RESUMO
5'-nucleotidases (5'-NTs) are enzymes that catalyze the hydrolysis of nucleoside monophosphates to produce nucleosides and phosphate. Since the identification of adenosine synthase A (AdsA) in Staphylococcus aureus in 2009, several other 5'-NTs have been discovered in Gram-positive cocci, mainly in streptococci. Despite some differences in substrate specificity, pH range and metal ion requirements, all characterized 5'-NTs use AMP and ADP, and in some cases ATP, to produce the immunosuppressive adenosine, which dampens pro-inflammatory immune responses. Several 5'-NTs are also able to use dAMP as substrate to generate deoxy-adenosine which is cytotoxic for macrophages. A synergy between 5'-NTs and exonucleases which are commonly expressed in Gram-positive cocci has been described, where the nucleases provide dAMP as a cleavage product from DNA. Some of these nucleases produce dAMP by degrading the DNA backbone of neutrophil extracellular traps (NETs) resulting in a "double hit" strategy of immune evasion. This Micro Review provides an overview of the biochemical properties of Gram-positive cell wall-anchored 5'-NTs and their role as virulence factors. A potential use of 5'-NTs for vaccine development is also briefly discussed.
Assuntos
5'-Nucleotidase , Infecções por Bactérias Gram-Positivas/microbiologia , Cocos Gram-Positivos/enzimologia , Fatores de Virulência , 5'-Nucleotidase/química , 5'-Nucleotidase/fisiologia , Animais , Parede Celular/enzimologia , Humanos , Evasão da Resposta Imune , Cinética , Especificidade por Substrato , Fatores de Virulência/química , Fatores de Virulência/fisiologiaRESUMO
Group A Streptococcus (GAS), or Streptococcus pyogenes, is a human pathogen that causes diseases ranging from skin and soft tissue infections to severe invasive diseases, such as toxic shock syndrome. Each GAS strain carries a particular pilus type encoded in the variable fibronectin-binding, collagen-binding, T antigen (FCT) genomic region. Here, we describe the functional analysis of the serotype M2 pilus encoded in the FCT-6 region. We found that, in contrast to other investigated GAS pili, the ancillary pilin 1 lacks adhesive properties. Instead, the backbone pilin is important for host cell adhesion and binds several host factors, including fibronectin and fibrinogen. Using a panel of recombinant pilus proteins, GAS gene deletion mutants and Lactococcus lactis gain-of-function mutants we show that, unlike other GAS pili, the FCT-6 pilus also contributes to immune evasion. This was demonstrated by a delay in blood clotting, increased intracellular survival of the bacteria in macrophages, higher bacterial survival rates in human whole blood and greater virulence in a Galleria mellonella infection model in the presence of fully assembled FCT-6 pili.
Assuntos
Aderência Bacteriana/fisiologia , Proteínas de Fímbrias/fisiologia , Streptococcus pyogenes/fisiologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Antígenos Virais de Tumores , Aderência Bacteriana/genética , Aderência Bacteriana/imunologia , Proteínas de Bactérias/metabolismo , Biofilmes , Fibronectinas/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/imunologia , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Humanos , Evasão da Resposta Imune , Mutação , Deleção de Sequência , Sorogrupo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/metabolismo , VirulênciaRESUMO
Streptococcus pyogenes is an important human pathogen that causes a wide range of diseases. Using bioinformatics analysis of the complete S. pyogenes strain SF370 genome, we have identified a novel S. pyogenes virulence factor, which we termed streptococcal 5'-nucleotidase A (S5nA). A recombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a Km of 169 µm and a Vmax of 7550 nmol/mg/min for the substrate AMP. Furthermore, recombinant S5nA acted synergistically with S. pyogenes nuclease A to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH 5 and pH 6.5 and between 37 and 47 °C. Like other 5'-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg(2+), Ca(2+), or Mn(2+). However, Zn(2+) inhibited the enzymatic activity. Structural modeling combined with mutational analysis revealed a highly conserved catalytic dyad as well as conserved substrate and cation-binding sites. Recombinant S5nA significantly increased the survival of the non-pathogenic bacterium Lactococcus lactis during a human whole blood killing assay in a dose-dependent manner, suggesting a role as an S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5'-nucleotidase activity and immune evasion properties.
