Matrix-assisted laser desorption ionization-time of flight and comparative genomic analysis of M-18 group a Streptococcus strains associated with an acute rheumatic fever outbreak in northeast Italy in 2012 and 2013.
ABSTRACT: Acute rheumatic fever (ARF) is a postsuppurative sequela caused by Streptococcus pyogenes infections affecting school-age children. We describe here the occurrence of an ARF outbreak that occurred in Bologna province, northeastern Italy, between November 2012 and May 2013. Molecular analysis revealed that ARF-related group A Streptococcus (GAS) strains belonged to the M-18 serotype, including subtypes emm18.29 and emm18.32. All M-18 GAS strains shared the same antigenic profile, including SpeA, SpeB, SpeC, SpeL, SpeM, and SmeZ. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis revealed that M-18 GAS strains grouped separately from other serotypes, suggesting a different S. pyogenes lineage. Single nucleotide polymorphisms and phylogenetic analysis based on whole-genome sequencing showed that emm18.29 and emm18.32 GAS strains clustered in two distinct groups, highlighting genetic variations between these subtypes. Comparative analysis revealed a similar genome architecture between emm18.29 and emm18.32 strains that differed from noninvasive emm18.0 strains. The major sources of differences between M-18 genomes were attributable to the prophage elements. Prophage regions contained several virulence factors that could have contributed to the pathogenic potential of emm18.29 and emm18.32 strains. Notably, phage ?SPBO.1 carried erythrogenic toxin A gene (speA1) in six ARF-related M-18 GAS strains but not in emm18.0 strains. In addition, a phage-encoded hyaluronidase gene (hylP.2) presented different variants among M-18 GAS strains by showing internal deletions located in the ?-helical and TS?H regions. In conclusion, our study yielded insights into the genome structure of M-18 GAS strains responsible for the ARF outbreak in Italy, thus expanding our knowledge of this serotype.
Project description:Acute rheumatic fever (ARF), a sequelae of group A Streptococcus (GAS) infection, is the most common cause of preventable childhood heart disease worldwide. The molecular basis of ARF and the subsequent rheumatic heart disease are poorly understood. Serotype M18 GAS strains have been associated for decades with ARF outbreaks in the U.S. As a first step toward gaining new insight into ARF pathogenesis, we sequenced the genome of strain MGAS8232, a serotype M18 organism isolated from a patient with ARF. The genome is a circular chromosome of 1,895,017 bp, and it shares 1.7 Mb of closely related genetic material with strain SF370 (a sequenced serotype M1 strain). Strain MGAS8232 has 178 ORFs absent in SF370. Phages, phage-like elements, and insertion sequences are the major sources of variation between the genomes. The genomes of strain MGAS8232 and SF370 encode many of the same proven or putative virulence factors. Importantly, strain MGAS8232 has genes encoding many additional secreted proteins involved in human-GAS interactions, including streptococcal pyrogenic exotoxin A (scarlet fever toxin) and two uncharacterized pyrogenic exotoxin homologues, all phage-associated. DNA microarray analysis of 36 serotype M18 strains from diverse localities showed that most regions of variation were phages or phage-like elements. Two epidemics of ARF occurring 12 years apart in Salt Lake City, UT, were caused by serotype M18 strains that were genetically identical, or nearly so. Our analysis provides a critical foundation for accelerated research into ARF pathogenesis and a molecular framework to study the plasticity of GAS genomes.
Project description:The 1,815,783-bp genome of a serotype M49 strain of Streptococcus pyogenes (group A streptococcus [GAS]), strain NZ131, has been determined. This GAS strain (FCT type 3; emm pattern E), originally isolated from a case of acute post-streptococcal glomerulonephritis, is unusually competent for electrotransformation and has been used extensively as a model organism for both basic genetic and pathogenesis investigations. As with the previously sequenced S. pyogenes genomes, three unique prophages are a major source of genetic diversity. Two clustered regularly interspaced short palindromic repeat (CRISPR) regions were present in the genome, providing genetic information on previous prophage encounters. A unique cluster of genes was found in the pathogenicity island-like emm region that included a novel Nudix hydrolase, and, further, this cluster appears to be specific for serotype M49 and M82 strains. Nudix hydrolases eliminate potentially hazardous materials or prevent the unbalanced accumulation of normal metabolites; in bacteria, these enzymes may play a role in host cell invasion. Since M49 S. pyogenes strains have been known to be associated with skin infections, the Nudix hydrolase and its associated genes may have a role in facilitating survival in an environment that is more variable and unpredictable than the uniform warmth and moisture of the throat. The genome of NZ131 continues to shed light upon the evolutionary history of this human pathogen. Apparent horizontal transfer of genetic material has led to the existence of highly variable virulence-associated regions that are marked by multiple rearrangements and genetic diversification while other regions, even those associated with virulence, vary little between genomes. The genome regions that encode surface gene products that will interact with host targets or aid in immune avoidance are the ones that display the most sequence diversity. Thus, while natural selection favors stability in much of the genome, it favors diversity in these regions.
