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1.
Vaccines (Basel) ; 12(4)2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38675764

RESUMO

Vaccine development against group A Streptococcus (GAS) has gained traction in the last decade, fuelled by recognition of the significant worldwide burden of the disease. Several vaccine candidates are currently being evaluated in preclinical and early clinical studies. Here, we investigate two conjugate vaccine candidates that have shown promise in mouse models of infection. Two antigens, the J8 peptide from the conserved C-terminal end of the M protein, and the group A carbohydrate lacking N-acetylglucosamine side chain (ΔGAC) were each conjugated to arginine deiminase (ADI), an anchorless surface protein from GAS. Both conjugate vaccine candidates combined with alum adjuvant were tested in a non-human primate (NHP) model of pharyngeal infection. High antibody titres were detected against J8 and ADI antigens, while high background antibody titres in NHP sera hindered accurate quantification of ΔGAC-specific antibodies. The severity of pharyngitis and tonsillitis signs, as well as the level of GAS colonisation, showed no significant differences in NHPs immunised with either conjugate vaccine candidate compared to NHPs in the negative control group.

2.
PLoS Pathog ; 18(1): e1010209, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35085362

RESUMO

Haemophilus influenzae (Hi) infections are associated with recurring acute exacerbations of chronic respiratory diseases in children and adults including otitis media, pneumonia, chronic obstructive pulmonary disease and asthma. Here, we show that persistence and recurrence of Hi infections are closely linked to Hi metabolic properties, where preferred growth substrates are aligned to the metabolome of human airway epithelial surfaces and include lactate, pentoses, and nucleosides, but not glucose that is typically used for studies of Hi growth in vitro. Enzymatic and physiological investigations revealed that utilization of lactate, the preferred Hi carbon source, required the LldD L-lactate dehydrogenase (conservation: 98.8% of strains), but not the two redox-balancing D-lactate dehydrogenases Dld and LdhA. Utilization of preferred substrates was directly linked to Hi infection and persistence. When unable to utilize L-lactate or forced to rely on salvaged guanine, Hi showed reduced extra- and intra-cellular persistence in a murine model of lung infection and in primary normal human nasal epithelia, with up to 3000-fold attenuation observed in competitive infections. In contrast, D-lactate dehydrogenase mutants only showed a very slight reduction compared to the wild-type strain. Interestingly, acetate, the major Hi metabolic end-product, had anti-inflammatory effects on cultured human tissue cells in the presence of live but not heat-killed Hi, suggesting that metabolic endproducts also influence HI-host interactions. Our work provides significant new insights into the critical role of metabolism for Hi persistence in contact with host cells and reveals for the first time the immunomodulatory potential of Hi metabolites.


Assuntos
Infecções por Haemophilus/metabolismo , Haemophilus influenzae/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/microbiologia , Animais , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Camundongos
3.
J Cell Signal ; 3(4): 193-206, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36777036

RESUMO

Oxidative stress (OS) in the airway epithelium is associated with inflammation, cell damage, and mitochondrial dysfunction that may initiate or worsen respiratory disease. Redox regulation maintains the equilibrium of pro-oxidant/antioxidant reactions but can be disturbed by environmental exposures. The mechanism(s) underlying the induction and impact of OS on airway epithelium and how these influences on respiratory disease is poorly understood. The aim of this study was to develop a stress response model in primary human nasal epithelial cells (NECs) grown at the air-liquid interface (ALI) into a well-differentiated epithelium and to use this model to investigate the mechanisms underlying OS. Hydrogen peroxide (H2O2) was used to induce acute OS and the responses were measured with trans epithelial electrical resistance (TEER), membrane permeability, cell death (LDH release), mitochondrial reactive oxygen species (mtROS) generation, redox status (GSH/GSSG ratio), cellular ATP, and signaling pathways (SIRT1, FOXO3, p53, p21, PINK1, PARKIN, NRF2). Following 25 mM (sensitive) or 50mM (resistant) H2O2 exposure, cell integrity decreased (p<0.05), GSH/GSSG ratio reduced (p<0.05), and ATP production declined by 83% (p<0.05) in the sensitive and 55% (p<0.05) in the resistant group; mtROS production increased 3.4-fold (p<0.001). Significant inter-individual differences between healthy humans with regards to susceptibility to OS, and differential activation of various pathways (FOXO3, PARKIN) were observed. These intra-individual differences in susceptibility to OS may be attributed to resistant individuals having more mitochondria or greater mitochondrial function.