Assuntos
Atividade Bactericida do Sangue/imunologia , Evasão da Resposta Imune , N-Glicosil Hidrolases/imunologia , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Humanos , Lactococcus lactis/genética , Lactococcus lactis/imunologia , Macrófagos , Viabilidade Microbiana/genética , Viabilidade Microbiana/imunologia , N-Glicosil Hidrolases/genética , Streptococcus pyogenes/genética , Fatores de Virulência/genéticaRESUMO
OBJECTIVE: To determine if multiple streptococcal species can be easily labeled for biophotonic imaging using a toxin-antitoxin stabilized reporter plasmid containing the native firefly luciferase gene, originally developed for use in Group A Streptococcus. RESULTS: A number of streptococcal species including Group B Streptococcus, Group C Streptococcus, Group G Streptococcus, S. iniae, S. vestibularis, and S. salivarius were successfully transformed with the reporter plasmid. In absence of antibiotic selection, the plasmid had variable stability amongst the six strains. The expression of firefly luciferase was highest in Group B Streptococcus and S. iniae, as observed by the brightest signal and lowest detection limits in vitro. CONCLUSION: Multiple streptococcal species can be easily transformed with our toxin-antitoxin stabilized bioluminescent reporter plasmid. However, this plasmid shows variable stability and signal in different species, restricting its use for certain applications.
Assuntos
Luciferases de Vaga-Lume/análise , Plasmídeos/genética , Streptococcus pyogenes/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Genes Reporter , Processamento de Imagem Assistida por Computador , Luciferases de Vaga-Lume/genética , Substâncias Luminescentes/análise , Plasmídeos/metabolismo , Coloração e Rotulagem/métodos , Streptococcus pyogenes/metabolismo , Transformação BacterianaRESUMO
NanoLuc luciferase (Nluc) is an engineered enzyme that catalyses the substrate, furimazine, to produce light. Nluc has higher sensitivity than the commonly used bioluminescent reporter, firefly luciferase (FFluc). We have introduced Nluc into a toxin-antitoxin stabilised plasmid for the efficient labeling of group A Streptococcus. Comparison of signal strength and kinetic properties between Nluc-labeled bacteria and similarly previously-labeled FFluc bacteria, showed that the bioluminescent signal produced by Nluc-labeled bacteria is up to 15-times higher than FFluc-labeled bacteria during the logarithmic phase. However, with Nluc we were unable to differentiate between bacteria that are metabolically active and inactive because of its ATP-independence. Nluc therefore offers a more sensitive reporter but, perhaps, one more restricted for downstream applications.
Assuntos
Genes Reporter , Luciferases/análise , Biologia Molecular/métodos , Coloração e Rotulagem/métodos , Streptococcus pyogenes/genética , Luciferases/genética , Medições Luminescentes , Sensibilidade e EspecificidadeRESUMO
Bioluminescence is a rapid and cost-efficient optical imaging technology that allows the detection of bacteria in real-time during disease development. Here, we report a novel strategy to generate a wide range of bioluminescent group A streptococcus (GAS) strains by using a toxin-antitoxin-stabilized plasmid. The bacterial luciferin-luciferase operon (lux) or the firefly luciferase gene (ffluc) was introduced into GAS via a stabilized plasmid. The FFluc reporter gave significantly stronger bioluminescent signals than the Lux reporter, and was generally more stable. Plasmid-based luciferase reporters could easily be introduced into a variety of GAS strains and the signals correlated linearly with viable cell counts. Co-expression of the streptococcal ω-ε-ζ toxin-antitoxin operon provided segregational stability in the absence of antibiotics for at least 17 passages in vitro and up to 7 days in a mouse infection model. In addition, genome-integrated reporter constructs were also generated by site-specific recombination, but were found to be technically more challenging. The quick and efficient generation of various M-type GAS strains expressing plasmid-based luciferase reporters with comparable and quantifiable bioluminescence signals allows for comparative analysis of different GAS strains in vitro and in vivo.
