RESUMO
Group A Streptococcal M-related proteins (Mrps) are dimeric α-helical-coiled-coil cell membrane-bound surface proteins. During infection, Mrp recruit the fragment crystallizable region of human immunoglobulin G via their A-repeat regions to the bacterial surface, conferring upon the bacteria enhanced phagocytosis resistance and augmented growth in human blood. However, Mrps show a high degree of sequence diversity, and it is currently not known whether this diversity affects the Mrp-IgG interaction. Herein, we report that diverse Mrps all bind human IgG subclasses with nanomolar affinity, with differences in affinity which ranged from 3.7 to 11.1 nM for mixed IgG. Using surface plasmon resonance, we confirmed Mrps display preferential IgG-subclass binding. All Mrps were found to have a significantly weaker affinity for IgG3 (p < 0.05) compared to all other IgG subclasses. Furthermore, plasma pulldown assays analyzed via Western blotting revealed that all Mrp were able to bind IgG in the presence of other serum proteins at both 25 °C and 37 °C. Finally, we report that dimeric Mrps bind to IgG with a 1:1 stoichiometry, enhancing our understanding of this important host-pathogen interaction.
Assuntos
Proteínas de Bactérias , Streptococcus pyogenes , Humanos , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Imunoglobulina G/metabolismo , Streptococcus pyogenes/metabolismoRESUMO
Group A Streptococcus (GAS) M and M-like proteins are essential virulence factors and represent the primary epidemiological marker of this pathogen. Protein sequences encoding 1054 M, Mrp and Enn proteins, from 1668 GAS genomes, were analysed by SplitsTree4, partitioning around medoids and co-occurrence. The splits network and groups-based analysis of all M and M-like proteins revealed four large protein groupings, with multiple evolutionary histories as represented by multiple edges for most splits, leading to 'M-family-groups' (FG) of protein sequences: FG I, Mrp; FG II, M protein and Protein H; FG III, Enn; and FG IV, M protein. M and Enn proteins formed two groups with nine sub-groups and Mrp proteins formed four groups with ten sub-groups. Discrete co-occurrence of M and M-like proteins were identified suggesting that while dynamic, evolution may be constrained by a combination of functional and virulence attributes. At a granular level, four distinct family-groups of M, Enn and Mrp proteins are observable, with Mrp representing the most genetically distinct of the family-group of proteins. While M and Enn protein families generally group into three distinct family-groups, horizontal and vertical gene flow between distinct GAS strains is ongoing.
Assuntos
Proteínas de Bactérias , Streptococcus pyogenes , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Fatores de Virulência/genéticaRESUMO
Antibiotic persistence is a phenomenon observed when genetically susceptible cells survive long-term exposure to antibiotics. These 'persisters' are an intrinsic component of bacterial populations and stem from phenotypic heterogeneity. Persistence to antibiotics is a concern for public health globally, as it increases treatment duration and can contribute to treatment failure. Furthermore, there is a growing array of evidence that persistence is a 'stepping-stone' for the development of genetic antimicrobial resistance. Urinary tract infections (UTIs) are a major contributor to antibiotic consumption worldwide, and are known to be both persistent (i.e. affecting the host for a prolonged period) and recurring. Currently, in clinical settings, routine laboratory screening of pathogenic isolates does not determine the presence or the frequency of persister cells. Furthermore, the majority of research undertaken on antibiotic persistence has been done on lab-adapted bacterial strains. In the study presented here, we characterized antibiotic persisters in a panel of clinical uropathogenic Escherichia coli isolates collected from hospitals in the UK and Australia. We found that a urine-pH mimicking environment not only induces higher levels of antibiotic persistence to meropenem and colistin than standard laboratory growth conditions, but also results in rapid development of transient colistin resistance, regardless of the genetic resistance profile of the isolate. Furthermore, we provide evidence for the presence of multiple virulence factors involved in stress resistance and biofilm formation in the genomes of these isolates, whose activities have been previously shown to contribute to the formation of persister cells.
