcal fitness to the same level in both wild-type and ComX-null genetic backgrounds, suggesting that the constitutive baseline expression of these genes was important for bacterial fitness. In contrast, some mutants were attenuated only in the wild-type genetic background but not in the ComX-null background, suggesting that specific expression of these genes during competence state contributed to pneumococcal fitness. Increased virulence during competence state was partially caused by the induction of allolytic enzymes that enhanced pneumolysin release. These results distinguish the role of basal expression versus competence induction in virulence functions encoded by ComX-regulated late competence genes.

Disentangling competence for genetic transformation and virulence in Streptococcus pneumoniae.

Horizontal gene transfer mediated by the competence regulon is a major driver of genome plasticity in Streptococcus pneumoniae. When pneumococcal cells enter the competent state, about 6% of the genes in the genome are up-regulated. Among these, some genes are essential for genetic transformation while others are dispensable for the process. Exhaustive deletion analyses show that some up-regulated genes dispensable for genetic transformation contribute to pneumococcal-mediated pneumonia and bacteremia infections. Interestingly, virulence functions of such genes are either dependent or independent of the competent state. Among the competent-state-dependent genes are those mediating allolysis, a process where small fraction of non-competent cells within the pneumococcal population are lysed by their competent counterparts, releasing DNA presumably for transformation. Inadvertently, the pore-forming toxin pneumolysin is also released during allolysis, contributing to virulence. In this review, we discuss recent advances in our understanding of pneumococcal virulence processes mediated by the competence regulon. We proposed that coupling of competence induction and bacterial fitness drives the natural selection to favor an intact competence regulon, which in turn, provides the long-term benefits of genetic plasticity.

Inhibition of competence development, horizontal gene transfer and virulence in Streptococcus pneumoniae by a modified competence stimulating peptide.

Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae.

Integrative Reverse Genetic Analysis Identifies Polymorphisms Contributing to Decreased Antimicrobial Agent Susceptibility in Streptococcus pyogenes.

Identification of genetic polymorphisms causing increased antibiotic resistance in bacterial pathogens traditionally has proceeded from observed phenotype to defined mutant genotype. The availability of large collections of microbial genome sequences that lack antibiotic susceptibility metadata provides an important resource and opportunity to obtain new information about increased antimicrobial resistance by a reverse genotype-to-phenotype bioinformatic and experimental workflow. We analyzed 26,465 genome sequences of Streptococcus pyogenes, a human pathogen causing 700 million infections annually. The population genomic data identified amino acid changes in penicillin-binding proteins 1A, 1B, 2A, and 2X with signatures of evolution under positive selection as potential candidates for causing decreased susceptibility to ß-lactam antibiotics. Construction and analysis of isogenic mutant strains containing individual amino acid replacements in penicillin-binding protein 2X (PBP2X) confirmed that the identified residues produced decreased susceptibility to penicillin. We also discovered the first chimeric PBP2X in S. pyogenes and show that strains containing it have significantly decreased ß-lactam susceptibility. The novel integrative reverse genotype-to-phenotype strategy presented is broadly applicable to other pathogens and likely will lead to new knowledge about antimicrobial agent resistance, a massive public health problem worldwide. IMPORTANCE The recent demonstration that naturally occurring amino acid substitutions in Streptococcus pyogenes PBP2X are sufficient to cause severalfold reduced susceptibility to multiple ß-lactam antibiotics in vitro raises the concern that these therapeutic agents may become compromised. Substitutions in PBP2X are common first-step mutations that, with the incremental accumulation of additional adaptive mutations within the PBPs, can result in high-level resistance. Because ß-lactam susceptibility testing is not routinely performed, the nature and extent of such substitutions within the PBPs of S. pyogenes are poorly characterized. To address this knowledge deficit, polymorphisms in the PBPs were identified among the most comprehensive cohort of S. pyogenes genome sequences investigated to date. The mutational processes and selective forces acting on the PBPs were assessed to identify specific substitutions likely to influence ß-lactam susceptibility and to evaluate factors posited to be impediments to resistance emergence.

Surfactant phospholipids act as molecular switches for premature induction of quorum sensing-dependent virulence in Pseudomonas aeruginosa.

