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1.
Nat Commun ; 12(1): 5469, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34552091

RESUMO

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.


Assuntos
Anticorpos Neutralizantes/farmacologia , COVID-19/tratamento farmacológico , Anticorpos de Domínio Único/farmacologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Administração Intranasal , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Epitopos/química , Epitopos/metabolismo , Feminino , Masculino , Mesocricetus , Testes de Neutralização , SARS-CoV-2/efeitos dos fármacos , Anticorpos de Domínio Único/administração & dosagem , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismo , Glicoproteína da Espícula de Coronavírus/química
2.
Nat Commun ; 12(1): 4349, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272394

RESUMO

Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.


Assuntos
Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Membrana/química , Periplasma/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteínas Tirosina Quinases/química , Motivos de Aminoácidos , Domínio Catalítico , Microscopia Crioeletrônica , Citoplasma/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Periplasma/química , Fosforilação , Conformação Proteica em alfa-Hélice , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Tirosina/química , Tirosina/metabolismo
4.
Pathogens ; 9(12)2020 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-33260788

RESUMO

Streptococcus uberis is a common cause of intramammary infection and mastitis in dairy cattle. Unlike other mammary pathogens, S. uberis evades detection by mammary epithelial cells, and the host-pathogen interactions during early colonisation are poorly understood. Intramammary challenge of dairy cows with S. uberis (strain 0140 J) or isogenic mutants lacking the surface-anchored serine protease, SUB1154, demonstrated that virulence was dependent on the presence and correct location of this protein. Unlike the wild-type strain, the mutant lacking SUB1154 failed to elicit IL-1ß from ex vivo CD14+ cells obtained from milk (bovine mammary macrophages, BMM), but this response was reinstated by complementation with recombinant SUB1154; the protein in isolation elicited no response. Production of IL-1ß was ablated in the presence of various inhibitors, indicating dependency on internalisation and activation of NLRP3 and caspase-1, consistent with inflammasome activation. Similar transcriptomic changes were detected in ex vivo BMM in response to the wild-type or the SUB1154 deletion mutant, consistent with S. uberis priming BMM, enabling the SUB1154 protein to activate inflammasome maturation in a transcriptionally independent manner. These data can be reconciled in a novel model of pathogenesis in which, paradoxically, early colonisation is dependent on the innate response to the initial infection.

6.
Nat Struct Mol Biol ; 27(9): 846-854, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32661423

RESUMO

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral , Receptores Virais/metabolismo , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/ultraestrutura , Afinidade de Anticorpos , Reações Antígeno-Anticorpo/imunologia , Betacoronavirus/metabolismo , Ligação Competitiva , COVID-19 , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Modelos Moleculares , Biblioteca de Peptídeos , Peptidil Dipeptidase A/ultraestrutura , Ligação Proteica , Conformação Proteica , Receptores Virais/ultraestrutura , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , SARS-CoV-2 , Homologia de Sequência de Aminoácidos , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/ultraestrutura , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura
7.
J Infect Dis ; 218(5): 801-808, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29701830

RESUMO

Background: Africa has the highest incidence of gonorrhea in the world. However, little is known about gonococcal populations in this continent or mechanisms of antimicrobial resistance (AMR). Methods: Whole-genome sequence data were analyzed from 103 Neisseria gonorrhoeae isolates from 73 patients, mainly men who have sex with men, from coastal Kenya. We annotated loci, defined the core genome, defined mechanisms of AMR, and performed phylogenetic analysis. For patients with multiple episodes of gonorrhea, we determined whether infections occurred with related strains. Results: We identified 3 clusters of isolates that are phylogenetically distinct from isolates found elsewhere. Plasmids were virtually ubiquitous: pTetM and pblaTEM were found in 97%, and 55% of isolates, respectively. This was associated with high doxycycline use for undiagnosed sexually transmitted infections. Twenty-three percent of multiple episodes of gonorrhea in the same individual were caused by a related strain, suggesting inadequate treatment or reinfection. Conclusions: The prevalence of plasmid-mediated AMR in Kenyan gonococci contrasts with that in wealthy countries, where AMR is largely chromosomally mediated. Antimicrobials have a profound effect on the maintenance of lineages harboring plasmids. Doxycycline can select for tetracycline and penicillin resistance, through plasmid cooperation. Understanding the mechanisms of AMR in high-risk groups is required to inform treatment strategies.