Assuntos
Infecções por Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Humanos , Colistina/farmacologia , Meropeném/farmacologia , Meropeném/uso terapêutico , Escherichia coli Uropatogênica/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia , Bactérias/genética , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologiaRESUMO
The opportunistic pathogen Pseudomonas aeruginosa is ubiquitous in the environment, and in humans, it is capable of causing acute or chronic infections. In the natural environment, predation by bacterivorous protozoa represents a primary threat to bacteria. Here, we determined the impact of long-term exposure of P. aeruginosa to predation pressure. P. aeruginosa persisted when coincubated with the bacterivorous Acanthamoeba castellanii for extended periods and produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors, and this correlated with a reduction in expression of virulence traits. Virulence for the nematode Caenorhabditis elegans was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early-stage amoeba-adapted and nonadapted counterparts. Further, late-stage amoeba-adapted P. aeruginosa showed increased competitive fitness and enhanced survival in amoebae as well as in macrophage and neutrophils. Interestingly, our findings indicate that the selection imposed by amoebae resulted in P. aeruginosa isolates with reduced virulence and enhanced fitness, similar to those recovered from chronic cystic fibrosis infections. Thus, predation by protozoa and long-term colonization of the human host may represent similar environments that select for similar losses of gene function. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans, and chronic infections in immunocompromised and cystic fibrosis patients. This bacterium is commonly found in soils and water, where bacteria are constantly under threat of being consumed by bacterial predators, e.g., protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here, we examined the effect of long-term predation on the genotypes and phenotypes expressed by P. aeruginosa. We show that long-term coincubation with protozoa gave rise to mutations that resulted in P. aeruginosa becoming less pathogenic. This is particularly interesting as similar mutations arise in bacteria associated with chronic infections. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to those observed in isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures among host cell types as well as similar adaptation strategies.
Assuntos
Amoeba , Fibrose Cística , Infecções por Pseudomonas , Animais , Fibrose Cística/microbiologia , Humanos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa , VirulênciaRESUMO
BACKGROUND & AIMS: The protease plasmin is an important wound healing factor, but it is not clear how it affects gastrointestinal infection-mediated damage, such as that resulting from Clostridioides difficile. We investigated the role of plasmin in C difficile-associated disease. This bacterium produces a spore form that is required for infection, so we also investigated the effects of plasmin on spores. METHODS: C57BL/6J mice expressing the precursor to plasmin, the zymogen human plasminogen (hPLG), or infused with hPLG were infected with C difficile, and disease progression was monitored. Gut tissues were collected, and cytokine production and tissue damage were analyzed by using proteomic and cytokine arrays. Antibodies that inhibit either hPLG activation or plasmin activity were developed and structurally characterized, and their effects were tested in mice. Spores were isolated from infected patients or mice and visualized using super-resolution microscopy; the functional consequences of hPLG binding to spores were determined. RESULTS: hPLG localized to the toxin-damaged gut, resulting in immune dysregulation with an increased abundance of cytokines (such as interleukin [IL] 1A, IL1B, IL3, IL10, IL12B, MCP1, MP1A, MP1B, GCSF, GMCSF, KC, TIMP-1), tissue degradation, and reduced survival. Administration of antibodies that inhibit plasminogen activation reduced disease severity in mice. C difficile spores bound specifically to hPLG and active plasmin degraded their surface, facilitating rapid germination. CONCLUSIONS: We found that hPLG is recruited to the damaged gut, exacerbating C difficile disease in mice. hPLG binds to C difficile spores, and, upon activation to plasmin, remodels the spore surface, facilitating rapid spore germination. Inhibitors of plasminogen activation might be developed for treatment of C difficile or other infection-mediated gastrointestinal diseases.
