RESUMO
The PspC and Hic proteins of Streptococcus pneumoniae are some of the most variable microbial immune evasion proteins identified to date. Due to structural similarities and conserved binding profiles, it was assumed for a long time that these pneumococcal surface proteins represent a protein family comprised of eleven subgroups. Recently, however, the evaluation of more proteins revealed a greater diversity of individual proteins. In contrast to previous assumptions a pattern evaluation of six PspC and five Hic variants, each representing one of the previously defined subgroups, revealed distinct structural and likely functionally regions of the proteins, and identified nine new domains and new domain alternates. Several domains are unique to PspC and Hic variants, while other domains are also present in other virulence factors encoded by pneumococci and other bacterial pathogens. This knowledge improved pattern evaluation at the level of full-length proteins, allowed a sequence comparison at the domain level and identified domains with a modular composition. This novel strategy increased understanding of individual proteins variability and modular domain composition, enabled a structural and functional characterization at the domain level and furthermore revealed substantial structural differences between PspC and Hic proteins. Given the exceptional genomic diversity of the multifunctional PspC and Hic proteins a detailed structural and functional evaluation need to be performed at the strain level. Such knowledge will also be useful for molecular strain typing and characterizing PspC and Hic proteins from new clinical S. pneumoniae strains.
Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Streptococcus pneumoniae/metabolismo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Humanos , Mutação , Filogenia , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/patologia , Ligação Proteica , Domínios Proteicos , Análise de Sequência de Proteína , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
The coenzyme-binding proteins play a vital role in the cellular metabolism processes, such as fatty acid biosynthesis, enzyme and gene regulation, lipid synthesis, particular vesicular traffic, and ß-oxidation donation of acyl-CoA esters. Based on the theory of Star Graph Topological Indices (SGTIs) of protein primary sequences, we proposed a method to develop a first classification model for predicting protein with coenzyme-binding properties. To simulate the properties of coenzyme-binding proteins, we created a dataset containing 2897 proteins, among 456 proteins functioned as coenzyme-binding activity. The SGTIs of peptide sequence were calculated with Sequence to Star Network (S2SNet) application. We used the SGTIs as inputs to several classification techniques with a machine learning software - Weka. A Random Forest classifier based on 3 features of the embedded and non-embedded graphs was identified as the best predictive model for coenzyme-binding proteins. This model developed was with the true positive (TP) rate of 91.7%, false positive (FP) rate of 7.6%, and Area Under the Receiver Operating Characteristic Curve (AUROC) of 0.971. The prediction of new coenzyme-binding activity proteins using this model could be useful for further drug development or enzyme metabolism researches.
Assuntos
Acil Coenzima A/química , Aprendizado de Máquina , Proteínas/química , Acil Coenzima A/metabolismo , Ésteres/química , Ésteres/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Proteínas/metabolismo , SoftwareRESUMO
Several pathogens including Gram-negative bacteria hijack complement regulators to escape host's innate response. Pathogenic Leptospira species bind Factor H, C4b binding protein and vitronectin from the complement system. We evaluated the ability of low passage (LP) and culture-attenuated (CA) pathogenic strains of Leptospira, to bind Factor H. We used LOCaS46 (Leptospira interrogans sv Canicola), LOVe30 (L. interrogans sv Icterohaemorrhagiae) and MOCA45 (L. santarosai sv Tarassovi), and ten high passage strains of Leptospira [used in the microscopic agglutination test (MAT)]. Afterwards, we assessed their survival in normal human serum (NHS). Interestingly, the ability in binding Factor H was higher for LOCaS46 and LOVe30 LP strains, than for the respective CA strains suggesting that the ability of evading the alternative complement pathway is lost after culture attenuation. Accordingly, the level of mRNA expression of the Factor H binding proteins, LigA, LigB and Lsa23 was higher in these LP strains than in the corresponding CA strains. Unexpectedly, no difference in Factor H binding and surviving was observed between LP and CA MOCA45 strains. The high passage MAT-reference strains showed variation in Factor H binding ability, but, in most cases, the ability for capturing Factor H by Leptospira strains correlated with their survival in NHS.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Regulação Bacteriana da Expressão Gênica , Leptospira/imunologia , Leptospira/patogenicidade , Proteínas de Transporte/genética , Fator H do Complemento/metabolismo , Humanos , Evasão da Resposta Imune/genética , Leptospira/genética , Leptospirose/microbiologia , Viabilidade Microbiana/genética , Viabilidade Microbiana/imunologia , Ligação Proteica , RNA Mensageiro/genéticaRESUMO
The opportunistic fungal pathogen Aspergillus fumigatus can cause severe infections, particularly in immunocompromised individuals. Upon infection, A. fumigatus faces the powerful and directly acting immune defense of the human host. The mechanisms on how A. fumigatus evades innate immune attack and complement are still poorly understood. Here, we identify A. fumigatus enolase, AfEno1, which was also characterized as fungal allergen, as a surface ligand for human plasma complement regulators. AfEno1 binds factor H, factor-H-like protein 1 (FHL-1), C4b binding protein (C4BP), and plasminogen. Factor H attaches to AfEno1 via two regions, via short conserved repeats (SCRs) 6-7 and 19-20, and FHL-1 contacts AfEno1 via SCRs 6-7. Both regulators when bound to AfEno1 retain cofactor activity and assist in C3b inactivation. Similarly, the classical pathway regulator C4BP binds to AfEno1 and bound to AfEno1; C4BP assists in C4b inactivation. Plasminogen which binds to AfEno1 via lysine residues is accessible for the tissue-type plasminogen activator (tPA), and active plasmin cleaves the chromogenic substrate S2251, degrades fibrinogen, and inactivates C3 and C3b. Plasmin attached to swollen A. fumigatus conidia damages human A549 lung epithelial cells, reduces the cellular metabolic activity, and induces cell retraction, which results in exposure of the extracellular matrix. Thus, A. fumigatus AfEno1 is a moonlighting protein and virulence factor which recruits several human regulators. The attached human regulators allow the fungal pathogen to control complement at the level of C3 and to damage endothelial cell layers and tissue components.