5.
Sci Rep ; 9(1): 2627, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30796268

RESUMO

Respiratory disease is a major cause of morbidity and mortality in patients with ataxia-telangiectasia (A-T) who are prone to recurrent sinopulmonary infections, bronchiectasis, pulmonary fibrosis, and pulmonary failure. Upper airway infections are common in patients and S. pneumoniae is associated with these infections. We demonstrate here that the upper airway microbiome in patients with A-T is different from that to healthy controls, with S. pneumoniae detected largely in patients only. Patient-specific airway epithelial cells and differentiated air-liquid interface cultures derived from these were hypersensitive to infection which was at least in part due to oxidative damage since it was partially reversed by catalase. We also observed increased levels of the pro-inflammatory cytokines IL-8 and TNF-α (inflammasome-independent) and a decreased level of the inflammasome-dependent cytokine IL-ß in patient cells. Further investigation revealed that the ASC-Caspase 1 signalling pathway was defective in A-T airway epithelial cells. These data suggest that the heightened susceptibility of these cells to S. pneumoniae infection is due to both increased oxidative damage and a defect in inflammasome activation, and has implications for lung disease in these patients.


Assuntos
Ataxia Telangiectasia/patologia , Células Epiteliais/patologia , Imunidade Inata , Pulmão/patologia , Estresse Oxidativo , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/patologia , Streptococcus pneumoniae/fisiologia , Adolescente , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Estudos de Casos e Controles , Diferenciação Celular , Células Cultivadas , Criança , Pré-Escolar , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Humanos , Inflamassomos/metabolismo , Inflamação/patologia , Pulmão/microbiologia , Masculino , Nariz/patologia
6.
PLoS Pathog ; 14(10): e1007348, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30321240

RESUMO

Human Group IIA secreted phospholipase A2 (hGIIA) is an acute phase protein with bactericidal activity against Gram-positive bacteria. Infection models in hGIIA transgenic mice have suggested the importance of hGIIA as an innate defense mechanism against the human pathogens Group A Streptococcus (GAS) and Group B Streptococcus (GBS). Compared to other Gram-positive bacteria, GAS is remarkably resistant to hGIIA activity. To identify GAS resistance mechanisms, we exposed a highly saturated GAS M1 transposon library to recombinant hGIIA and compared relative mutant abundance with library input through transposon-sequencing (Tn-seq). Based on transposon prevalence in the output library, we identified nine genes, including dltA and lytR, conferring increased hGIIA susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included two genes, gacH and gacI that are located within the Group A Carbohydrate (GAC) gene cluster. Using GAS 5448 wild-type and the isogenic gacI mutant and gacI-complemented strains, we demonstrate that loss of the GAC N-acetylglucosamine (GlcNAc) side chain in the ΔgacI mutant increases hGIIA resistance approximately 10-fold, a phenotype that is conserved across different GAS serotypes. Increased resistance is associated with delayed penetration of hGIIA through the cell wall. Correspondingly, loss of the Lancefield Group B Carbohydrate (GBC) rendered GBS significantly more resistant to hGIIA-mediated killing. This suggests that the streptococcal Lancefield antigens, which are critical determinants for streptococcal physiology and virulence, are required for the bactericidal enzyme hGIIA to exert its bactericidal function.


Assuntos
Antibacterianos/farmacologia , Parede Celular/metabolismo , Fosfolipases A2 do Grupo II/imunologia , Imunidade Inata/efeitos dos fármacos , Polissacarídeos Bacterianos/farmacologia , Infecções Estreptocócicas/microbiologia , Streptococcus/imunologia , Atividade Bactericida do Sangue , Fosfolipases A2 do Grupo II/sangue , Fosfolipases A2 do Grupo II/genética , Interações Hospedeiro-Patógeno , Humanos , Infecções Estreptocócicas/sangue , Infecções Estreptocócicas/enzimologia , Streptococcus/patogenicidade
7.
mBio ; 9(1)2018 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-29382733