Assuntos
Clostridioides difficile/efeitos dos fármacos , Enterocolite Pseudomembranosa/etiologia , Enterocolite Pseudomembranosa/patologia , Plasminogênio/farmacologia , Esporos Bacterianos/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Humanos , Intestino Delgado , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Colonization of the oropharynx is the initial step in Group A Streptococcus (GAS) pharyngeal infection. We have previously reported that the highly virulent M1T1 GAS clone attaches to oral epithelial cells via M1 protein interaction with blood group antigen carbohydrate structures. Here, we have identified that colonization of human oral epithelial cells by GAS serotypes M3 and M12 is mediated by human blood group antigens [ABO(H)] and Lewis (Le) antigen expression. Removal of linkage-specific fucose, galactose, N-acetylgalactosamine, and sialic acid modulated GAS colonization, dependent on host ABO(H) blood group and Le expression profile. Furthermore, N-linked glycans from human salivary glycoproteins, when released and purified, were potent inhibitors of M1, M3, and M12 GAS colonization ex vivo. These data highlight the important role played by human protein glycosylation patterns in GAS attachment to oral epithelial cell surfaces.-De Oliveira, D. M. P., Everest-Dass, A., Hartley-Tassell, L., Day, C. J., Indraratna, A., Brouwer, S., Cleary, A., Kautto, L., Gorman, J., Packer, N. H., Jennings, M. P., Walker, M. J., Sanderson-Smith, M. L. Human glycan expression patterns influence Group A streptococcal colonization of epithelial cells.
Assuntos
Interações entre Hospedeiro e Microrganismos/fisiologia , Polissacarídeos/metabolismo , Streptococcus pyogenes/patogenicidade , Antígenos de Bactérias/fisiologia , Aderência Bacteriana/imunologia , Aderência Bacteriana/fisiologia , Proteínas da Membrana Bacteriana Externa/fisiologia , Antígenos de Grupos Sanguíneos/química , Proteínas de Transporte/fisiologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Glicosilação , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Técnicas In Vitro , Polissacarídeos/química , Polissacarídeos/imunologia , Ligação Proteica , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/imunologia , Proteínas e Peptídeos Salivares/metabolismo , Infecções Estreptocócicas/etiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/crescimento & desenvolvimento , Streptococcus pyogenes/fisiologia , Virulência/fisiologiaRESUMO
OBJECTIVE: Constipation is common in patients with end-stage kidney disease. Nondrug strategies to manage constipation are challenging because of dietary potassium, phosphate, and fluid restrictions. Nuts are a high-fiber food but are excluded from the diet because of the high potassium and phosphate content. The aim of this study was to examine the safety and efficacy of using nuts to improve constipation in adults undertaking hemodialysis (HD). DESIGN AND METHODS: Adult patients undertaking HD were recruited to this nonrandomized, 10-week repeated measures, within-subject, pragmatic clinical trial, conducted in two HD units. The intervention consisted of consumption of 40g of raw almonds daily for four weeks, followed by a two-week washout and four-week control period. The primary safety outcome measures were change in predialysis serum potassium and phosphate levels. The primary efficacy outcome was reduction in constipation, measured using the Bristol Stool Form Scale and Palliative Care Outcome Scale (POS-S) renal symptom score. Secondary outcomes included quality of life, selected uremic toxins, cognition, gut microbiota profile, and symptom burden. RESULTS: Twenty patients completed the trial (median age: 67 [interquartile range: 57.5-77.8] years, 51% male). After controlling for dialysis adequacy, anuria, dietary intake, bicarbonate, and parathyroid hormone, there were no statistically significant changes in serum potassium (P = 0.21) or phosphate (P = 0.16) associated with daily consumption of almonds. However, statistically significant improvements in constipation were seen at weeks 2, 3, 4, and 10. There were statistically significant improvements in quality of life (P = 0.030), overall symptom burden (P = 0.002), vomiting (P = 0.020), itching (P = 0.006), and skin changes (P = 0.002). CONCLUSION: Daily consumption of almonds for four weeks was safe, effective, and well tolerated. Improvements in quality of life and symptom burden warrant further research to elucidate potential mechanisms. The findings support the potential reinclusion of foods such as nuts into the diet of patients who underwent HD.
