Search

Humans commonly carry pathogenic bacteria asymptomatically, but despite decades of study, the underlying molecular contributors remain poorly understood. Here, we show that a group A streptococcus carriage strain contains a frameshift mutation in the hasA gene resulting in loss of hyaluronic acid capsule biosynthesis. This mutation was repaired by allelic replacement, resulting in restoration of capsule production in the isogenic derivative strain. The "repaired" isogenic strain was significantly more virulent than the carriage strain in a mouse model of necrotizing fasciitis and had enhanced growth ex vivo in human blood. Importantly, the repaired isogenic strain colonized the mouse oropharynx with significantly greater bacterial burden and had significantly reduced ability to internalize into cultured epithelial cells than the acapsular carriage strain. We conducted full-genome sequencing of 81 strains cultured serially from 19 epidemiologically unrelated human subjects and discovered the common theme that mutations negatively affecting capsule biosynthesis arise in vivo in the has operon. The significantly decreased capsule production is a key factor contributing to the molecular détente between pathogen and host. Our discoveries suggest a general model for bacterial pathogens in which mutations that downregulate or ablate virulence factor production contribute to carriage., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Infection with different strains of the same species of bacteria often results in vastly different clinical outcomes. Despite extensive investigation, the genetic basis of microbial strain-specific virulence remains poorly understood. Recent whole-genome sequencing has revealed that SNPs are the most prevalent form of genetic diversity among different strains of the same species of bacteria. For invasive serotype M3 group A streptococci (GAS) strains, the gene encoding regulator of proteinase B (RopB) has the highest frequency of SNPs. Here, we have determined that ropB polymorphisms alter RopB function and modulate GAS host-pathogen interactions. Sequencing of ropB in 171 invasive serotype M3 GAS strains identified 19 distinct ropB alleles. Inactivation of the ropB gene in strains producing distinct RopB variants had dramatically divergent effects on GAS global gene expression. Additionally, generation of isoallelic GAS strains differing only by a single amino acid in RopB confirmed that variant proteins affected transcript levels of the gene encoding streptococcal proteinase B, a major RopB-regulated virulence factor. Comparison of parental, RopB-inactivated, and RopB isoallelic strains in mouse infection models demonstrated that ropB polymorphisms influence GAS virulence and disease manifestations. These data detail a paradigm in which unbiased, whole-genome sequence analysis of populations of clinical bacterial isolates creates new avenues of productive investigation into the pathogenesis of common human infections.

Genome sequences are available for many bacterial strains, but there has been little progress in using these data to understand the molecular basis of pathogen emergence and differences in strain virulence. Serotype M3 strains of group A Streptococcus (GAS) are a common cause of severe invasive infections with unusually high rates of morbidity and mortality. To gain insight into the molecular basis of this high-virulence phenotype, we sequenced the genome of strain MGAS315, an organism isolated from a patient with streptococcal toxic shock syndrome. The genome is composed of 1,900,521 bp, and it shares approximately 1.7 Mb of related genetic material with genomes of serotype M1 and M18 strains. Phage-like elements account for the great majority of variation in gene content relative to the sequenced M1 and M18 strains. Recombination produces chimeric phages and strains with previously uncharacterized arrays of virulence factor genes. Strain MGAS315 has phage genes that encode proteins likely to contribute to pathogenesis, such as streptococcal pyrogenic exotoxin A (SpeA) and SpeK, streptococcal superantigen (SSA), and a previously uncharacterized phospholipase A(2) (designated Sla). Infected humans had anti-SpeK, -SSA, and -Sla antibodies, indicating that these GAS proteins are made in vivo. SpeK and SSA were pyrogenic and toxic for rabbits. Serotype M3 strains with the phage-encoded speK and sla genes increased dramatically in frequency late in the 20th century, commensurate with the rise in invasive disease caused by M3 organisms. Taken together, the results show that phage-mediated recombination has played a critical role in the emergence of a new, unusually virulent clone of serotype M3 GAS.