RESUMO

Classification of streptococci is based upon expression of unique cell wall carbohydrate antigens. All serotypes of group A Streptococcus (GAS; Streptococcus pyogenes), a leading cause of infection-related mortality worldwide, express the group A carbohydrate (GAC). GAC, the classical Lancefield antigen, is comprised of a polyrhamnose backbone with N-acetylglucosamine (GlcNAc) side chains. The immunodominant GlcNAc epitope of GAC is the basis of all rapid diagnostic testing for GAS infection. We previously identified the 12-gene GAC biosynthesis gene cluster and determined that the glycosyltransferase GacI was required for addition of the GlcNAc side chain to the polyrhamnose core. Loss of the GAC GlcNAc epitope in serotype M1 GAS resulted in attenuated virulence in two animal infection models and increased GAS sensitivity to killing by whole human blood, serum, neutrophils, and antimicrobial peptides. Here, we report that the GAC biosynthesis gene cluster is ubiquitous among 520 GAS isolates from global sources, representing 105 GAS emm serotypes. Isogenic ΔgacI mutants were constructed in M2, M3, M4, M28, and M89 backgrounds and displayed an array of phenotypes in susceptibility to killing by whole human blood, baby rabbit serum, human platelet releasate, human neutrophils, and antimicrobial peptide LL-37. The contribution of the GlcNAc side chain to GAS survival in vivo also varied by strain, demonstrating that it is not a prerequisite for virulence in the murine infection model. Thus, the relative contribution of GAC to virulence in non-M1 serotypes appears to depend on the quorum of other virulence factors that each strain possesses.IMPORTANCE The Lancefield group A carbohydrate (GAC) is the species-defining antigen for group A Streptococcus (GAS), comprising ~50% of the cell wall of this major human pathogen. We previously showed that the GlcNAc side chain of GAC contributes to the innate immune resistance and animal virulence phenotypes of the globally disseminated strain of serotype M1 GAS. Here, we use isogenic mutagenesis to examine the role of GAC GlcNAc in five additional medically relevant GAS serotypes. Overall, the GlcNAc side chain of GAC contributes to the innate immune resistance of GAS, but the relative contribution varies among individual strains. Moreover, the GAC GlcNAc side chain is not a universal prerequisite for GAS virulence in the animal model.


Assuntos
Acetilglucosamina/metabolismo , Antígenos de Bactérias/metabolismo , Parede Celular/metabolismo , Polissacarídeos Bacterianos/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/metabolismo , Animais , Antígenos de Bactérias/genética , Atividade Bactericida do Sangue , Modelos Animais de Doenças , Deleção de Genes , Humanos , Camundongos , Polissacarídeos Bacterianos/genética , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/genética , Análise de Sobrevida , Virulência , Fatores de Virulência/genética
8.
Proc Natl Acad Sci U S A ; 113(34): 9515-20, 2016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27512043

RESUMO

The sequences of M proteins, the major surface-associated virulence factors of the widespread bacterial pathogen group A Streptococcus, are antigenically variable but have in common a strong propensity to form coiled coils. Paradoxically, these sequences are also replete with coiled-coil destabilizing residues. These features are evident in the irregular coiled-coil structure and thermal instability of M proteins. We present an explanation for this paradox through studies of the B repeats of the medically important M1 protein. The B repeats are required for interaction of M1 with fibrinogen (Fg) and consequent proinflammatory activation. The B repeats sample multiple conformations, including intrinsically disordered, dissociated, as well as two alternate coiled-coil conformations: a Fg-nonbinding register 1 and a Fg-binding register 2. Stabilization of M1 in the Fg-nonbinding register 1 resulted in attenuation of Fg binding as expected, but counterintuitively, so did stabilization in the Fg-binding register 2. Strikingly, these register-stabilized M1 proteins gained the ability to bind Fg when they were destabilized by a chaotrope. These results indicate that M1 stability is antithetical to Fg interaction and that M1 conformational dynamics, as specified by destabilizing residues, are essential for interaction. A "capture-and-collapse" model of association accounts for these observations, in which M1 captures Fg through a dynamic conformation and then collapses into a register 2-coiled coil as a result of stabilization provided by binding energy. Our results support the general conclusion that destabilizing residues are evolutionarily conserved in M proteins to enable functional interactions necessary for pathogenesis.