Assuntos
Constipação Intestinal/dietoterapia , Constipação Intestinal/etiologia , Dieta/métodos , Falência Renal Crônica/complicações , Nozes , Diálise Renal , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Constipação Intestinal/fisiopatologia , Feminino , Humanos , Intestinos/fisiopatologia , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Group A Streptococcus (GAS) skin infections are particularly prevalent in developing nations. The GAS M protein, by which strains are differentiated into >220 different emm types, is immunogenic and elicits protective antibodies. A major obstacle for vaccine development has been the traditional understanding that immunity following infection is restricted to a single emm type. However, recent evidence has led to the hypothesis of immune cross-reactivity between emm types. METHODS: We investigated the human serological response to GAS impetigo in Fijian schoolchildren, focusing on 3 major emm clusters (E4, E6, and D4). Pre- and postinfection sera were assayed by enzyme-linked immunosorbent assay with N-terminal M peptides and bactericidal assays using the infecting-type strain, emm cluster-related strains, and nonrelated strains. RESULTS: Twenty of the 53 paired sera demonstrated a ≥4-fold increase in antibody titer against the infecting type. When tested against all cluster-related M peptides, we found that 9 of 17 (53%) paired sera had a ≥4-fold increase in antibody titer to cluster-related strains as well. When grouped by cluster, the mean change to cluster-related emm types in E4 and E6 was >4-fold (5.9-fold and 19.5-fold, respectively) but for D4 was 3.8-fold. The 17 paired sera were tested in bactericidal assays against selected cluster-related and nonrelated strains. While the responses were highly variable, numerous instances of cross-reactive killing were observed. CONCLUSIONS: These data demonstrate that M type-specific and cross-reactive immune responses occur following skin infection. The cross-reactive immune responses frequently align with emm clusters, raising new opportunities to design multivalent vaccines with broad coverage.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Transporte/imunologia , Dermatopatias Bacterianas/epidemiologia , Dermatopatias Bacterianas/imunologia , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Adolescente , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Fiji/epidemiologia , Humanos , Estudos Longitudinais , EstudantesRESUMO
Infections caused by group A Streptococcus (GAS) are characterized by robust inflammatory responses and can rapidly lead to life-threatening disease manifestations. However, host mechanisms that respond to GAS, which may influence disease pathology, are understudied. Recent works indicate that GAS infection is recognized by multiple extracellular and intracellular receptors and activates cell signalling via discrete pathways. Host leukocyte receptor binding to GAS-derived products mediates release of inflammatory mediators associated with severe GAS disease. GAS induces divergent phagocyte programmed cell death responses and has inflammatory implications. Epithelial cell apoptotic and autophagic components are mobilized by GAS infection, but can be subverted to ensure bacterial survival. Examination of host interactions with GAS and consequences of GAS infection in the context of cellular receptors responsible for GAS recognition, inflammatory mediator responses, and cell death mechanisms, highlights potential avenues for diagnostic and therapeutic intervention. Understanding the molecular and cellular basis of host symptoms during severe GAS disease will assist the development of improved treatment regimens for this formidable pathogen.
Assuntos
Interações Hospedeiro-Parasita/fisiologia , Inflamação/microbiologia , Infecções Estreptocócicas/microbiologia , Animais , Morte Celular/fisiologia , Humanos , Inflamação/imunologia , Inflamação/fisiopatologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/fisiopatologia , Streptococcus pyogenesRESUMO
Streptococcus pyogenes, also known as group A Streptococcus (GAS), causes mild human infections such as pharyngitis and impetigo and serious infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. Furthermore, repeated GAS infections may trigger autoimmune diseases, including acute poststreptococcal glomerulonephritis, acute rheumatic fever, and rheumatic heart disease. Combined, these diseases account for over half a million deaths per year globally. Genomic and molecular analyses have now characterized a large number of GAS virulence determinants, many of which exhibit overlap and redundancy in the processes of adhesion and colonization, innate immune resistance, and the capacity to facilitate tissue barrier degradation and spread within the human host. This improved understanding of the contribution of individual virulence determinants to the disease process has led to the formulation of models of GAS disease progression, which may lead to better treatment and intervention strategies. While GAS remains sensitive to all penicillins and cephalosporins, rising resistance to other antibiotics used in disease treatment is an increasing worldwide concern. Several GAS vaccine formulations that elicit protective immunity in animal models have shown promise in nonhuman primate and early-stage human trials. The development of a safe and efficacious commercial human vaccine for the prophylaxis of GAS disease remains a high priority.