The identification of the molecular events responsible for strain emergence, enhanced virulence, and epidemicity has been a long-pursued goal in infectious diseases research. A recent analysis of 3,615 genomes of serotype M1 group A Streptococcus strains (the so-called 'flesh-eating' bacterium) identified a recombination event that coincides with the global M1 pandemic beginning in the early 1980s. Here, we have shown that the allelic variation that results from this recombination event, which replaces the chromosomal region encoding secreted NADase and streptolysin O, is the key driver of increased toxin production and enhanced infection severity of the M1 pandemic strains. Using isoallelic mutant strains, we found that 3 polymorphisms in this toxin gene region increase resistance to killing by human polymorphonuclear leukocytes, increase bacterial proliferation, and increase virulence in animal models of pharyngitis and necrotizing fasciitis. Genome sequencing of an additional 1,125 streptococcal strains and virulence studies revealed that a highly similar recombinational replacement event underlies an ongoing intercontinental epidemic of serotype M89 group A Streptococcus infections. By identifying the molecular changes that enhance upper respiratory tract fitness, increased resistance to innate immunity, and increased tissue destruction, we describe a mechanism that underpins epidemic streptococcal infections, which have affected many millions of people., Introduction One elusive goal in infectious diseases research is identification of the genetic changes and molecular mechanisms responsible for strain emergence, enhanced virulence, and epidemicity. This goal has practical importance [...]

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. [ABSTRACT FROM AUTHOR]

Background. Invasive group A Streptococcus (iGAS) disease caused by type emm89 strains has been increasing worldwide, driven by the emergence of an epidemic clonal variant (clade 3 emm89). The clinical characteristics of patients with emm89 iGAS disease, and in particular with clade 3 emm89 iGAS disease, are poorly described. Methods. We used population-based iGAS surveillance data collected in metropolitan Toronto, Ontario, Canada during the period 2000-2014. We sequenced the genomes of 105 emm89 isolates representing all emm89 iGAS disease cases in the area during the period and 138 temporally matched emm89 iGAS isolates collected elsewhere in Ontario. Results. Clades 1 and 2 and clade O, a newly discovered emm89 genetic variant, caused most cases of emm89 iGAS disease in metropolitan Toronto before 2008. After rapid emergence of new clade 3, previously circulating clades were purged from the population and the incidence of emm89 iGAS disease significantly increased from 0.14 per 100 000 in 2000-2007 to 0.22 per 100 000 in 2008-2014. Overall, emm89 organisms caused significantly more arthritis but less necrotizing fasciitis than strains of the more common type emm1. Other clinical presentations were soft tissue and severe respiratory tract infections. Clinical outcomes did not differ significantly between emm89 clades overall. However, clade 3 emm89 iGAS disease was more common in youth and middle-aged individuals. Conclusions. The rapid shift in emm89 iGAS strain genetics in metropolitan Toronto has resulted in a significant increase in the incidence of emm89 iGAS disease, with noticeably higher rates of clade 3 disease in younger patients. [ABSTRACT FROM AUTHOR]

Recent advances in the scale and diversity of population genomic datasets for bacteria now provide the potential for genome-wide patterns of co-evolution to be studied at the resolution of individual bases. Here we describe a new statistical method, genomeDCA, which uses recent advances in computational structural biology to identify the polymorphic loci under the strongest co-evolutionary pressures. We apply genomeDCA to two large population data sets representing the major human pathogens Streptococcus pneumoniae (pneumococcus) and Streptococcus pyogenes (group A Streptococcus). For pneumococcus we identified 5,199 putative epistatic interactions between 1,936 sites. Over three-quarters of the links were between sites within the pbp2x, pbp1a and pbp2b genes, the sequences of which are critical in determining non-susceptibility to beta-lactam antibiotics. A network-based analysis found these genes were also coupled to that encoding dihydrofolate reductase, changes to which underlie trimethoprim resistance. Distinct from these antibiotic resistance genes, a large network component of 384 protein coding sequences encompassed many genes critical in basic cellular functions, while another distinct component included genes associated with virulence. The group A Streptococcus (GAS) data set population represents a clonal population with relatively little genetic variation and a high level of linkage disequilibrium across the genome. Despite this, we were able to pinpoint two RNA pseudouridine synthases, which were each strongly linked to a separate set of loci across the chromosome, representing biologically plausible targets of co-selection. The population genomic analysis method applied here identifies statistically significantly co-evolving locus pairs, potentially arising from fitness selection interdependence reflecting underlying protein-protein interactions, or genes whose product activities contribute to the same phenotype. This discovery approach greatly enhances the future potential of epistasis analysis for systems biology, and can complement genome-wide association studies as a means of formulating hypotheses for targeted experimental work. [ABSTRACT FROM AUTHOR]