Assuntos
Farmacorresistência Bacteriana , Gonorreia/microbiologia , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/isolamento & purificação , Plasmídeos/análise , Adolescente , Adulto , Antibacterianos/uso terapêutico , Análise por Conglomerados , Biologia Computacional , Uso de Medicamentos , Feminino , Gonorreia/tratamento farmacológico , Gonorreia/epidemiologia , Humanos , Quênia/epidemiologia , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Anotação de Sequência Molecular , Neisseria gonorrhoeae/genética , Filogenia , Prevalência , Análise de Sequência de DNA , Sequenciamento Completo do Genoma , Adulto Jovem
8.
Structure ; 24(6): 926-34, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27161979

RESUMO

DNA transformation is a widespread process allowing bacteria to capture free DNA by using filamentous nano-machines composed of type IV pilins. These proteins can act as DNA receptors as demonstrated by the finding that Neisseria meningitidis ComP minor pilin has intrinsic DNA-binding ability. ComP binds DNA better when it contains the DNA-uptake sequence (DUS) motif abundant in this species genome, playing a role in its trademark ability to selectively take up its own DNA. Here, we report high-resolution structures for meningococcal ComP and Neisseria subflava ComPsub, which recognize different DUS motifs. We show that they are structurally identical type IV pilins that pack readily into filament models and display a unique DD region delimited by two disulfide bonds. Functional analysis of ComPsub defines a new mode of DNA binding involving the DD region, adapted for exported DNA receptors.


Assuntos
DNA Bacteriano/metabolismo , Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Neisseria/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Fímbrias Bacterianas/metabolismo , Modelos Moleculares , Neisseria/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína
9.
BMC Genomics ; 16: 334, 2015 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-25898893

RESUMO

BACKGROUND: Streptococcus uberis, a Gram-positive, catalase-negative member of the family Streptococcaceae is an important environmental pathogen responsible for a significant proportion of subclinical and clinical bovine intramammary infections. Currently, the genome of only a single reference strain (0140J) has been described. Here we present a comparative analysis of complete draft genome sequences of an additional twelve S. uberis strains. RESULTS: Pan and core genome analysis revealed the core genome common to all strains to be 1,550 genes in 1,509 orthologous clusters, complemented by 115-246 accessory genes present in one or more S. uberis strains but absent in the reference strain 0140J. Most of the previously predicted virulent genes were present in the core genome of all 13 strains but gene gain/loss was observed between the isolates in CDS associated with clustered regularly interspaced short palindromic repeats (CRISPRs), prophage and bacteriocin production. Experimental challenge experiments confirmed strain EF20 as non-virulent; only able to infect in a transient manner that did not result in clinical mastitis. Comparison of the genome sequence of EF20 with the validated virulent strain 0140J identified genes associated with virulence, however these did not relate clearly with clinical/non-clinical status of infection. CONCLUSION: The gain/loss of mobile genetic elements such as CRISPRs and prophage are a potential driving force for evolutionary change. This first "whole-genome" comparison of strains isolated from clinical vs non-clinical intramammary infections including the type virulent vs non-virulent strains did not identify simple gene gain/loss rules that readily explain, or be confidently associated with, differences in virulence. This suggests that a more complex dynamic determines infection potential and clinical outcome not simply gene content.


Assuntos
Genoma Bacteriano , Streptococcus/genética , Virulência/genética , Animais , Bacteriocinas/metabolismo , Sequência de Bases , Bovinos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Hibridização Genômica Comparativa , Feminino , Mastite Bovina/genética , Mastite Bovina/microbiologia , Mastite Bovina/patologia , Leite/microbiologia , Dados de Sequência Molecular , Filogenia , Polimorfismo de Nucleotídeo Único , Alinhamento de Sequência , Análise de Sequência de DNA , Streptococcus/classificação , Streptococcus/patogenicidade
10.
Elife ; 32014 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-25534642