Assuntos
Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/metabolismo , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Interações Hospedeiro-Patógeno , Humanos , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/mortalidade , Vacinas Estreptocócicas/administração & dosagem , Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/genética , Virulência , Fatores de Virulência/genéticaRESUMO
Plasminogen (Plg) circulates in the host as two predominant glycoforms. Glycoform I Plg (GI-Plg) contains glycosylation sites at Asn289 and Thr346, whereas glycoform II Plg (GII-Plg) is exclusively glycosylated at Thr346. Surface plasmon resonance experiments demonstrated that Plg binding group A streptococcal M protein (PAM) exhibits comparative equal affinity for GI- and GII-Plg in the "closed" conformation (for GII-Plg, KD = 27.4 nM; for GI-Plg, KD = 37.0 nM). When Plg was in the "open" conformation, PAM exhibited an 11-fold increase in affinity for GII-Plg (KD = 2.8 nM) compared with that for GI-Plg (KD = 33.2 nM). The interaction of PAM with Plg is believed to be mediated by lysine binding sites within kringle (KR) 2 of Plg. PAM-GI-Plg interactions were fully inhibited with 100 mM lysine analogue ε-aminocaproic acid (εACA), whereas PAM-GII-Plg interactions were shown to be weakened but not inhibited in the presence of 400 mM εACA. In contrast, binding to the KR1-3 domains of GII-Plg (angiostatin) by PAM was completely inhibited in the presence 5 mM εACA. Along with PAM, emm pattern D GAS isolates express a phenotypically distinct SK variant (type 2b SK) that requires Plg ligands such as PAM to activate Plg. Type 2b SK was able to generate an active site and activate GII-Plg at a rate significantly higher than that of GI-Plg when bound to PAM. Taken together, these data suggest that GAS selectively recruits and activates GII-Plg. Furthermore, we propose that the interaction between PAM and Plg may be partially mediated by a secondary binding site outside of KR2, affected by glycosylation at Asn289.
Assuntos
Proteínas de Bactérias/metabolismo , Plasminogênio/metabolismo , Infecções Estreptocócicas/enzimologia , Streptococcus pyogenes/metabolismo , Aminocaproatos/química , Aminocaproatos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Ativação Enzimática , Glicosilação , Humanos , Kringles , Plasminogênio/química , Plasminogênio/genética , Ligação Proteica , Conformação Proteica , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/química , Streptococcus pyogenes/genética , Streptococcus pyogenes/isolamento & purificaçãoRESUMO
Recruitment of the serine protease plasmin is central to the pathogenesis of many bacterial species, including Group A streptococcus (GAS), a leading cause of morbidity and mortality globally. A key process in invasive GAS disease is the ability to accumulate plasmin at the cell surface, however the role of host activators of plasminogen in this process is poorly understood. Here, we demonstrate for the first time that the urokinase-type plasminogen activator (uPA) contributes to plasmin recruitment and subsequent invasive disease initiation in vivo. In the absence of a source of host plasminogen activators, streptokinase (Ska) was required to facilitate cell surface plasmin acquisition by GAS. However, in the absence of Ska, host activators were sufficient to promote cell surface plasmin acquisition by GAS strain 5448 during incubation with plasminogen or human plasma. Furthermore, GAS were able mediate a significant increase in the activation of zymogen pro-uPA in human plasma. In order to assess the contribution of uPA to invasive GAS disease, a previously undescribed transgenic mouse model of infection was employed. Both C57/black 6J, and AlbPLG1 mice expressing the human plasminogen transgene, were significantly more susceptible to invasive GAS disease than uPA-/- mice. The observed decrease in virulence in uPA-/-mice was found to correlate directly with a decrease in bacterial dissemination and reduced cell surface plasmin accumulation by GAS. These findings have significant implications for our understanding of GAS pathogenesis, and research aimed at therapeutic targeting of plasminogen activation in invasive bacterial infections.