Assuntos
Aminoácidos/química , Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Transporte/química , Fibrinogênio/química , Streptococcus pyogenes/química , Sequência de Aminoácidos , Aminoácidos/metabolismo , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fibrinogênio/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica
9.
PLoS One ; 11(6): e0156639, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27310707

RESUMO

The C-terminal region of the M-protein of Streptococcus pyogenes is a major target for vaccine development. The major feature is the C-repeat region, consisting of 35-42 amino acid repeat units that display high but not perfect identity. SV1 is a S. pyogenes vaccine candidate that incorporates five 14mer amino acid sequences (called J14i variants) from differing C-repeat units in a single recombinant construct. Here we show that the J14i variants chosen for inclusion in SV1 are the most common variants in a dataset of 176 unique M-proteins. Murine antibodies raised against SV1 were shown to bind to each of the J14i variants present in SV1, as well as variants not present in the vaccine. Antibodies raised to the individual J14i variants were also shown to bind to multiple but different combinations of J14i variants, supporting the underlying rationale for the design of SV1. A Lewis Rat Model of valvulitis was then used to assess the capacity of SV1 to induce deleterious immune response associated with rheumatic heart disease. In this model, both SV1 and the M5 positive control protein were immunogenic. Neither of these antibodies were cross-reactive with cardiac myosin or collagen. Splenic T cells from SV1/CFA and SV1/alum immunized rats did not proliferate in response to cardiac myosin or collagen. Subsequent histological examination of heart tissue showed that 4 of 5 mice from the M5/CFA group had valvulitis and inflammatory cell infiltration into valvular tissue, whereas mice immunised with SV1/CFA, SV1/alum showed no sign of valvulitis. These results suggest that SV1 is a safe vaccine candidate that will elicit antibodies that recognise the vast majority of circulating GAS M-types.


Assuntos
Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/imunologia , Cardiopatia Reumática/prevenção & controle , Infecções Estreptocócicas/prevenção & controle , Vacinas Estreptocócicas/administração & dosagem , Streptococcus pyogenes/imunologia , Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen/administração & dosagem , Animais , Antígenos de Bactérias/genética , Colágeno/genética , Colágeno/metabolismo , Feminino , Expressão Gênica , Valvas Cardíacas/efeitos dos fármacos , Valvas Cardíacas/imunologia , Valvas Cardíacas/microbiologia , Valvas Cardíacas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Miosinas/genética , Miosinas/metabolismo , Ratos , Ratos Endogâmicos Lew , Sequências Repetitivas de Aminoácidos , Cardiopatia Reumática/imunologia , Cardiopatia Reumática/microbiologia , Cardiopatia Reumática/patologia , Baço/efeitos dos fármacos , Baço/imunologia , Baço/microbiologia , Baço/patologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/patologia , Vacinas Estreptocócicas/biossíntese , Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/efeitos dos fármacos , Streptococcus pyogenes/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/microbiologia , Linfócitos T/patologia , Vacinas Sintéticas
10.
mBio ; 7(3)2016 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-27302756

RESUMO

UNLABELLED: Group A Streptococcus (GAS) is an important human pathogen responsible for both superficial infections and invasive diseases. Autoimmune sequelae may occur upon repeated infection. For this reason, development of a vaccine against GAS represents a major challenge, since certain GAS components may trigger autoimmunity. We formulated three combination vaccines containing the following: (i) streptolysin O (SLO), interleukin 8 (IL-8) protease (Streptococcus pyogenes cell envelope proteinase [SpyCEP]), group A streptococcal C5a peptidase (SCPA), arginine deiminase (ADI), and trigger factor (TF); (ii) the conserved M-protein-derived J8 peptide conjugated to ADI; and (iii) group A carbohydrate lacking the N-acetylglucosamine side chain conjugated to ADI. We compared these combination vaccines to a "gold standard" for immunogenicity, full-length M1 protein. Vaccines were adjuvanted with alum, and mice were immunized on days 0, 21, and 28. On day 42, mice were challenged via cutaneous or subcutaneous routes. High-titer antigen-specific antibody responses with bactericidal activity were detected in mouse serum samples for all vaccine candidates. In comparison with sham-immunized mice, all vaccines afforded protection against cutaneous challenge. However, only full-length M1 protein provided protection in the subcutaneous invasive disease model. IMPORTANCE: This set of experiments demonstrates the inherent variability of mouse models for the characterization of GAS vaccine candidate protective efficacy. Such variability poses an important challenge for GAS vaccine development, as advancement of candidates to human clinical trials requires strong evidence of efficacy. This study highlights the need for an open discussion within the field regarding standardization of animal models for GAS vaccine development.