Project description:The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 (emm12) Streptococcus pyogenes (group A Streptococcus, GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage ?HKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here, we report the molecular characterization of ?HKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for resistance to neutrophil killing. Investigating single, double and triple isogenic knockout mutants of the ?HKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the pathogenesis of scarlet fever-causing GAS mediated by prophage ?HKU.vir exotoxins.
Project description:The pathogenesis of acute rheumatic fever (ARF) is poorly understood. We identified two contiguous bacteriophage genes, designated speL and speM, encoding novel inferred superantigens in the genome sequence of an ARF strain of serotype M18 group A streptococcus (GAS). speL and speM were located at the same genomic site in 33 serotype M18 isolates, and no nucleotide sequence diversity was observed in the 33 strains analyzed. Furthermore, the genes were absent in 13 non-M18 strains tested. These data indicate a recent acquisition event by a distinct clone of serotype M18 GAS. speL and speM were transcribed in vitro and upregulated in the exponential phase of growth. Purified SpeL and SpeM were pyrogenic and mitogenic for rabbit splenocytes and human peripheral blood mononuclear cells in picogram amounts. SpeL preferentially expanded human T cells expressing T-cell receptors Vbeta1, Vbeta5.1, and Vbeta23, and SpeM had specificity for Vbeta1 and Vbeta23 subsets, indicating that both proteins had superantigen activity. SpeL was lethal in two animal models of streptococcal toxic shock, and SpeM was lethal in one model. Serologic studies indicated that ARF patients were exposed to serotype M18 GAS, SpeL, and SpeM. The data demonstrate that SpeL and SpeM are pyrogenic toxin superantigens and suggest that they may participate in the host-pathogen interactions in some ARF patients.
Project description:Streptococcus pyogenes (group A streptococcus [GAS]) is a frequent cause of purulent infections in humans. As potentially important aspects of its pathogenicity, GAS was recently shown to aggregate, form intratissue microcolonies, and potentially participate in multispecies biofilms. In this study, we show that GAS in fact forms monospecies biofilms in vitro, and we analyze the basic parameters of S. pyogenes in vitro biofilm formation, using Streptococcus epidermidis as a biofilm-positive control. Of nine clinically important serotype strains, M2, M6, M14, and M18 were found to significantly adhere to coated and uncoated polystyrene surfaces. Fibronectin and collagen types I and IV best supported primary adherence of serotype M2 and M18 strains, respectively, whereas serotype M6 and M14 strains strongly bound to uncoated polystyrene surfaces. Absorption measurements of safranin staining, as well as electron scanning and confocal laser scanning microscopy, documented that primary adherence led to subsequent formation of three-dimensional biofilm structures consisting of up to 46 bacterial layers. Of note, GAS isolates belonging to the same serotype were found to be very heterogeneous in their biofilm-forming behavior. Biofilm formation was equally efficient under static and continuous flow conditions and consisted of the classical three steps, including partial disintegration after long-term incubation. Activity of the SilC signaling peptide as a component of a putative quorum-sensing system was found to influence the biofilm structure and density of serotype M14 and M18 strains. Based on the presented methods and results, standardized analyses of GAS biofilms and their impact on GAS pathogenicity are now feasible.
Project description:Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (KanR) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (SmS), into a targeted prophage on the chromosome of a streptomycin resistant (SmR) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the KanS/SmR phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1??, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1?? to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and further elucidate how the presence of prophage may affect overall streptococcal survival, pathogenicity, and evolution.
Project description:In recent years we have studied the relationship between strain genotypes and patient phenotypes in group A Streptococcus (GAS), a model human bacterial pathogen that causes extensive morbidity and mortality worldwide. We have concentrated our efforts on serotype M3 organisms because these strains are common causes of pharyngeal and invasive infections, produce unusually severe invasive infections, and can exhibit epidemic behavior. Our studies have been hindered by the lack of genome-scale phylogenies of multiple GAS strains and whole-genome sequences of multiple serotype M3 strains recovered from individuals with defined clinical phenotypes. To remove some of these impediments, we sequenced to closure the genome of four additional GAS strains and conducted comparative genomic resequencing of 12 contemporary serotype M3 strains representing distinct genotypes and phenotypes. Serotype M3 strains are a single phylogenetic lineage. Strains from asymptomatic throat carriers were significantly less virulent for mice than sterile-site isolates and evolved to a less virulent phenotype by multiple genetic pathways. Strain persistence or extinction between epidemics was strongly associated with presence or absence, respectively, of the prophage encoding streptococcal pyrogenic exotoxin A. A serotype M3 clone significantly underrepresented among necrotizing fasciitis cases has a unique frameshift mutation that truncates MtsR, a transcriptional regulator controlling expression of genes encoding iron-acquisition proteins. Expression microarray analysis of this clone confirmed significant alteration in expression of genes encoding iron metabolism proteins. Our analysis provided unprecedented detail about the molecular anatomy of bacterial strain genotype-patient phenotype relationships.