Assuntos
Aspergillus fumigatus/enzimologia , Proteína de Ligação ao Complemento C4b/metabolismo , Fator H do Complemento/metabolismo , Proteínas Fúngicas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/metabolismo , Fosfopiruvato Hidratase/metabolismo , Plasminogênio/metabolismo , Células Epiteliais Alveolares/imunologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/microbiologia , Aspergilose/imunologia , Aspergilose/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/imunologia , Linhagem Celular , Fator H do Complemento/imunologia , Proteínas Fúngicas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Evasão da Resposta Imune , Cinética , Fosfopiruvato Hidratase/imunologia , Ligação ProteicaRESUMO
Ficolins recognize pathogen associated molecular patterns and activate the lectin pathway of complement system. However, our knowledge regarding pathogen recognition of human ficolins is still limited. We therefore set out to explore and investigate the possible interactions of the two main serum ficolins, ficolin-2 and ficolin-3 with different Gram-negative bacteria. We used recombinant ficolin molecules and normal human serum, which were detected with anti-ficolin monoclonal antibodies. In addition we investigated the capacity of these pathogens to activate the lectin pathway of complement system. We show for the first time that human ficolin-2 recognizes the nonpathogenic spirochete Leptospira biflexa serovar Patoc, but not the pathogenic Leptospira interrogans serovar Kennewicki strain Fromm. Additionally, human ficolin-2 and ficolin-3 recognize pathogenic Pasteurella pneumotropica, enteropathogenic Escherichia coli (EPEC) serotype O111ab:H2 and enteroaggregative E. coli (EAEC) serogroup O71 but not four enterohemorrhagic E. coli, three EPEC, three EAEC and two nonpathogenic E. coli strains (DH5α and HB101). The lectin pathway was activated by Pasteurella pneumotropica, EPEC O111ab:H2 and EAEC O71 after incubation with C1q depleted human serum. In conclusion, this study provide novel insight in the binding and complement activating capacity of the lectin pathway initiation molecules ficolin-2 and ficolin-3 towards relevant Gram-negative pathogens of pathophysiological relevance.
Assuntos
Lectina de Ligação a Manose da Via do Complemento/imunologia , Escherichia coli/imunologia , Glicoproteínas/imunologia , Lectinas/imunologia , Leptospira/imunologia , Pasteurella pneumotropica/imunologia , Humanos , Proteínas Recombinantes/imunologia , FicolinasRESUMO
Neonatal calf colibacillosis caused by enterotoxigenic Escherichia coli (ETEC) is an economically significant problem in most parts of the world. The most common ETEC found in calves express the F5 (K99) fimbriae, which are necessary for the attachment of the bacteria to the ganglioside receptors on enterocytes. It is known that prevention of ETEC F5(+) adhesion to its ganglioside receptors with specific antibodies protects calves from colibacillosis. Previously we have described the development and characterization of a mouse recombinant antibody fragment (moRAb) that prevents F5 fimbrial protein induced agglutination of horse red blood cells (HRBC), which exhibit the same gangloside receptor for F5 fimbriae. Here we demonstrate that this recombinant antibody fragment inhibits in vitro the attachment of ETEC F5(+) bacteria to HRBC as well as isolated calf enterocytes, and in vivo it decreases fluid accumulation in intestinal loops of calves. Thus, correct oral administration of this anti-F5 moRAb may serve as an immunoprophylactic for cost effective control of colibacillosis in calves.