Background:  Many countries worldwide have reported increasing numbers of emm89 group A Streptococcus (GAS) infections during last decade. Pathogen genetic factors linked to this increase need assessment.Methods:  We investigated epidemiological characteristics of emm89 GAS bacteremic infections, including 7-day and 30-day case-fatality rates, in Finland during 2004-2014 and linked them to whole-genome sequencing data obtained from corresponding strains. The Fisher exact test and exact logistic regression were used to compare differences between bacteremic infections due to emm89 GAS belonging to different genetic clades and subclades.Results:  Out of 1928 cases of GAS bacteremic infection, 278 were caused by emm89 GAS. We identified 2 genetically distinct clades, arbitrarily designated clade 2 and clade 3. Both clades were present during 2004-2008, but clade 3 increased rapidly from 2009 onward. Six subclades (designated subclades A-F) were identified within clade 3, based on phylogenetic core genome analysis. The case-fatality rate differed significantly between subclades (P < .05), with subclade D having the highest 30-day estimated case-fatality rate (19% vs 3%-14%).Conclusions:  A new emm89 clone, clade 3, emerged in 2009 and spread rapidly in Finland. Patients infected with certain subclades of clade 3 were significantly more likely to die. A specific polymerase chain reaction assay was developed to follow the spread of subclade D in 2015. [ABSTRACT FROM AUTHOR]

Group A Streptococcus (GAS) causes a wide range of human infections, ranging from simple pharyngitis to life-threatening necrotizing fasciitis and toxic shock syndrome. A globally disseminated clone of M1T1 GAS has been associated with an increase in severe, invasive GAS infections in recent decades. The secreted GAS pore-forming toxin streptolysin O (SLO), which induces eukaryotic cell lysis in a cholesterol-dependent manner, is highly upregulated in the GAS M1T1 clone during bloodstream dissemination. SLO is known to promote GAS resistance to phagocytic clearance by neutrophils, a critical first element of host defense against invasive bacterial infection. Here, we examine the role of SLO in modulating specific neutrophil functions during their early interaction with GAS. We find that SLO at subcytotoxic concentrations and early time points is necessary and sufficient to suppress neutrophil oxidative burst, in a manner reversed by free cholesterol and anti-SLO blocking antibodies. In addition, SLO at subcytotoxic concentrations blocked neutrophil degranulation, interleukin-8 secretion and responsiveness, and elaboration of DNA-based neutrophil extracellular traps, cumulatively supporting a key role for SLO in GAS resistance to immediate neutrophil killing. A non-toxic SLO derivate elicits protective immunity against lethal GAS challenge in a murine infection model. We conclude that SLO exerts a novel cytotoxic-independent function at early stages of invasive infections (<30 min), contributing to GAS escape from neutrophil clearance. [ABSTRACT FROM AUTHOR]

Whole-genome sequencing of serotype M3 group A streptococci (GAS) from oropharyngeal and invasive infections in Ontario recently showed that the gene encoding regulator of protease B (RopB) is highly polymorphic in this population. To test the hypothesis that ropB is under diversifying selective pressure among all serotype M3 GAS strains, we sequenced this gene in 1178 strains collected from different infection types, geographic regions, and time periods. The results confirmed our hypothesis and discovered a significant association between mutant ropB alleles, decreased activity of its major regulatory target SpeB, and pharyngitis. Additionally, isoallelic strains with ropB polymorphisms were significantly less virulent in a mouse model of necrotizing fasciitis. These studies provide a model strategy for applying whole-genome sequencing followed by deep single-gene sequencing to generate new insight to the rapid evolution and virulence regulation of human pathogens. [ABSTRACT FROM AUTHOR]