Assuntos
Toxinas Bacterianas/metabolismo , Instabilidade Genômica , Processamento de Imagem Assistida por Computador , Luciferases/análise , Plasmídeos , Coloração e Rotulagem/métodos , Streptococcus pyogenes/enzimologia , Animais , Toxinas Bacterianas/genética , Técnicas Bacteriológicas/métodos , Genes Reporter , Luciferases/genética , Medições Luminescentes , Camundongos , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismoRESUMO
Group A Streptococcus (GAS, Streptococcus pyogenes) is an exclusively human pathogen that causes a range of diseases, including pharyngitis, tonsillitis, impetigo, erysipelas, necrotizing fasciitis, and toxic shock syndrome. Post-streptococcal sequelae include acute rheumatic fever and rheumatic heart disease. The bacterium produces a large arsenal of virulence factors that contribute to host tissue adhesion/colonization, bacterial spread, and host immune evasion. Immune evasion factors include proteins that interfere with complement, a system of plasma proteins that are activated by pathogens resulting in a variety of reactions on the surface of the pathogen. This leads to the activation of active components with a variety of effector functions, such as cell lysis, opsonization, and chemotaxis of phagocytes to the site of infection. We have recently identified a novel "complement evasion factor" (CEF) in S. pyogenes. CEF directly interacts with complement proteins C1r, C1s, C3, and C5, interrupts all three complement pathways, and prevents opsonization of the bacterial surface with C3b. We here present methods used to analyze the complement interference of CEF.
Assuntos
Infecções Estreptocócicas , Streptococcus pyogenes , Humanos , Streptococcus pyogenes/metabolismo , Virulência , Proteínas do Sistema Complemento , Fatores de Virulência/metabolismo , Fatores Imunológicos , Proteínas de Bactérias/metabolismoRESUMO
INTRODUCTION: The unique mucosal immune system allows the generation of robust protective immune responses at the front line of pathogen encounters. The needle-free delivery route and cold chain-free logistic requirements also provide additional advantages in ease and economy. However, the development of mucosal vaccines faces several challenges, and only a handful of mucosal vaccines are currently licensed. These vaccines are all in the form of live attenuated or inactivated whole organisms, whereas no subunit-based mucosal vaccine is available. AREAS COVERED: The selection of antigen, delivery vehicle, route and adjuvants for mucosal vaccination are highly important. This is particularly crucial for subunit vaccines, as they often fail to elicit strong immune responses. Emerging research is providing new insights into the biological and immunological uniqueness of mucosal tissues. However, many aspects of the mucosal immunology still await to be investigated. EXPERT OPINION: This article provides an overview of the current understanding of mucosal vaccination and discusses the remaining knowledge gaps. We emphasize that because of the potential benefits mucosal vaccines can bring from the biomedical, social and economic standpoints, the unmet goal to achieve mucosal vaccine success is worth the effort.
Assuntos
Vacinação , Vacinas , Humanos , Mucosa , Imunidade , Adjuvantes Imunológicos , Imunidade nas MucosasRESUMO
Group A Streptococcus (GAS) is a globally important pathogen causing a broad range of human diseases. GAS pili are elongated proteins with a backbone comprised repeating T-antigen subunits, which extend from the cell surface and have important roles in adhesion and establishing infection. No GAS vaccines are currently available, but T-antigen-based candidates are in pre-clinical development. This study investigated antibody-T-antigen interactions to gain molecular insight into functional antibody responses to GAS pili. Large, chimeric mouse/human Fab-phage libraries generated from mice vaccinated with the complete T18.1 pilus were screened against recombinant T18.1, a representative two-domain T-antigen. Of the two Fab identified for further characterization, one (designated E3) was cross-reactive and also recognized T3.2 and T13, while the other (H3) was type-specific reacting with only T18.1/T18.2 within a T-antigen panel representative of the major GAS T-types. The epitopes for the two Fab, determined by x-ray crystallography and peptide tiling, overlapped and mapped to the N-terminal region of the T18.1 N-domain. This region is predicted to be buried in the polymerized pilus by the C-domain of the next T-antigen subunit. However, flow cytometry and opsonophagocytic assays showed that these epitopes were accessible in the polymerized pilus at 37°C, though not at lower temperature. This suggests that there is motion within the pilus at physiological temperature, with structural analysis of a covalently linked T18.1 dimer indicating "knee-joint" like bending occurs between T-antigen subunits to expose this immunodominant region. This temperature dependent, mechanistic flexing provides new insight into how antibodies interact with T-antigens during infection.
