ABSTRACT
Bacterial species often consist of strains with variable gene content, collectively referred to as the pangenome. Variations in the genetic makeup of strains can alter bacterial physiology and fitness. To define biologically relevant genes of a genome, genome-wide transposon mutant libraries have been used to identify genes essential for survival or virulence in a given strain. Such phenotypic studies have been conducted in four different genotypes of the human pathogen Streptococcus pyogenes, yet challenges exist in comparing results across studies conducted in different genetic backgrounds and conditions. To advance genotype to phenotype inferences across different S. pyogenes strains, we built a pangenome database of 249 S. pyogenes reference genomes. We systematically re-analyzed publicly available transposon sequencing datasets from S. pyogenes using a transposon sequencing-specific analysis pipeline, Transit. Across four genetic backgrounds and nine phenotypic conditions, 355 genes were essential for survival, corresponding to ~24% of the core genome. Clusters of Orthologous Genes (COG) categories related to coenzyme and lipid transport and growth functions were overrepresented as essential. Finally, essential operons across S. pyogenes genotypes were defined, with an increased number of essential operons detected under in vivo conditions. This study provides an extendible database to which new studies can be added, and a searchable html-based resource to direct future investigations into S. pyogenes biology.IMPORTANCEStreptococcus pyogenes is a human-adapted pathogen occupying restricted ecological niches. Understanding the essentiality of genes across different strains and experimental conditions is important to direct research questions and efforts to prevent the large burden of disease caused by S. pyogenes. To this end we systematically reanalyzed transposon sequencing studies in S. pyogenes using transposon sequencing-specific methods, integrating them into an extendible meta-analysis framework. This provides a repository of gene essentiality in S. pyogenes which was used to highlight specific genes of interest and for the community to guide future phenotypic studies.
Subject(s)
DNA Transposable Elements , Genes, Essential , Genome, Bacterial , Streptococcus pyogenes , Streptococcus pyogenes/genetics , Genes, Essential/genetics , Genome, Bacterial/genetics , DNA Transposable Elements/genetics , Humans , Genotype , Virulence/genetics , Streptococcal Infections/microbiology , Phenotype , Operon/geneticsABSTRACT
Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.
Subject(s)
Streptococcal Infections , Streptococcus , Vaccines , Humans , Streptococcus pyogenes/genetics , Gene FlowABSTRACT
Streptococcus pyogenes (Group A Streptococcus; GAS) is exquisitely adapted to the human host, resulting in asymptomatic infection, pharyngitis, pyoderma, scarlet fever or invasive diseases, with potential for triggering post-infection immune sequelae. GAS deploys a range of virulence determinants to allow colonization, dissemination within the host and transmission, disrupting both innate and adaptive immune responses to infection. Fluctuating global GAS epidemiology is characterized by the emergence of new GAS clones, often associated with the acquisition of new virulence or antimicrobial determinants that are better adapted to the infection niche or averting host immunity. The recent identification of clinical GAS isolates with reduced penicillin sensitivity and increasing macrolide resistance threatens both frontline and penicillin-adjunctive antibiotic treatment. The World Health Organization (WHO) has developed a GAS research and technology road map and has outlined preferred vaccine characteristics, stimulating renewed interest in the development of safe and effective GAS vaccines.
Subject(s)
Anti-Bacterial Agents , Streptococcal Infections , Humans , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Macrolides/pharmacology , Macrolides/therapeutic use , Drug Resistance, Bacterial , Streptococcal Infections/drug therapy , Streptococcal Infections/epidemiology , Streptococcal Infections/prevention & control , Streptococcus pyogenes/genetics , Penicillins/therapeutic useABSTRACT
A new variant of Streptococcus pyogenes serotype M1 (designated 'M1UK') has been reported in the United Kingdom, linked with seasonal scarlet fever surges, marked increase in invasive infections, and exhibiting enhanced expression of the superantigen SpeA. The progenitor S. pyogenes 'M1global' and M1UK clones can be differentiated by 27 SNPs and 4 indels, yet the mechanism for speA upregulation is unknown. Here we investigate the previously unappreciated expansion of M1UK in Australia, now isolated from the majority of serious infections caused by serotype M1 S. pyogenes. M1UK sub-lineages circulating in Australia also contain a novel toxin repertoire associated with epidemic scarlet fever causing S. pyogenes in Asia. A single SNP in the 5' transcriptional leader sequence of the transfer-messenger RNA gene ssrA drives enhanced SpeA superantigen expression as a result of ssrA terminator read-through in the M1UK lineage. This represents a previously unappreciated mechanism of toxin expression and urges enhanced international surveillance.