Group A Streptococcus (GAS) is a human-adapted pathogen that causes a variety of diseases, including pharyngitis and invasive infections. GAS strains are categorized by variation in the nucleotide sequence of the gene (emm) that encodes the M protein. To identify the emm types of GAS strains causing pharyngitis in Ontario, Canada, we sequenced the hypervariable region of the emm gene in 4,635 pharyngeal GAS isolates collected during 2002-2010. The most prevalent emm types varied little from year to year. In contrast, fine-scale geographic analysis identified inter-site variability in the most common emm types. Additionally, we observed fluctuations in yearly frequency of emm3 strains from pharyngitis patients that coincided with peaks of emm3 invasive infections. We also discovered a striking increase in frequency of emm89 strains among isolates from patients with pharyngitis and invasive disease. These findings about the epidemiology of GAS are potentially useful for vaccine research. [ABSTRACT FROM AUTHOR]

α-Glucans such as starch and glycogen are abundant in the human oropharynx, the main site of group A Streptococcus (GAS) infection. However, the role in pathogenesis of GAS extracellular α-glucan binding and degrading enzymes is unknown. The serotype M1 GAS genome encodes two extracellular proteins putatively involved in α-glucan binding and degradation; pulA encodes a cell wall anchored pullulanase and amyA encodes a freely secreted putative cyclomaltodextrin α-glucanotransferase. Genetic inactivation of amyA, but not pulA, abolished GAS α-glucan degradation. The Δ amyA strain had a slower rate of translocation across human pharyngeal epithelial cells. Consistent with this finding, the Δ amyA strain was less virulent following mouse mucosal challenge. Recombinant AmyA degraded α-glucans into β-cyclomaltodextrins that reduced pharyngeal cell transepithelial resistance, providing a physiologic explanation for the observed transepithelial migration phenotype. Higher amyA transcript levels were present in serotype M1 GAS strains causing invasive infection compared with strains causing pharyngitis. GAS proliferation in a defined α-glucan-containing medium was dependent on the presence of human salivary α-amylase. These data delineate the molecular mechanisms by which α-glucan degradation contributes to GAS host–pathogen interaction, including how GAS uses human salivary α-amylase for its own metabolic benefit. [ABSTRACT FROM AUTHOR]

Outbreaks of disease attributable to human error or natural causes can provide unique opportunities to gain new information about host-pathogen interactions and new leads for pathogenesis research. Poststreptococcal glomerulonephritis (PSGN), a sequela of infection with pathogenic streptococci, is a common cause of preventable kidney disease worldwide. Although PSGN usually occurs after infection with group A streptococci, organisms of Lancefield group C and G also can be responsible. Despite decades of study, the molecular pathogenesis of PSGN is poorly understood. As a first step toward gaining new information about PSGN pathogenesis, we sequenced the genome of Streptococcus equi subsp. zooepidemicus strain MGCS10565, a group C organism that caused a very large and unusually severe epidemic of nephritis in Brazil. The genome is a circular chromosome of 2,024,171 bp. The genome shares extensive gene content, including many virulence factors, with genetically related group A streptococci, but unexpectedly lacks prophages. The genome contains many apparently foreign genes interspersed around the chromosome, consistent with the presence of a full array of genes required for natural competence. An inordinately large family of genes encodes secreted extracellular collagen-like proteins with multiple integrin-binding motifs. The absence of a gene related to speB rules out the long-held belief that streptococcal pyrogenic exotoxin B or antibodies reacting with it singularly cause PSGN. Many proteins previously implicated in GAS PSGN, such as streptokinase, are either highly divergent in strain MGCS10565 or are not more closely related between these species than to orthologs present in other streptococci that do not commonly cause PSGN. Our analysis provides a comparative genomics framework for renewed appraisal of molecular events underlying APSGN pathogenesis. [ABSTRACT FROM AUTHOR]