RESUMO

Genome-wide association studies have found variation within the complement factor H gene family links to host susceptibility to meningococcal disease caused by infection with Neisseria meningitidis (Davila et al., 2010). Mechanistic insights have been challenging since variation within this locus is complex and biological roles of the factor H-related proteins, unlike factor H, are incompletely understood. N. meningitidis subverts immune responses by hijacking a host-immune regulator, complement factor H (CFH), to the bacterial surface (Schneider et al., 2006; Madico et al., 2007; Schneider et al., 2009). We demonstrate that complement factor-H related 3 (CFHR3) promotes immune activation by acting as an antagonist of CFH. Conserved sequences between CFH and CFHR3 mean that the bacterium cannot sufficiently distinguish between these two serum proteins to allow it to hijack the regulator alone. The level of protection from complement attack achieved by circulating N. meningitidis therefore depends on the relative levels of CFH and CFHR3 in serum. These data may explain the association between genetic variation in both CFH and CFHR3 and susceptibility to meningococcal disease.


Assuntos
Proteínas de Bactérias/metabolismo , Fator H do Complemento/metabolismo , Meningites Bacterianas/genética , Neisseria meningitidis/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Fator H do Complemento/química , Fator H do Complemento/genética , Predisposição Genética para Doença , Células HEK293 , Humanos , Meningites Bacterianas/imunologia , Dados de Sequência Molecular , Neisseria meningitidis/patogenicidade , Homologia de Sequência de Aminoácidos
11.
PLoS Pathog ; 8(10): e1002981, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23133374

RESUMO

Neisseria meningitis remains a leading cause of sepsis and meningitis, and vaccines are required to prevent infections by this important human pathogen. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against the meningococcus and recruits the host complement regulator, fH. As the high affinity interaction between fHbp and fH could impair immune responses, we sought to identify non-functional fHbps that could act as effective immunogens. This was achieved by alanine substitution of fHbps from all three variant groups (V1, V2 and V3 fHbp) of the protein; while some residues affected fH binding in each variant group, the distribution of key amino underlying the interaction with fH differed between the V1, V2 and V3 proteins. The atomic structure of V3 fHbp in complex with fH and of the C-terminal barrel of V2 fHbp provide explanations to the differences in the precise nature of their interactions with fH, and the instability of the V2 protein. To develop transgenic models to assess the efficacy of non-functional fHbps, we determined the structural basis of the low level of interaction between fHbp and murine fH; in addition to changes in amino acids in the fHbp binding site, murine fH has a distinct conformation compared with the human protein that would sterically inhibit binding to fHbp. Non-functional V1 fHbps were further characterised by binding and structural studies, and shown in non-transgenic and transgenic mice (expressing chimeric fH that binds fHbp and precisely regulates complement system) to retain their immunogenicity. Our findings provide a catalogue of non-functional fHbps from all variant groups that can be included in new generation meningococcal vaccines, and establish proof-in-principle for clinical studies to compare their efficacy with wild-type fHbps.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Meningite Meningocócica/prevenção & controle , Vacinas Meningocócicas/imunologia , Neisseria meningitidis/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Antibacterianos/imunologia , Sítios de Ligação , Fator H do Complemento/imunologia , Fator H do Complemento/metabolismo , Feminino , Humanos , Meningite Meningocócica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica/imunologia , Isoformas de Proteínas/genética , Estrutura Secundária de Proteína
12.
Microbiology (Reading) ; 158(Pt 6): 1581-1592, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22383474

RESUMO

The regulation and control of gene expression in response to differing environmental stimuli is crucial for successful pathogen adaptation and persistence. The regulatory gene vru of Streptococcus uberis encodes a stand-alone response regulator with similarity to the Mga of group A Streptococcus. Mga controls expression of a number of important virulence determinants. Experimental intramammary challenge of dairy cattle with a mutant of S. uberis carrying an inactivating lesion in vru showed reduced ability to colonize the mammary gland and an inability to induce clinical signs of mastitis compared with the wild-type strain. Analysis of transcriptional differences of gene expression in the mutant, determined by microarray analysis, identified a number of coding sequences with altered expression in the absence of Vru. These consisted of known and putative virulence determinants, including Lbp (Sub0145), SclB (Sub1095), PauA (Sub1785) and hasA (Sub1696).