Assuntos
Anticorpos Antibacterianos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Doenças dos Bovinos/prevenção & controle , Enterócitos/efeitos dos fármacos , Infecções por Escherichia coli/veterinária , Animais , Animais Recém-Nascidos , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/uso terapêutico , Bovinos , Doenças dos Bovinos/tratamento farmacológico , Doenças dos Bovinos/patologia , Enterotoxinas/toxicidade , Eritrócitos/efeitos dos fármacos , Escherichia coli , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/patologia , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/imunologia , Fímbrias Bacterianas/patologia , Cavalos , Íleo/patologia , Masculino , Proteínas Recombinantes/imunologiaRESUMO
Pasteurella pneumotropica is an opportunist Gram negative bacterium responsible for rodent pasteurellosis that affects upper respiratory, reproductive and digestive tracts of mammals. In animal care facilities the presence of P. pneumotropica causes severe to lethal infection in immunodeficient mice, being also a potential source for human contamination. Indeed, occupational exposure is one of the main causes of human infection by P. pneumotropica. The clinical presentation of the disease includes subcutaneous abscesses, respiratory tract colonization and systemic infections. Given the ability of P. pneumotropica to fully disseminate in the organism, it is quite relevant to study the role of the complement system to control the infection as well as the possible evasion mechanisms involved in bacterial survival. Here, we show for the first time that P. pneumotropica is able to survive the bactericidal activity of the human complement system. We observed that host regulatory complement C4BP and Factor H bind to the surface of P. pneumotropica, controlling the activation pathways regulating the formation and maintenance of C3-convertases. These results show that P. pneumotropica has evolved mechanisms to evade the human complement system that may increase the efficiency by which this pathogen is able to gain access to and colonize inner tissues where it may cause severe infections.
Assuntos
Proteína de Ligação ao Complemento C4b/imunologia , Fator H do Complemento/imunologia , Pasteurella pneumotropica/imunologia , Convertases de Complemento C3-C5/metabolismo , HumanosRESUMO
Many herpesviruses, poxviruses and retroviruses encode proteins related to chemokines and chemokine receptors. The first one discovered, US28 of human cytomegalovirus (HCMV), is a 7-transmembrane domain G protein-coupled chemokine receptor able to activate diverse cellular responses, including cell migration and gene expression. A related ORF named US27 is adjacent to US28, but no functions have been defined yet. Recently ORFs 3-7, a cluster of five concatenated ORFs with highest homology to US28 and mammalian chemokine receptors, were sequenced from a prototype "stealth virus", an African green monkey simian CMV (SCMV)-related entity with unusual fungal, bacterial and mammalian gene homologues. Stealth viruses have not yet been independently replicated in tissue culture, and therefore their biological significance remains unclear. ORF3, ORF4, ORF5 and ORF6 are complete ORFs whereas the sequence of ORF7 is incomplete. In the present study, we identified five corresponding ORFs in the genome of a clinical isolate of bonafide simian CMV (SCMV), strain 9610. We found substantial differences between the SCMV and "stealth virus" ORFs, especially for ORF5 where there are 31% non-identities at the amino acid level. Four conserved genes unrelated to chemokines (64K/CAP, DNBI, UL32, and IE2) in SCMV and HCMV had on average 52% identity at the deduced amino acid level, whereas the corresponding values for the SCMV ORFs versus US28 ranged from 21% to 30%, suggesting rapid gene diversification in this cluster. Consistent with this, the amino acid identity for any pairwise comparison among the SCMV ORFs is only 21-52%. The chemokine receptor homologues are estimated to comprise approximately 2-3% of the SCMV genome. HCMV US27 and US28 homologues have also been identified in the chimpanzee CMV genome, whereas mouse and rat CMV lack chemokine receptor homologues. This genomic analysis indicates that SCMV has an unusually high concentration of US28-related chemokine receptor homologues that have arisen by gene duplication and have diverged extensively from their closest relatives in mammals and other beta herpesviruses. The rate of divergence appears to be very rapid compared to other known SCMV genes, suggesting strong positive selection.
Assuntos
Citomegalovirus/genética , Genoma Viral , Receptores de Quimiocinas/genética , Sequência de Aminoácidos , Animais , Chlorocebus aethiops/virologia , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Pan troglodytes/virologia , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
Chemokines regulate the trafficking of leukocytes in immunity and inflammation and have been implicated in mouse models in acute cardiac and renal allograft rejection; however, their significance to human transplantation is not yet defined. The association of human chemokine receptor genetic variants, CCR5-Delta32, CCR5-59029-A/G, CCR2-V64I, CX3CR1-V249I, and CX3CR1-T280M, with outcome in 163 renal transplant recipients was examined here. Significant reductions were found in risk of acute renal transplant rejection in recipients who possessed the CCR2-64I allele (odds ratio [OR], 0.30; 95% confidence interval [CI], 0.12 to 0.78; P = 0.014) or who were homozygous for the 59029-A allele (OR, 0.37; 95% CI, 0.16 to 0.85; P = 0.016). There were no significant differences in the incidence of rejection among patients stratified as with or without CCR5-Delta32 or by the CX3CR1-V249I or CX3CR1-T280M genotypes. Adjustment for known risk factors for transplant rejection confirmed the univariate findings for possession of the CCR2-64I allele (OR, 0.20; P = 0.032) and homozygosity for the 59029-A allele (OR, 0.26; P = 0.027). It was concluded that the risk of acute rejection in renal transplantation is associated with genetic variation in the chemokine receptors CCR2 and CCR5.