The human bacterial pathogen group A Streptococcus (GAS) causes many different diseases including pharyngitis, tonsillitis, impetigo, scarlet fever, streptococcal toxic shock syndrome, necrotizing fasciitis and myositis, and the post-infection sequelae glomerulonephritis and rheumatic fever. The frequency and severity of GAS infections increased in the 1980s and 1990s, but the cause of this increase is unknown. Recently, genome sequencing of serotype M1, M3 and M18 strains revealed many new proven or putative virulence factors that are encoded by phages or phage-like elements. Importantly, these genetic elements account for an unexpectedly large proportion of the difference in gene content between the three strains. These new genome-sequencing studies have provided evidence that temporally and geographically distinct epidemics, and the complex array of GAS clinical presentations, might be related in part to the acquisition or evolution of phage-encoded virulence factors. We anticipate that new phage-encoded virulence factors will be identified by sequencing the genomes of additional GAS strains, including organisms non-randomly associated with particular clinical syndromes. [Copyright &y& Elsevier]

The article discusses a study that identified a monoclonal cluster of invasive type emm59 group A streptococcus (GAS) infections in the San Francisco Bay area in California. The genomes of all available invasive emm59 GAS strains collected from 2000 to 2009 by the Active Bacterial Core surveillance arm of the U.S. Centers for Disease Control and Prevention were sequenced. It was shown that the level of genetic diversity seen among emm59 GAS strains in the U.S. was greater than that among strains from an epidemic in Canada. The authors also call for more intense monitoring for a potential shift in the epidemic behavior of emm59 GAS.

The identification of the molecular events responsible for strain emergence, enhanced virulence, and epidemicity has been a long-pursued goal in infectious diseases research. A recent analysis of 3,615 genomes of serotype M1 group A Streptococcus strains (the so-called "flesh-eating" bacterium) identified a recombination event that coincides with the global M1 pandemic beginning in the early 1980s. Here, we have shown that the allelic variation that results from this recombination event, which replaces the chromosomal region encoding secreted NADase and streptolysin O, is the key driver of increased toxin production and enhanced infection severity of the M1 pandemic strains. Using isoallelic mutant strains, we found that 3 polymorphisms in this toxin gene region increase resistance to killing by human polymorphonuclear leukocytes, increase bacterial proliferation, and increase virulence in animal models of pharyngitis and necrotizing fasciitis. Genome sequencing of an additional 1,125 streptococcal strains and virulence studies revealed that a highly similar recombinational replacement event underlies an ongoing intercontinental epidemic of serotype M89 group A Streptococcus infections. By identifying the molecular changes that enhance upper respiratory tract fitness, increased resistance to innate immunity, and increased tissue destruction, we describe a mechanism that underpins epidemic streptococcal infections, which have affected many millions of people. [ABSTRACT FROM AUTHOR]

Group A Streptococcus (GAS) is an important human pathogen that causes many types of infections, including pharyngitis and severe invasive diseases. We recently sequenced the genome of a serotype M3 strain and identified a prophage-encoded secreted phospholipase A2 designated SlaA. To study SlaA structure-activity relationships, 20 site-specific mutants were constructed by alanine-replacement mutagenesis and purified to apparent homogeneity. Enzymatic activity was greatly reduced by alanine replacement of amino acid residues previously described as crucial in the catalytic mechanism of secreted phospholipase A2. Similarly, substitution of five residues in an inferred Ca2+binding loop and three residues in the inferred active site region resulted in loss of activity of 76.5% or greater relative to the wild-type enzyme. Analysis of enzyme substrate specificity confirmed SlaA as a phospholipase A2, with activity against multiple phospholipid head groups and acyl chains located at the sn-2 position. PCR analysis of 1,189 GAS strains representing 48 M protein serotypes commonly causing human infections identified the slaA gene in 129 strains of nine serotypes (M1, M2, M3, M4, M6, M22, M28, M75, and st3757). Expression of SlaA by strains of these serotypes was confirmed by Western immunoblot. SlaA production increased rapidly and substantially on co-culture with Detroit 562 human pharyngeal epithelial cells. Together, these data provide new information about a novel extracellular enzyme that participates in GAS-human interactions. [ABSTRACT FROM AUTHOR]