Assuntos
Bacteriemia/prevenção & controle , Dermatopatias Bacterianas/prevenção & controle , Infecções Estreptocócicas/prevenção & controle , Vacinas Estreptocócicas/administração & dosagem , Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen/administração & dosagem , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Bacteriemia/imunologia , Atividade Bactericida do Sangue , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , Dermatopatias Bacterianas/imunologia , Infecções Estreptocócicas/imunologia , Resultado do Tratamento , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/imunologia , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologia
11.
J Mol Med (Berl) ; 94(2): 219-33, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26411873

RESUMO

UNLABELLED: Inhibitory CD33-related Siglec receptors regulate immune cell activation upon engaging ubiquitous sialic acids (Sias) on host cell surface glycans. Through molecular mimicry, Sia-expressing pathogen group B Streptococcus binds inhibitory human Siglec-9 (hSiglec-9) to blunt neutrophil activation and promote bacterial survival. We unexpectedly discovered that hSiglec-9 also specifically binds high molecular weight hyaluronan (HMW-HA), another ubiquitous host glycan, through a region of its terminal Ig-like V-set domain distinct from the Sia-binding site. HMW-HA recognition by hSiglec-9 limited neutrophil extracellular trap (NET) formation, oxidative burst, and apoptosis, defining HMW-HA as a regulator of neutrophil activation. However, the pathogen group A Streptococcus (GAS) expresses a HMW-HA capsule that engages hSiglec-9, blocking NET formation and oxidative burst, thereby promoting bacterial survival. Thus, a single inhibitory lectin receptor detects two distinct glycan "self-associated molecular patterns" to maintain neutrophil homeostasis, and two leading human bacterial pathogens have independently evolved molecular mimicry to exploit this immunoregulatory mechanism. KEY MESSAGE: HMW-HA is the first example of a non-sialic acid containing glycan to be recognized by CD33-related Siglecs. HMW-HA engagement of hSiglec-9 attenuates neutrophil activation. Group A Streptococcus exploits hSiglec-9 recognition via its polysaccharide HMW-HA capsule to subvert neutrophil killing.


Assuntos
Antígenos CD/metabolismo , Interações Hospedeiro-Patógeno , Ácido Hialurônico/metabolismo , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Animais , Antígenos CD/química , Antígenos CD/genética , Apoptose/genética , Apoptose/imunologia , Bactérias/imunologia , Bactérias/metabolismo , Quimiotaxia de Leucócito/imunologia , Armadilhas Extracelulares/genética , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Ácido Hialurônico/química , Imunidade Inata , Fragmentos Fc das Imunoglobulinas/metabolismo , Peso Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão , Explosão Respiratória/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/química , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Streptococcus/fisiologia
12.
Cell Microbiol ; 17(12): 1721-41, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26433203

RESUMO

Group A Streptococcus (Streptococcus pyogenes), group B Streptococcus (Streptococcus agalactiae) and Streptococcus pneumoniae (pneumococcus) are host-adapted bacterial pathogens among the leading infectious causes of human morbidity and mortality. These microbes and related members of the genus Streptococcus produce an array of toxins that act against human cells or tissues, resulting in impaired immune responses and subversion of host physiological processes to benefit the invading microorganism. This toxin repertoire includes haemolysins, proteases, superantigens and other agents that ultimately enhance colonization and survival within the host and promote dissemination of the pathogen.


Assuntos
Toxinas Bacterianas/metabolismo , Interações Hospedeiro-Patógeno , Infecções Estreptocócicas/patologia , Streptococcus agalactiae/patogenicidade , Streptococcus pneumoniae/patogenicidade , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/metabolismo , Humanos , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/metabolismo , Streptococcus pneumoniae/metabolismo , Streptococcus pyogenes/metabolismo
13.
FEMS Microbiol Rev ; 39(4): 488-508, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25670736

RESUMO

Streptococcus pyogenes, also known as group A Streptococcus (GAS), is an exclusively human Gram-positive bacterial pathogen ranked among the 'top 10' causes of infection-related deaths worldwide. GAS commonly causes benign and self-limiting epithelial infections (pharyngitis and impetigo), and less frequent severe invasive diseases (bacteremia, toxic shock syndrome and necrotizing fasciitis). Annually, GAS causes 700 million infections, including 1.8 million invasive infections with a mortality rate of 25%. In order to establish an infection, GAS must counteract the oxidative stress conditions generated by the release of reactive oxygen species (ROS) at the infection site by host immune cells such as neutrophils and monocytes. ROS are the highly reactive and toxic byproducts of oxygen metabolism, including hydrogen peroxide (H2O2), superoxide anion (O2•(-)), hydroxyl radicals (OH•) and singlet oxygen (O2*), which can damage bacterial nucleic acids, proteins and cell membranes. This review summarizes the enzymatic and regulatory mechanisms utilized by GAS to thwart ROS and survive under conditions of oxidative stress.