Assuntos
Resistência à Doença , Plasminogênio/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Cruzamentos Genéticos , Suscetibilidade a Doenças , Precursores Enzimáticos/sangue , Precursores Enzimáticos/metabolismo , Fibrinolisina/metabolismo , Heterozigoto , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Plasminogênio/genética , Proteólise , Infecções Estreptocócicas/sangue , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes/patogenicidade , Estreptoquinase/metabolismo , Propriedades de Superfície , Ativador de Plasminogênio Tipo Uroquinase/sangue , Ativador de Plasminogênio Tipo Uroquinase/genética , VirulênciaRESUMO
SK (streptokinase) is a secreted plasminogen activator and virulence factor of GAS (group A Streptococcus). Among GAS isolates, SK gene sequences are polymorphic and are grouped into two sequence clusters (cluster type-1 and cluster type-2) with cluster type-2 being further classified into subclusters (type-2a and type-2b). In the present study, we examined the role of bacterial and host-derived cofactors in SK-mediated plasminogen activation. All SK variants, apart from type-2b, can form an activator complex with Glu-Plg (Glu-plasminogen). Specific ligand-binding-induced conformational changes in Glu-Plg mediated by fibrinogen, PAM (plasminogen-binding group A streptococcal M protein), fibrinogen fragment D or fibrin, were required for type-2b SK to form a functional activator complex with Glu-Plg. In contrast with type-1 and type-2a SK, type-2b SK activator complexes were inhibited by α2-antiplasmin unless bound to fibrin or to the GAS cell-surface via PAM in combination with fibrinogen. Taken together, these data suggest that type-2b SK plasminogen activation may be restricted to specific microenvironments within the host such as fibrin deposits or the bacterial cell surface through the action of α2-antiplasmin. We conclude that phenotypic SK variation functionally underpins a pathogenic mechanism whereby SK variants differentially focus plasminogen activation, leading to specific niche adaption within the host.
Assuntos
Plasminogênio/metabolismo , Streptococcus pyogenes/enzimologia , Estreptoquinase/metabolismo , Domínio CatalíticoRESUMO
Streptococcus pyogenes ranks among the main causes of mortality from bacterial infections worldwide. Currently there is no vaccine to prevent diseases such as rheumatic heart disease and invasive streptococcal infection. The streptococcal M protein that is used as the substrate for epidemiological typing is both a virulence factor and a vaccine antigen. Over 220 variants of this protein have been described, making comparisons between proteins difficult, and hindering M protein-based vaccine development. A functional classification based on 48 emm-clusters containing closely related M proteins that share binding and structural properties is proposed. The need for a paradigm shift from type-specific immunity against S. pyogenes to emm-cluster based immunity for this bacterium should be further investigated. Implementation of this emm-cluster-based system as a standard typing scheme for S. pyogenes will facilitate the design of future studies of M protein function, streptococcal virulence, epidemiological surveillance, and vaccine development.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Clonagem Molecular , Dados de Sequência Molecular , Filogenia , Proteínas RecombinantesRESUMO
Diseases caused by Streptococcus pyogenes (Group A streptococcus, GAS) range from superficial infections such as pharyngitis and impetigo to potentially fatal rheumatic heart disease and invasive disease. Studies spanning emm-typing surveillance to population genomics are providing new insights into the epidemiology, pathogenesis, and biology of this organism. Such studies have demonstrated the differences that exist in the epidemiology of streptococcal disease between developing and developed nations. In developing nations, where streptococcal disease is endemic, the diversity of GAS emm-types circulating is much greater than that found in developed nations. An association between emm-type and disease, as observed in developed countries is also lacking. Intriguingly, comparative genetic studies suggest that emm-type is not always a good predictor of the evolutionary relatedness of geographically distant isolates. A view of GAS as a highly dynamic organism, in possession of a core set of virulence genes that contribute to host niche specialization and common pathogenic processes, augmented by accessory genes that change the relative virulence of specific lineages is emerging. Our inability to definitively identify genetic factors that contribute to specific disease outcome underscores the complex nature of streptococcal diseases.