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mastite Bovina/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus/metabolismo , Streptococcus/patogenicidade , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Bovinos , Feminino , Infecções Estreptocócicas/microbiologia , Streptococcus/genética , Virulência , Fatores de Virulência/genética
13.
Vet Res ; 41(5): 63, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20519112

RESUMO

Streptococcus uberis, strain 0140J, contains a single copy sortase A (srtA), encoding a transamidase capable of covalently anchoring specific proteins to peptidoglycan. Unlike the wild-type, an isogenic mutant carrying an inactivating ISS1 insertion within srtA was only able to infect the bovine mammary gland in a transient fashion. For the first 24 h post challenge, the srtA mutant colonised at a similar rate and number to the wild type strain, but unlike the wild type did not subsequently colonise in higher numbers. Similar levels of host cell infiltration were detected in response to infection with both strains, but only in those mammary quarters infected with the wild type strain were clinical signs of disease evident. Mutants that failed to express individual sortase substrate proteins (sub0135, sub0145, sub0207, sub0241, sub0826, sub0888, sub1095, sub1154, sub1370, and sub1730) were isolated and their virulence determined in the same challenge model. This revealed that mutants lacking sub0145, sub1095 and sub1154 were attenuated in cattle. These data demonstrate that a number of sortase anchored proteins each play a distinct, non-redundant and important role in pathogenesis of S. uberis infection within the lactating bovine mammary gland.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Mastite Bovina/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus/classificação , Streptococcus/patogenicidade , Aminoaciltransferases/genética , Animais , Proteínas de Bactérias/genética , Bovinos , Cisteína Endopeptidases/genética , DNA Bacteriano/genética , Indústria de Laticínios , Feminino , Regulação Bacteriana da Expressão Gênica/fisiologia , Lactação , Mutação , Infecções Estreptocócicas/microbiologia
14.
J Proteome Res ; 9(2): 1088-95, 2010 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-20038184

RESUMO

Sortase (a transamidase) has been shown to be responsible for the covalent attachment of proteins to the bacterial cell wall. Anchoring is effected on secreted proteins containing a specific cell wall motif toward their C-terminus; that for sortase A (SrtA) in Gram-positive bacteria often incorporates the sequence LPXTG. Such surface proteins are often characterized as virulence determinants and play important roles during the establishment and persistence of infection. Intramammary infection with Streptococcus uberis is a common cause of bovine mastitis, which impacts on animal health and welfare and the economics of milk production. Comparison of stringently produced cell wall fractions from S. uberis and an isogenic mutant strain lacking SrtA permitted identification of 9 proteins likely to be covalently anchored at the cell surface. Analysis of these sequences implied the presence of two anchoring motifs for S. uberis, the classical LPXTG motif and an additional LPXXXD motif.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Dados de Sequência Molecular , Oligopeptídeos/química , Homologia de Sequência de Aminoácidos , Streptococcus , Especificidade por Substrato
15.
PLoS One ; 4(7): e6072, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19603075

RESUMO

BACKGROUND: Streptococcus suis is a zoonotic pathogen that infects pigs and can occasionally cause serious infections in humans. S. suis infections occur sporadically in human Europe and North America, but a recent major outbreak has been described in China with high levels of mortality. The mechanisms of S. suis pathogenesis in humans and pigs are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: The sequencing of whole genomes of S. suis isolates provides opportunities to investigate the genetic basis of infection. Here we describe whole genome sequences of three S. suis strains from the same lineage: one from European pigs, and two from human cases from China and Vietnam. Comparative genomic analysis was used to investigate the variability of these strains. S. suis is phylogenetically distinct from other Streptococcus species for which genome sequences are currently available. Accordingly, approximately 40% of the approximately 2 Mb genome is unique in comparison to other Streptococcus species. Finer genomic comparisons within the species showed a high level of sequence conservation; virtually all of the genome is common to the S. suis strains. The only exceptions are three approximately 90 kb regions, present in the two isolates from humans, composed of integrative conjugative elements and transposons. Carried in these regions are coding sequences associated with drug resistance. In addition, small-scale sequence variation has generated pseudogenes in putative virulence and colonization factors. CONCLUSIONS/SIGNIFICANCE: The genomic inventories of genetically related S. suis strains, isolated from distinct hosts and diseases, exhibit high levels of conservation. However, the genomes provide evidence that horizontal gene transfer has contributed to the evolution of drug resistance.