Assuntos
Interações Hospedeiro-Patógeno/imunologia , Estresse Oxidativo/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus/efeitos dos fármacos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Infecções Estreptocócicas/imunologia , Streptococcus/enzimologia , Streptococcus/imunologia
14.
J Biol Chem ; 289(46): 32303-32315, 2014 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-25266727

RESUMO

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.


Assuntos
Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/enzimologia , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases , Membrana Celular/microbiologia , Biologia Computacional , Exotoxinas/metabolismo , Feminino , Teste de Complementação Genética , Histidina Quinase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Dados de Sequência Molecular , Neutrófilos/microbiologia , Mutação Puntual , Polissacarídeo-Liases/metabolismo , Polissacarídeos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Virulência
15.
Cell Host Microbe ; 15(6): 729-740, 2014 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-24922575

RESUMO

Group A Streptococcus (GAS) is a leading cause of infection-related mortality in humans. All GAS serotypes express the Lancefield group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N-acetylglucosamine (GlcNAc) side chain, which is the basis of rapid diagnostic tests. No biological function has been attributed to this conserved antigen. Here we identify and characterize the GAC biosynthesis genes, gacA through gacL. An isogenic mutant of the glycosyltransferase gacI, which is defective for GlcNAc side-chain addition, is attenuated for virulence in two infection models, in association with increased sensitivity to neutrophil killing, platelet-derived antimicrobials in serum, and the cathelicidin antimicrobial peptide LL-37. Antibodies to GAC lacking the GlcNAc side chain and containing only polyrhamnose promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge after passive immunization. Thus, the Lancefield antigen plays a functional role in GAS pathogenesis, and a deeper understanding of this unique polysaccharide has implications for vaccine development.


Assuntos
Infecções Estreptocócicas/virologia , Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/patogenicidade , Acetilglucosamina/imunologia , Acetilglucosamina/metabolismo , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Peptídeos Catiônicos Antimicrobianos , Proteínas de Bactérias/genética , Carboidratos/imunologia , Catelicidinas/farmacologia , Epitopos , Feminino , Glicosiltransferases/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Masculino , Camundongos Endogâmicos , Mutagênese , Neutrófilos/microbiologia , Coelhos , Infecções Estreptocócicas/imunologia , Vacinas Estreptocócicas/genética , Streptococcus pyogenes/efeitos dos fármacos , Fatores de Virulência/genética
16.
J Infect Dis ; 210(8): 1325-38, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-24799598

RESUMO

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 Recombinantes
17.
Clin Microbiol Rev ; 27(2): 264-301, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24696436

RESUMO

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/genética
18.
mBio ; 4(4)2013 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-23919999