Project description:Group Astreptococcus (GAS) is a gram-positive bacterial pathogen that causes various suppurative infections and nonsuppurative sequelae. Since the late 1980s, streptococcal toxic-shock like syndrome (STSS) and severe invasive GAS infections have been reported globally. Here we sequenced the genome of serotype M3 strain SSI-1, isolated from an STSS patient in Japan, and compared it with those of other GAS strains. The SSI-1 genome is composed of 1,884,275 bp, and 1.7 Mb of the sequence is highly conserved relative to strain SF370 (serotype M1) and MGAS8232 (serotype M18), and almost completely conserved relative to strain MGAS315 (serotype M3). However, a large genomic rearrangement has been shown to occur across the replication axis between the homologous rrn-comX1 regions and between two prophage-coding regions across the replication axis. Atotal of 1 Mb of chromosomal DNA is inverted across the replication axis. Interestingly, the recombinations between the prophage regions are within the phage genes, and the genes encoding superantigens and mitogenic factors are interchanged between two prophages. This genomic rearrangement occurs in 65% of clinical isolates (64/94) collected after 1990, whereas it is found in only 25% of clinical isolates (7/28) collected before 1985. These observations indicate that streptococcal phages represent important plasticity regions in the GAS chromosome where recombination between homologous phage genes can occur and result not only in new phage derivatives, but also in large chromosomal rearrangements.
Project description:Streptococcus pyogenes strains producing pyrogenic exotoxin A (scarlet fever toxin) have recently caused episodes of streptococcal toxic-shock-like syndrome (TSLS). We exploited knowledge of genetic diversity and relationships among exotoxin A-producing patient strains provided by multilocus enzyme electrophoresis to select strains for comparative sequencing of toxin genes. Our analysis identified four alleles of speA in natural populations, one of which (speA1) occurs in many distinct clonal lineages and is probably old. Two other alleles (speA2 and speA3), characterized solely by single amino acid substitutions, were each identified in single clones that together have caused the majority of TSLS episodes. It is unlikely that these alleles have had a long association with S. pyogenes clones. A fourth allele (speA4) also is present in a single phylogenetic lineage and is 9% divergent from the other three toxin alleles. An absence of synonomous (silent) nucleotide changes in speA2 and speA3 is unusual and suggests that the allelic variation is not selectively neutral, which implies that the toxins are not functionally equivalent. These results may be important in helping to understand the recent increase in frequency and severity of disease caused by S. pyogenes.
Project description:Despite the public health challenges associated with the emergence of new pathogenic bacterial strains and/or serotypes, there is a dearth of information regarding the molecular mechanisms that drive this variation. Here, we began to address the mechanisms behind serotype-specific variation between serotype M1 and M3 strains of the human pathogen Streptococcus pyogenes (the group A Streptococcus [GAS]). Spatially diverse contemporary clinical serotype M3 isolates were discovered to contain identical inactivating mutations within genes encoding two regulatory systems that control the expression of important virulence factors, including the thrombolytic agent streptokinase, the protease inhibitor-binding protein-G-related ?2-macroglobulin-binding (GRAB) protein, and the antiphagocytic hyaluronic acid capsule. Subsequent analysis of a larger collection of isolates determined that M3 GAS, since at least the 1920s, has harbored a 4-bp deletion in the fasC gene of the fasBCAX regulatory system and an inactivating polymorphism in the rivR regulator-encoding gene. The fasC and rivR mutations in M3 isolates directly affect the virulence factor profile of M3 GAS, as evident by a reduction in streptokinase expression and an enhancement of GRAB expression. Complementation of the fasC mutation in M3 GAS significantly enhanced levels of the small regulatory RNA FasX, which in turn enhanced streptokinase expression. Complementation of the rivR mutation in M3 GAS restored the regulation of grab mRNA abundance but did not alter capsule mRNA levels. While important, the fasC and rivR mutations do not provide a full explanation for why serotype M3 strains are associated with unusually severe invasive infections; thus, further investigation is warranted.