Assuntos
Resistência Microbiana a Medicamentos/genética , Streptococcus suis/patogenicidade , Virulência/genética , Zoonoses/microbiologia , Animais , DNA Bacteriano/genética , Surtos de Doenças , Genoma Bacteriano , Humanos , Filogenia , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/microbiologia , Streptococcus suis/classificação , Streptococcus suis/efeitos dos fármacos , Streptococcus suis/genética
16.
BMC Genomics ; 10: 54, 2009 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-19175920

RESUMO

BACKGROUND: Streptococcus uberis, a Gram positive bacterial pathogen responsible for a significant proportion of bovine mastitis in commercial dairy herds, colonises multiple body sites of the cow including the gut, genital tract and mammary gland. Comparative analysis of the complete genome sequence of S. uberis strain 0140J was undertaken to help elucidate the biology of this effective bovine pathogen. RESULTS: The genome revealed 1,825 predicted coding sequences (CDSs) of which 62 were identified as pseudogenes or gene fragments. Comparisons with related pyogenic streptococci identified a conserved core (40%) of orthologous CDSs. Intriguingly, S. uberis 0140J displayed a lower number of mobile genetic elements when compared with other pyogenic streptococci, however bacteriophage-derived islands and a putative genomic island were identified. Comparative genomics analysis revealed most similarity to the genomes of Streptococcus agalactiae and Streptococcus equi subsp. zooepidemicus. In contrast, streptococcal orthologs were not identified for 11% of the CDSs, indicating either unique retention of ancestral sequence, or acquisition of sequence from alternative sources. Functions including transport, catabolism, regulation and CDSs encoding cell envelope proteins were over-represented in this unique gene set; a limited array of putative virulence CDSs were identified. CONCLUSION: S. uberis utilises nutritional flexibility derived from a diversity of metabolic options to successfully occupy a discrete ecological niche. The features observed in S. uberis are strongly suggestive of an opportunistic pathogen adapted to challenging and changing environmental parameters.


Assuntos
Adaptação Biológica/genética , Genoma Bacteriano , Streptococcus/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bovinos , Hibridização Genômica Comparativa , DNA Bacteriano/genética , Evolução Molecular , Perfilação da Expressão Gênica , Genes Bacterianos , Ilhas Genômicas , Mastite Bovina/microbiologia , Filogenia , Análise de Sequência de DNA , Streptococcus/metabolismo , Streptococcus/patogenicidade , Virulência
17.
J Mol Biol ; 381(3): 734-47, 2008 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-18588895

RESUMO

The characteristics of a streptococcal plasminogen activator (PA) displaying specificity for ruminant plasminogen (Plg) were defined using molecular approaches. The 16-kDa secreted protein PadA was found to be prevalent in Streptococcus dysgalactiae subspecies dysgalactiae isolated from cases of bovine mastitis and septic arthritis in lambs. PadA was able to activate bovine, ovine and caprine Plg, but not human Plg. Amino acid sequence analysis identified a limited level of homology to other streptococcal PAs, including streptokinase; however, PadA was found to align well with and match in size the staphylococcal PA, staphylokinase. Recombinant PadA was used to investigate interaction with bovine Plg, leading to formation of an activator complex that was capable of recruiting and converting further substrate Plg into plasmin. Individual non-overlapping peptides of PadA or bovine microplasminogen were found to block the interaction between PadA and bovine Plg, preventing the formation of the activation complex. Homology modelling based upon structures of staphylokinase complexed with human microplasminogen supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex.