RESUMO

UNLABELLED: Streptococcus pyogenes (group A Streptococcus [GAS]) causes ~700 million human infections/year, resulting in >500,000 deaths. There is no commercial GAS vaccine available. The GAS surface protein arginine deiminase (ADI) protects mice against a lethal challenge. ADI is an enzyme that converts arginine to citrulline and ammonia. Administration of a GAS vaccine preparation containing wild-type ADI, a protein with inherent enzymatic activity, may present a safety risk. In an approach intended to maximize the vaccine safety of GAS ADI, X-ray crystallography and structural immunogenic epitope mapping were used to inform vaccine design. This study aimed to knock out ADI enzyme activity without disrupting the three-dimensional structure or the recognition of immunogenic epitopes. We determined the crystal structure of ADI at 2.5 Å resolution and used it to select a number of amino acid residues for mutagenesis to alanine (D166, E220, H275, D277, and C401). Each mutant protein displayed abrogated activity, and three of the mutant proteins (those with the D166A, H275A, and D277A mutations) possessed a secondary structure and oligomerization state equivalent to those of the wild type, produced high-titer antisera, and avoided disruption of B-cell epitopes of ADI. In addition, antisera raised against the D166A and D277A mutant proteins bound to the GAS cell surface. The inactivated D166A and D277A mutant ADIs are ideal for inclusion in a GAS vaccine preparation. There is no human ortholog of ADI, and we confirm that despite limited structural similarity in the active-site region to human peptidyl ADI 4 (PAD4), ADI does not functionally mimic PAD4 and antiserum raised against GAS ADI does not recognize human PAD4. IMPORTANCE: We present an example of structural biology informing human vaccine design. We previously showed that the administration of the enzyme arginine deiminase (ADI) to mice protected the mice against infection with multiple GAS serotypes. In this study, we determined the structure of GAS ADI and used this information to improve the vaccine safety of GAS ADI. Catalytically inactive mutant forms of ADI retained structure, recognition by antisera, and immunogenic epitopes, rendering them ideal for inclusion in GAS vaccine preparations. This example of structural biology informing vaccine design may underpin the formulation of a safe and efficacious GAS vaccine.


Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Hidrolases/química , Hidrolases/metabolismo , Vacinas Estreptocócicas/química , Vacinas Estreptocócicas/metabolismo , Streptococcus pyogenes/enzimologia , Substituição de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Cristalografia por Raios X , Mapeamento de Epitopos , Humanos , Hidrolases/genética , Hidrolases/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Proteínas Mutantes/metabolismo , Conformação Proteica , Multimerização Proteica , Vacinas Estreptocócicas/genética , Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/química , Streptococcus pyogenes/genética , Streptococcus pyogenes/imunologia
19.
Nanomedicine ; 9(7): 935-44, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23428988

RESUMO

A novel vaccine development platform that enables the site-specific conjugation of synthetic lipid adjuvants to recombinant proteins was produced. This technology facilitates the simple and efficient production of homogeneous, chemically-defined, semisynthetic lipoprotein vaccines. Using a polytope 'string-of-beads' approach, a synthetic gene incorporating seven Streptococcus pyogenes M protein strain-specific antigens, and a conserved M protein antigen (J14) was produced, expressed, and attached to a lipoamino acid based adjuvant (lipid core peptide; LCP). Nanoparticles (40 nm diameter) of an optimal size for stimulating antibody-mediated immunity were formed upon the addition of these lipoproteins to aqueous buffer (PBS). Systemic antigen-specific IgG antibodies were raised against all eight antigens in C57BL/6J mice, without the need to formulate with additional adjuvant. These antibodies bound cell surface M proteins of S. pyogenes strains represented within the polytope sequence, with higher antibody levels observed where a dendritic cell targeting peptide (DCpep) was incorporated within the LCP adjuvant. FROM THE CLINICAL EDITOR: In this study, a novel vaccine development system is presented, combining adjuvants with recombinant protein antigens, and presenting the antigen in a nanoparticle system optimized for antibody production. They demonstrate efficient vaccination in a murine model system without the need for additional adjuvants.


Assuntos
Adjuvantes Imunológicos/química , Lipídeos/química , Nanopartículas/química , Vacinas Estreptocócicas/imunologia , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Imunofluorescência , Imunidade , Lipoproteínas/química , Maleimidas/química , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/prevenção & controle , Vacinas Estreptocócicas/síntese química , Vacinas Estreptocócicas/química , Streptococcus pyogenes/imunologia
20.
Curr Top Microbiol Immunol ; 368: 207-42, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23250780

RESUMO

Currently there is no commercial Group A Streptococcus (GAS; S. pyogenes) vaccine available. The development of safe GAS vaccines is challenging, researchers are confronted with obstacles such as the occurrence of many unique serotypes (there are greater than 150 M types), antigenic variation within the same serotype, large variations in the geographical distribution of serotypes, and the production of antibodies cross-reactive with human tissue which can lead to host auto-immune disease. Cell wall anchored, cell membrane associated, secreted and anchorless proteins have all been targeted as GAS vaccine candidates. As GAS is an exclusively human pathogen, the quest for an efficacious vaccine is further complicated by the lack of an animal model which mimics human disease and can be consistently and reproducibly colonized by multiple GAS strains.


Assuntos
Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Animais , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Transporte/imunologia , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Humanos
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