Assuntos
Antígenos Virais de Tumores , Epitopos Imunodominantes , Animais , Humanos , Camundongos , Epitopos Imunodominantes/metabolismo , Antígenos Virais de Tumores/metabolismo , Temperatura , Fímbrias Bacterianas/metabolismo , Proteínas de Fímbrias/metabolismo , Proteínas de Bactérias/metabolismo , Epitopos , StreptococcusRESUMO
Group A Streptococcus (GAS) is a leading human pathogen for which there is no licensed vaccine. Infections are most common in young children and the elderly suggesting immunity accumulates with exposure until immune senescence in older age. Though protection has been postulated to be strain type specific, based on the M-protein (emm-type), the antigenic basis of population-level immunity remains poorly understood. Naturally acquired GAS antibody responses were investigated using intravenous immunoglobulin (IVIG), which contains pooled immunoglobulins from thousands of healthy human donors, as a surrogate for population immunity. Functional opsonophagocytic killing assays were conducted with GAS strains (n = 6) representing the three major emm-pattern types (emm12, A-C pattern; emm53, D-pattern; and emm75, E-pattern). While IVIG induced opsonophagocytic killing of all GAS strains tested, specificity assays showed the profile of protective antibodies differed considerably between emm-types. Antibodies targeting the M-protein were a major component of the functional IVIG antibody response for emm12 and emm53 strains but not for emm75 strains. The striking differences in the contribution of M-protein specific antibodies to killing suggest naturally acquired immunity differs between strains from the major emm-patterns. This challenges the dogma that M-protein is the primary protective antigen across all GAS straintypes. IMPORTANCE Group A Streptococcus (GAS) is a globally important pathogen. With the surge of invasive GAS infections that have occurred in multiple countries, contemporaneous with the relaxation of COVID-19 pandemic restrictions, there is increased interest in the mechanisms underpinning GAS immunity. We utilized intravenous immunoglobulin (IVIG), pooled immunoglobulins from thousands of healthy donors, as a surrogate for population-level immunity to GAS, and explored the contribution of strain-specific (M-type specific) antibodies to GAS immunity using functional killing assays. This revealed striking differences between major strain types as to the contribution of strain specific antibodies to killing. For GAS strains belonging to the E pattern group, M-type specific antibodies do not mediate killing and immunity, which contrasts with strains belonging to pattern A-C and D groups. This challenges the historical dogma, originally proposed by Rebecca Lancefield in the 1950-1960s, that the M-protein is the major protective antigen across all GAS strain types.
Assuntos
Antígenos de Bactérias , Imunoglobulinas Intravenosas , Criança , Humanos , Pré-Escolar , Idoso , Formação de Anticorpos , Pandemias , Streptococcus pyogenesRESUMO
In the elderly, immunosenescence and malnourishment can contribute to increased risk and severity of upper respiratory tract infections (URTI). Gold kiwifruit (Actinidia chinensis 'Hort16A') contains nutrients important for immune function and mitigation of symptoms of infection, including vitamins C and E, folate, polyphenols and carotenoids. The objective of the present study was to evaluate whether regular consumption of gold kiwifruit reduces symptoms of URTI in older people, and determine the effect it has on plasma antioxidants, and markers of oxidative stress, inflammation and immune function. A total of thirty-two community-dwelling people (≥65 years) participated in a randomised crossover study, consuming the equivalent of four kiwifruit or two bananas daily for 4 weeks, with treatments separated by a 4-week washout period. Participants completed the Wisconsin Upper Respiratory Symptom Survey-21 daily, and blood samples were collected at baseline and at the end of each treatment and washout period. Gold kiwifruit did not significantly reduce the overall incidence of URTI compared with banana, but significantly reduced the severity and duration of head congestion, and the duration of sore throat. Gold kiwifruit significantly increased plasma vitamin C, α-tocopherol and lutein/zeaxanthin concentrations, and erythrocyte folate concentrations, and significantly reduced plasma lipid peroxidation. No changes to innate immune function (natural killer cell activity, phagocytosis) or inflammation markers (high-sensitivity C-reactive protein, homocysteine) were detected. Consumption of gold kiwifruit enhanced the concentrations of several dietary plasma analytes, which may contribute to reduced duration and severity of selected URTI symptoms, offering a novel tool for reducing the burden of URTI in older individuals.