Assuntos
Proteínas de Bactérias/metabolismo , Ativadores de Plasminogênio/metabolismo , Plasminogênio/metabolismo , Streptococcus/enzimologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Bovinos , Ativação Enzimática , Metaloendopeptidases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Plasminogênio/química , Ativadores de Plasminogênio/química , Ligação Proteica , Proteínas Recombinantes/metabolismo , Especificidade da Espécie
18.
Vet Immunol Immunopathol ; 104(3-4): 155-62, 2005 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-15734536

RESUMO

Streptococcus (S.) uberis is a common cause of mastitis in cattle. A protein (PauA) secreted by this bacterium is capable of activating plasminogen from sheep and cattle. The PauA first binds to bovine plasminogen (b-plg) to form a PauA-plasminogen complex that subsequently binds to and activates b-plg to form plasmin. We have identified several linear epitopes of PauA that are recognized by murine monoclonal antibodies to PauA. Two of the monoclonal antibodies which neutralized the enzymatic activity of PauA, EC3 and 2.22, recognized common linear peptide sequences with similar charge and spacing patterns. These neutralization epitopes are located in the predicted alpha-domain of the PauA molecule. Further, these same epitopes are in critical structure/function domains identified in other studies. These characterizations may facilitate the design of an efficacious vaccine for streptococcal mastitis in the dairy cow.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/imunologia , Doenças das Cabras/microbiologia , Mastite Bovina/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus/imunologia , Sequência de Aminoácidos , Animais , Bovinos , Ensaio de Imunoadsorção Enzimática/veterinária , Epitopos/análise , Feminino , Cabras , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia
19.
J Mol Biol ; 342(4): 1101-14, 2004 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-15351638

RESUMO

The interactions between bovine plasminogen and the streptococcal plasminogen activator PauA that culminate in the generation of plasmin are not fully understood. Formation of an equimolar activation complex comprising PauA and plasminogen by non-proteolytic means is a prerequisite to the recruitment of substrate plasminogen; however the determinants that facilitate these interactions have yet to be defined. A mutagenesis strategy comprising nested deletions and random point substitutions indicated roles for both amino and carboxyl-terminal regions of PauA and identified further essential residues within the alpha domain of the plasminogen activator. A critical region within the alpha domain was identified using non-overlapping PauA peptides to block the interaction between PauA and bovine plasminogen, preventing formation of the activation complex. Homology modelling of the activation complex based upon the known structures of streptokinase complexed with human plasmin supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex.


Assuntos
Proteínas de Bactérias/metabolismo , Plasminogênio/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Bovinos , Primers do DNA , Dados de Sequência Molecular , Mutagênese , Ligação Proteica , Deleção de Sequência , Homologia de Sequência de Aminoácidos
20.
Indian J Med Res ; 119 Suppl: 136-40, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15232179

RESUMO

BACKGROUND & OBJECTIVES: Streptococci produce a diverse range of secreted plasminogen activators capable of converting mammalian plasminogen to plasmin in a species-specific manner. In all examples to date, the host animal's plasminogen and that of a number of additional species have been shown to interact with these molecules leading to the conclusion that the pathogenesis of streptococci is in some way dependent upon activation of host plasminogen. PauA was the first plasminogen activator described from Streptococcus uberis, a pathogen frequently isolated from cases of bovine mastitis. Recently, a second S. uberis plasminogen activator (PauB) was identified from a Danish mastitis isolate. Interestingly, the pauB open reading frame occupied the locus normally filled by pauA. In the present study a genetic screen of streptococcal and field isolates frequently associated with mastitis was undertaken to assess the distribution, chromosomal location and sequence variation of these putative virulence factors. METHODS: Southern analysis of a diverse panel of streptococci and additional bacterial isolates frequently associated with bovine mastitis was performed using pauA and pauB probes. Sequence variation of PauA was assessed at the protein level following nucleotide sequence analysis of pauA alleles amplified from isolates picked from different geographical locations. RESULTS: We observed plasminogen activators to be universally distributed amongst S. uberis. A pauA allele was identified in all but one strain of S. uberis. This strain had a pauB allele substituted for pauA at the same locus. The remarkably low level of sequence variation demonstrated by PauA was further restricted to a limited number of residues within the molecule. INTERPRETATION & CONCLUSION: The high prevalence of PauA alleles in field isolates of S.uberis supported the observation that plasminogen activators are likely to confer an advantage with respect to colonization and growth. The findings of the present study support the theory that PauA plays a critical role in the pathogenesis of S. uberis.


Assuntos
Ativadores de Plasminogênio/genética , Streptococcus/genética , Sequência de Bases , Southern Blotting , Primers do DNA , Fases de Leitura Aberta
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