Assuntos
Infecções Estreptocócicas/epidemiologia , Streptococcus pyogenes/genética , Humanos , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/genéticaRESUMO
In Western countries, invasive infections caused by M1T1 serotype group A Streptococcus (GAS) are epidemiologically linked to mutations in the control of virulence regulatory 2-component operon (covRS). In indigenous communities and developing countries, severe GAS disease is associated with genetically diverse non-M1T1 GAS serotypes. Hypervirulent M1T1 covRS mutant strains arise through selection by human polymorphonuclear cells for increased expression of GAS virulence factors such as the DNase Sda1, which promotes neutrophil resistance. The GAS bacteremia isolate NS88.2 (emm 98.1) is a covS mutant that exhibits a hypervirulent phenotype and neutrophil resistance yet lacks the phage-encoded Sda1. Here, we have employed a comprehensive systems biology (genomic, transcriptomic, and proteomic) approach to identify NS88.2 virulence determinants that enhance neutrophil resistance in the non-M1T1 GAS genetic background. Using this approach, we have identified streptococcal collagen-like protein A and general stress protein 24 proteins as NS88.2 determinants that contribute to survival in whole blood and neutrophil resistance in non-M1T1 GAS. This study has revealed new factors that contribute to GAS pathogenicity that may play important roles in resisting innate immune defenses and the development of human invasive infections.
Assuntos
Proteínas de Bactérias/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Animais , Aderência Bacteriana/genética , Aderência Bacteriana/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Genômica/métodos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Camundongos , Viabilidade Microbiana/genética , Viabilidade Microbiana/imunologia , Mutação , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Infecções Estreptocócicas/sangue , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Virulência/genética , Virulência/imunologiaRESUMO
Most invasive bacterial infections are caused by species that more commonly colonize the human host with minimal symptoms. Although phenotypic or genetic correlates underlying a bacterium's shift to enhanced virulence have been studied, the in vivo selection pressures governing such shifts are poorly understood. The globally disseminated M1T1 clone of group A Streptococcus (GAS) is linked with the rare but life-threatening syndromes of necrotizing fasciitis and toxic shock syndrome. Mutations in the GAS control of virulence regulatory sensor kinase (covRS) operon are associated with severe invasive disease, abolishing expression of a broad-spectrum cysteine protease (SpeB) and allowing the recruitment and activation of host plasminogen on the bacterial surface. Here we describe how bacteriophage-encoded GAS DNase (Sda1), which facilitates the pathogen's escape from neutrophil extracellular traps, serves as a selective force for covRS mutation. The results provide a paradigm whereby natural selection exerted by the innate immune system generates hypervirulent bacterial variants with increased risk of systemic dissemination.
Assuntos
Desoxirribonuclease I/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/patogenicidade , Animais , Sobrevivência Celular , Desoxirribonuclease I/genética , Humanos , Imunidade Inata , Camundongos , Neutrófilos/citologia , Neutrófilos/microbiologia , Fenótipo , Seleção Genética , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/genética , VirulênciaRESUMO
Ulcerative Colitis (UC), a type of Inflammatory Bowel Disease (IBD), is a chronic, relapsing gastrointestinal condition with increasing global prevalence. The gut microbiome profile of people living with UC differs from healthy controls and this may play a role in the pathogenesis and clinical management of UC. Probiotics have been shown to induce remission in UC; however, their impact on the gut microbiome and inflammation is less clear. Anthocyanins, a flavonoid subclass, have shown anti-inflammatory and microbiota-modulating properties; however, this evidence is largely preclinical. To explore the combined effect and clinical significance of anthocyanins and a multi-strain probiotic, a 3-month randomised controlled trial will be conducted in 100 adults with UC. Participants will be randomly assigned to one of four groups: anthocyanins (blackcurrant powder) + placebo probiotic, probiotic + placebo fruit powder, anthocyanin + probiotic, or double placebo. The primary outcome is a clinically significant change in the health-related quality-of-life measured with the Inflammatory Bowel Disease Questionnaire-32. Secondary outcomes include shotgun metagenomic sequencing of the faecal microbiota, faecal calprotectin, symptom severity, and mood and cognitive tests. This research will identify the role of adjuvant anti-inflammatory dietary treatments in adults with UC and elucidate the relationship between the gut microbiome and inflammatory biomarkers in this disease, to help identify targeted individualised microbial therapies. ANZCTR registration ACTRN12623000630617.