Assuntos
Actinidia , Envelhecimento/imunologia , Ácido Ascórbico/sangue , Frutas , Imunidade Inata , Infecções Respiratórias/sangue , Infecções Respiratórias/prevenção & controle , Idoso , Antioxidantes/análise , Biomarcadores/sangue , Estudos Cross-Over , Eritrócitos/metabolismo , Feminino , Ácido Fólico/sangue , Humanos , Incidência , Peroxidação de Lipídeos , Masculino , Nova Zelândia/epidemiologia , Estresse Oxidativo , Pigmentação , Projetos Piloto , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/fisiopatologia , Índice de Gravidade de DoençaRESUMO
Peptide vaccines offer an attractive strategy to induce highly specific immune responses while reducing potential side effects. However, peptides are often poorly immunogenic and unstable on their own, requiring the need for potentially toxic adjuvants or expensive chemical coupling. The novel peptide delivery platform PilVax utilizes the rigid pilus structure from Group A Streptococcus (GAS) to stabilize and amplify the peptide, and present it on the surface of the non-pathogenic food-grade bacterium Lactococcus lactis. Upon intranasal immunization, PilVax vaccines have proven to induce peptide-specific systemic and mucosal responses. PilVax provides an alternative method to develop mucosal vaccines that are inexpensive to produce and easy to administer.
Assuntos
Imunidade nas Mucosas , Administração Intranasal , Animais , Imunização , Lactococcus lactis , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos , Vacinas de Subunidades AntigênicasRESUMO
Streptococcus pyogenes, a leading human pathogen, is responsible for a wide range of diseases, including skin and soft tissue infections and severe invasive diseases. S. pyogenes produces a large arsenal of virulence factors, including several immune evasion factors. We have identified an open reading frame (spy0136) in the S. pyogenes SF370 genome encoding a protein of unknown function. Using recombinant Spy0136 in a pull-down assay with human plasma and ELISA, we have identified four complement proteins (C1r, C1s, C3, and C5) as binding partners. Treatment of the complement proteins with PNGase F abrogated binding to C1s, C3, and C5, indicating glycan-dependent interactions. rSpy0136 inhibited complement-mediated hemolysis and interfered with all three complement pathways in a Wieslab complement assay. Furthermore, rSpy0136 inhibited deposition of the C3b opsonin and the membrane attack complex (MAC) on the surface of S. pyogenes. We therefore named the previously unknown protein 'complement evasion factor' (CEF).An S. pyogenes Δspy0136/cef deletion mutant showed decreased virulence in an in-vitro whole blood killing assay and a Galleria mellonella (wax moth) infection model. Furthermore, an L. lactis spy0136/cef gain-of-function mutant showed increased survival during growth in whole human blood. Analysis of serum samples from patients with invasive S. pyogenes revealed Spy0136/CEF sero-conversion indicating expression during disease. In summary, we have identified a novel S. pyogenes immune evasion factor that binds to several complement proteins to interfere with complement function. This is the first example of a S. pyogenes virulence factor binding to several different target proteins via glycan-dependent interactions.
Assuntos
Infecções Estreptocócicas , Streptococcus pyogenes , Proteínas de Bactérias/metabolismo , Proteínas do Sistema Complemento , Humanos , Evasão da Resposta Imune , Streptococcus pyogenes/genética , Fatores de Virulência/metabolismoRESUMO
The number of sugar-sweetened beverages consumed per day has been associated with an increased risk of acute rheumatic fever, an autoimmune disease triggered by superficial Streptococcus pyogenes infection. To explore if there could be a biological basis for this association, we used a mouse model of S. pyogenes nasopharyngeal colonisation combined with a dietary intervention. We observed an increased bacterial load in the nasopharynx of mice receiving sucrose drinking water post-infection, suggesting that high sucrose intake promotes S. pyogenes growth and/or survival. This provides new insight into the potential biological basis behind the association seen in humans.
Assuntos
Água Potável , Streptococcus pyogenes , Humanos , Camundongos , Animais , Açúcares , Nasofaringe/microbiologia , Sacarose , Bebidas , Ingestão de LíquidosRESUMO
Pili of Group A Streptococcus (GAS) are surface-exposed structures involved in adhesion and colonisation of the host during infection. The major protein component of the GAS pilus is the T-antigen, which multimerises to form the pilus shaft. There are currently no licenced vaccines against GAS infections and the T-antigen represents an attractive target for vaccination. We have generated a multivalent vaccine called TeeVax1, a recombinant protein that consists of a fusion of six T-antigen domains. Vaccination with TeeVax1 produces opsonophagocytic antibodies in rabbits and confers protective efficacy in mice against invasive disease. Two further recombinant proteins, TeeVax2 and TeeVax3 were constructed to cover 12 additional T-antigens. Combining TeeVax1-3 produced a robust antibody response in rabbits that was cross-reactive to a full panel of 21 T-antigens, expected to provide over 95% vaccine coverage. These results demonstrate the potential for a T-antigen-based vaccine to prevent GAS infections.