Subject(s)
Scarlet Fever , Streptococcal Infections , Humans , Streptococcus pyogenes/genetics , Scarlet Fever/epidemiology , Superantigens , Bacterial Proteins/genetics , United Kingdom , Exotoxins/genetics , Mutation , AustraliaABSTRACT
Free online tools for bacterial genome analyses are available for local infection surveillance at hospitals. The tools do not require bioinformatic expertise and provide rapid actionable results. Within half a year carbapenemase producing Enterobacter cloacae was reported in clinical samples from three patients who had been hospitalized at the same ward. The aim of this outbreak investigation was to characterize and compare genomes of the isolated bacteria in order to determine molecular evidence of hospital transmission. The three isolates and two isolates reported as susceptible to carbapenems were locally analyzed by whole genome sequencing (WGS). Draft genome assembly, species identification, phylogenetic analyses, typing, resistance gene determination, and plasmid analyses were carried out using free online tools from the Center for Genomic Epidemiology (CGE). Genome analyses identified all three suspected outbreak isolates as E. hormaechei carrying bla OXA-436 gene. Two of the suspected outbreak isolates were closely related, while one was substantially different from them. Horizontal transfer of plasmid may have taken place in the ward. Detailed knowledge on the genomic composition of bacteria in suspected hospital outbreaks can be obtained by free online tools and may reveal transfer of resistance genes between different strains in addition to dissemination of specific clones.
ABSTRACT
The nasopharynx and the skin are the major oxygen-rich anatomical sites for colonization by the human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]). To establish infection, GAS must survive oxidative stress generated during aerobic metabolism and the release of reactive oxygen species (ROS) by host innate immune cells. Glutathione is the major host antioxidant molecule, while GAS is glutathione auxotrophic. Here, we report the molecular characterization of the ABC transporter substrate binding protein GshT in the GAS glutathione salvage pathway. We demonstrate that glutathione uptake is critical for aerobic growth of GAS and that impaired import of glutathione induces oxidative stress that triggers enhanced production of the reducing equivalent NADPH. Our results highlight the interrelationship between glutathione assimilation, carbohydrate metabolism, virulence factor production, and innate immune evasion. Together, these findings suggest an adaptive strategy employed by extracellular bacterial pathogens to exploit host glutathione stores for their own benefit. IMPORTANCE During infection, microbes must escape host immune responses and survive exposure to reactive oxygen species produced by immune cells. Here, we identify the ABC transporter substrate binding protein GshT as a key component of the glutathione salvage pathway in glutathione-auxotrophic GAS. Host-acquired glutathione is crucial to the GAS antioxidant defense system, facilitating escape from the host innate immune response. This study demonstrates a direct link between glutathione assimilation, aerobic metabolism, and virulence factor production in an important human pathogen. Our findings provide mechanistic insight into host adaptation that enables extracellular bacterial pathogens such as GAS to exploit the abundance of glutathione in the host cytosol for their own benefit.
Subject(s)
Streptococcal Infections , Streptococcus pyogenes , ATP-Binding Cassette Transporters/metabolism , Antioxidants/metabolism , Bacterial Proteins/metabolism , Glutathione/metabolism , Humans , Immune Evasion , Reactive Oxygen Species/metabolism , Streptococcal Infections/microbiology , Streptococcus pyogenes/metabolism , Virulence Factors/metabolismABSTRACT
Streptococcus pyogenes is one of the Top 10 human infectious disease killers worldwide causing a range of clinical manifestations in humans. Colonizing a range of ecological niches within its sole host, the human, is key to the ability of this opportunistic pathogen to cause direct and post-infectious manifestations. The expansion of genome sequencing capabilities and data availability over the last decade has led to an improved understanding of the evolutionary dynamics of this pathogen within a global framework where epidemiological relationships and evolutionary mechanisms may not be universal. This review uses the recent publication by Davies et al., 2019 as an updated global framework to address S. pyogenes population genomics, highlighting how genomics is being used to gain new insights into evolutionary processes, transmission pathways, and vaccine design.
Subject(s)
Genome, Bacterial , Genomics , Streptococcal Infections/epidemiology , Streptococcal Infections/microbiology , Streptococcus pyogenes/genetics , Genomics/methods , Global Health , Humans , Molecular Epidemiology , Population Surveillance , Streptococcus pyogenes/classificationABSTRACT
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 ΦHKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here, we report the molecular characterization of ΦHKU.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 ΦHKU.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 ΦHKU.vir exotoxins.