The virulence behaviors of many Gram-negative bacterial pathogens are governed by quorum-sensing (QS), a hierarchical system of gene regulation that relies on population density by producing and detecting extracellular signaling molecules. Although extensively studied under in vitro conditions, adaptation of QS system to physiologically relevant host environment is not fully understood. In this study, we investigated the influence of lung environment on the regulation of Pseudomonas aeruginosa virulence factors by QS in a mouse model of acute pneumonia. When cultured under laboratory conditions in lysogeny broth, wild-type P. aeruginosa strain PAO1 began to express QS-regulated virulence factors elastase B (LasB) and rhamnolipids (RhlA) during transition from late-exponential into stationary growth phase. In contrast, during acute pneumonia as well as when cultured in mouse bronchial alveolar lavage fluids (BALF), exponential phase PAO1 bacteria at low population density prematurely expressed QS regulatory genes lasI-lasR and rhlI-rhlR and their downstream virulence genes lasB and rhlA. Further analysis indicated that surfactant phospholipids were the primary components within BALF that induced the synthesis of N-(3-oxododecanoyl)-L-homoserine lactone (C12-HSL), which triggered premature expression of LasB and RhlA. Both phenol extraction and phospholipase A2 digestion abolished the ability of mouse BALF to promote LasB and RhlA expression. In contrast, provision of the major surfactant phospholipid dipalmitoylphosphatidylcholine (DPPC) restored the expression of both virulence factors. Collectively, our study demonstrates P. aeruginosa modulates its QS to coordinate the expression of virulence factors during acute pneumonia by recognizing pulmonary surfactant phospholipids.

New Pathogenesis Mechanisms and Translational Leads Identified by Multidimensional Analysis of Necrotizing Myositis in Primates.

A fundamental goal of contemporary biomedical research is to understand the molecular basis of disease pathogenesis and exploit this information to develop targeted and more-effective therapies. Necrotizing myositis caused by the bacterial pathogen Streptococcus pyogenes is a devastating human infection with a high mortality rate and few successful therapeutic options. We used dual transcriptome sequencing (RNA-seq) to analyze the transcriptomes of S. pyogenes and host skeletal muscle recovered contemporaneously from infected nonhuman primates. The in vivo bacterial transcriptome was strikingly remodeled compared to organisms grown in vitro, with significant upregulation of genes contributing to virulence and altered regulation of metabolic genes. The transcriptome of muscle tissue from infected nonhuman primates (NHPs) differed significantly from that of mock-infected animals, due in part to substantial changes in genes contributing to inflammation and host defense processes. We discovered significant positive correlations between group A streptococcus (GAS) virulence factor transcripts and genes involved in the host immune response and inflammation. We also discovered significant correlations between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness, as assessed by previously conducted genome-wide transposon-directed insertion site sequencing (TraDIS). By integrating the bacterial RNA-seq data with the fitness data generated by TraDIS, we discovered five new pathogen genes, namely, S. pyogenes 0281 (Spy0281 [dahA]), ihk-irr, slr, isp, and ciaH, that contribute to necrotizing myositis and confirmed these findings using isogenic deletion-mutant strains. Taken together, our study results provide rich new information about the molecular events occurring in severe invasive infection of primate skeletal muscle that has extensive translational research implications.IMPORTANCE Necrotizing myositis caused by Streptococcus pyogenes has high morbidity and mortality rates and relatively few successful therapeutic options. In addition, there is no licensed human S. pyogenes vaccine. To gain enhanced understanding of the molecular basis of this infection, we employed a multidimensional analysis strategy that included dual RNA-seq and other data derived from experimental infection of nonhuman primates. The data were used to target five streptococcal genes for pathogenesis research, resulting in the unambiguous demonstration that these genes contribute to pathogen-host molecular interactions in necrotizing infections. We exploited fitness data derived from a recently conducted genome-wide transposon mutagenesis study to discover significant correlation between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness. Collectively, our findings have significant implications for translational research, potentially including vaccine efforts.

Genetic heterogeneity of the Spy1336/R28-Spy1337 virulence axis in Streptococcus pyogenes and effect on gene transcript levels and pathogenesis.

Streptococcus pyogenes is a strict human pathogen responsible for more than 700 million infections annually worldwide. Strains of serotype M28 S. pyogenes are typically among the five more abundant types causing invasive infections and pharyngitis in adults and children. Type M28 strains also have an unusual propensity to cause puerperal sepsis and neonatal disease. We recently discovered that a one-nucleotide indel in an intergenic homopolymeric tract located between genes Spy1336/R28 and Spy1337 altered virulence in a mouse model of infection. In the present study, we analyzed size variation in this homopolymeric tract and determined the extent of heterogeneity in the number of tandemly-repeated 79-amino acid domains in the coding region of Spy1336/R28 in large samples of strains recovered from humans with invasive infections. Both repeat sequence elements are highly polymorphic in natural populations of M28 strains. Variation in the homopolymeric tract results in (i) changes in transcript levels of Spy1336/R28 and Spy1337 in vitro, (ii) differences in virulence in a mouse model of necrotizing myositis, and (iii) global transcriptome changes as shown by RNAseq analysis of isogenic mutant strains. Variation in the number of tandem repeats in the coding sequence of Spy1336/R28 is responsible for size variation of R28 protein in natural populations. Isogenic mutant strains in which genes encoding R28 or transcriptional regulator Spy1337 are inactivated are significantly less virulent in a nonhuman primate model of necrotizing myositis. Our findings provide impetus for additional studies addressing the role of R28 and Spy1337 variation in pathogen-host interactions.