Assuntos
Antígenos de Bactérias/imunologia , Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Linhagem Celular Tumoral , Humanos , Imunogenicidade da Vacina , Camundongos , Coelhos , Vacinas Combinadas/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Bioluminescence is a rapid and cost-saving technology that enables monitoring of bacteria in real time in animal infection models. Here, we report a method for labeling GAS with a set of plasmids we have developed and deposited with Addgene. These plasmids can be used to either integrate firefly luciferase (Ffluc) or red-shifted firefly luciferase (FflucRT) into the GAS genome or to introduce the reporters on plasmids which have been stabilized with a toxin-antitoxin system to prevent loss of plasmid in the absence of antibiotic selection.
Assuntos
Engenharia Genética/métodos , Medições Luminescentes/métodos , Proteínas Luminescentes/genética , Antibacterianos , Diagnóstico por Imagem , Testes Diagnósticos de Rotina , Genes Reporter/genética , Testes Imunológicos , Luciferases de Vaga-Lume/genética , Proteínas Luminescentes/biossíntese , Plasmídeos/genética , Streptococcus pyogenes/genéticaRESUMO
The critical first step of Group A Streptococcus (GAS) pathogenesis is adhesion to the host pharyngeal and skin epithelial cell surfaces (Brouwer et al., FEBS Lett 590:3739-3757, 2016). Host-cell adhesion assays provide a straightforward model to study these host-pathogen interactions. Here, we describe the culturing of immortalized cell lines into monolayers to mimic host epithelia. Various GAS strains can then be added to study their adhesion properties. In addition, we describe the use of antibodies raised against the cell-surface components of GAS to study if these are able to neutralize the binding of GAS to the cell lines. This provides an indication if these cell-surface components are involved in adhesion and if antibodies generated against them function through neutralization.
Assuntos
Aderência Bacteriana/imunologia , Aderência Bacteriana/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Proteínas de Bactérias/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Epitélio/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Modelos Biológicos , Faringe , Pele/metabolismo , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/metabolismoRESUMO
Recently, the use of Galleria mellonella larvae as a nonmammalian model to simulate bacterial infectious diseases has shown to be a rapid, simple, and cost-effective alternative. The insect's innate immune response is remarkably similar to that of the vertebrates, and consists of both the cellular and the humoral immune response. Here, we provide a protocol for using G. mellonella larvae to study virulence of GAS, including the use of a health score system for quantitative analysis and the methods for assessing post-infection bacterial burden in vivo.
Assuntos
Mariposas/microbiologia , Infecções Estreptocócicas/imunologia , Animais , Infecções Bacterianas/imunologia , Modelos Animais de Doenças , Larva/microbiologia , Mariposas/imunologia , Mariposas/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade , Virulência/imunologia , Fatores de Virulência/imunologiaRESUMO
Group A Streptococcus (GAS) often exists as an asymptomatic colonizer of the upper respiratory tract in humans. Unsurprisingly, a high proportion of symptomatic infections caused by GAS include pharyngitis. While not usually life-threatening, these infections cause significant morbidity and economic burden/loss of productivity, and can have downstream life-threatening autoimmune consequences. Modeling asymptomatic colonization in animals is, therefore, a useful tool to dissect host-bacteria interactions and to evaluate efficacy of vaccines aimed at reducing the burden of carriage. Here we describe a mouse model of nasopharyngeal colonization using nasal challenge of susceptible mice and the evaluation of subsequent bacterial burden.
Assuntos
Nasofaringe/microbiologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Animais , Antígenos de Bactérias/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Doenças Nasofaríngeas/microbiologia , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes/patogenicidadeRESUMO
The isolation of a single Group A Streptococcus (GAS) virulence determinant in functional investigations is challenging, as GAS employs a multitude of virulence factors. The redundancy between many surface proteins such as adhesins also adds complexity and difficulty. Lactococcus lactis is a non-pathogenic Gram-positive species related to GAS that can be an ideal surrogate organism to circumvent this problem. Genetic manipulation in L. lactis is easy, and the mechanisms for processing and cell wall-anchoring of surface proteins are similar to GAS. Lactococci have been extensively used to express heterologous surface proteins from other bacterial species, and modern molecular cloning tools and protocols have been developed. This chapter describes the workflow of generating recombinant L. lactis strains expressing GAS surface proteins and the validation and quantification of their surface expression.