Assuntos
Colite Ulcerativa , Doenças Inflamatórias Intestinais , Probióticos , Adulto , Humanos , Antocianinas/farmacologia , Anti-Inflamatórios , Colite Ulcerativa/terapia , Doenças Inflamatórias Intestinais/terapia , Pós , Probióticos/farmacologia , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Streptococcus pyogenes (Group A Streptococcus; GAS) is a Gram-positive bacterium responsible for substantial human mortality and morbidity. Conventional diagnosis of GAS pharyngitis relies on throat swab culture, a low-throughput, slow, and relatively invasive 'gold standard'. While molecular approaches are becoming increasingly utilized, the potential of saliva as a diagnostic fluid for GAS infection remains largely unexplored. Here, we present a novel, high-throughput, sensitive, and robust speB qPCR assay that reliably detects GAS in saliva using innovative 3base™ technology (Genetic Signatures Limited, Sydney, Australia). The assay has been validated on baseline, acute, and convalescent saliva samples generated from the Controlled Human Infection for Vaccination Against Streptococcus (CHIVAS-M75) trial, in which healthy adult participants were challenged with emm75 GAS. In these well-defined samples, our high-throughput assay outperforms throat culture and conventional qPCR in saliva respectively, affirming the utility of the 3base™ platform, demonstrating the feasibility of saliva as a diagnostic biofluid, and paving the way for the development of novel non-invasive approaches for the detection of GAS and other oropharyngeal pathogens.
Assuntos
Faringite , Saliva , Streptococcus pyogenes , Humanos , Streptococcus pyogenes/isolamento & purificação , Saliva/microbiologia , Faringite/microbiologia , Faringite/diagnóstico , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/microbiologia , Adulto , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
Wildlife are implicated in the dissemination of antimicrobial resistance, but their roles as hosts for Escherichia coli that pose a threat to human and animal health is limited. Gulls (family Laridae) in particular, are known to carry diverse lineages of multiple-antibiotic resistant E. coli, including extra-intestinal pathogenic E. coli (ExPEC). Whole genome sequencing of 431 E. coli isolates from 69 healthy Australian silver gulls (Chroicocephalus novaehollandiae) sampled during the 2019 breeding season, and without antibiotic selection, was undertaken to assess carriage in an urban wildlife population. Phylogenetic analysis and genotyping resolved 123 sequence types (STs) representing most phylogroups, and identified diverse ExPEC, including an expansive phylogroup B2 cluster comprising 103 isolates (24 %; 31 STs). Analysis of the mobilome identified: i) widespread carriage of the Yersinia High Pathogenicity Island (HPI), a key ExPEC virulence determinant; ii) broad distribution of two novel phage elements, each carrying sitABCD and iii) carriage of the transmissible locus of stress tolerance (tLST), an element linked to sanitation resistance. Of the 169 HPI carrying isolates, 49 (48 %) represented diverse B2 isolates hosting FII-64 ColV-like plasmids that lacked iutABC and sitABC operons typical of ColV plasmids, but carried the serine protease autotransporter gene, sha. Diverse E. coli also carried archetypal ColV plasmids (52 isolates; 12 %). Clusters of closely related E. coli (<50 SNVs) from ST58, ST457 and ST746, sourced from healthy gulls, humans, and companion animals, were frequently identified. In summary, anthropogenically impacted gulls host an expansive E. coli population, including: i) putative ExPEC that carry ColV virulence gene cargo (101 isolates; 23.4 %) and HPI (169 isolates; 39 %); ii) atypical enteropathogenic E. coli (EPEC) (17 isolates; 3.9 %), and iii) E. coli that carry the tLST (20 isolates; 4.6 %). Gulls play an important role in the evolution and transmission of E. coli that impact human health.