RESUMO
Salmonella enterica infections remain a challenging health issue, causing significant morbidity and mortality worldwide. Current vaccines against typhoid fever display moderate efficacy whilst no licensed vaccines are available for paratyphoid fever or invasive non-typhoidal salmonellosis. Therefore, there is an urgent need to develop high efficacy broad-spectrum vaccines that can protect against typhoidal and non-typhoidal Salmonella. The Salmonella outer membrane porins OmpC and OmpF, have been shown to be highly immunogenic antigens, efficiently eliciting protective antibody, and cellular immunity. Furthermore, enterobacterial porins, particularly the OmpC, have a high degree of homology in terms of sequence and structure, thus making them a suitable vaccine candidate. However, the degree of the amino acid conservation of OmpC among typhoidal and non-typhoidal Salmonella serovars is currently unknown. Here we used a bioinformatical analysis to classify the typhoidal and non-typhoidal Salmonella OmpC amino acid sequences into different clades independently of their serological classification. Further, our analysis determined that the porin OmpC contains various amino acid sequences that are highly conserved among both typhoidal and non-typhoidal Salmonella serovars. Critically, some of these highly conserved sequences were located in the transmembrane ß-sheet within the porin ß-barrel and have immunogenic potential for binding to MHC-II molecules, making them suitable candidates for a broad-spectrum Salmonella vaccine. Collectively, these findings suggest that these highly conserved sequences may be used for the rational design of an effective broad-spectrum vaccine against Salmonella.
Assuntos
Proteínas de Bactérias/genética , Porinas/genética , Salmonella/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência Conservada , Humanos , Filogenia , Porinas/química , Porinas/metabolismo , Conformação Proteica em alfa-Hélice , Salmonella/química , Salmonella/classificação , Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhi/química , Salmonella typhi/classificação , Salmonella typhi/genética , Salmonella typhi/metabolismo , Alinhamento de Sequência , Febre Tifoide/microbiologiaRESUMO
The interaction between CD40, and its ligand, CD154, is essential for the development of humoral and cellular immune responses. The selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically based diseases and malignancies. We are developing a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Tyr140-Ser-149 amino acid strand. This OmpC-CD154 binds CD40 and activates B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth, proliferation and induced apoptosis in the B-NHL cell lines Raji and Ramos. The Bcl-2 family proteins were regulated and the Bcl-6 and YY1 oncoproteins were inhibited. p38 MAPK activation is an important mechanism underlying the effect on proliferation and apoptosis mediated by this fusion protein. This study establishes a basis for the possible use of fusion protein OmpC-CD154 as an alternative treatment for B-NHL.
Assuntos
Caspases/metabolismo , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Fator de Transcrição YY1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Antígenos CD40/metabolismo , Ligante de CD40/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Mimetismo Molecular , Peptídeos/química , Porinas/químicaRESUMO
The emergence of infections associated to new antimicrobial resistance in Acinetobacter baumannii (Ab) genotypes represents a major challenge. In this context, this study aimed to determine the diversity of resistance mechanisms and investigate clonal dissemination and predominant sequence types (STs) in multidrug-resistant Ab strains of clinical (tracheal aspirate, n = 17) and environmental (surface, n = 6) origins. Additionally, the major clones found in clinical (A) and environmental (H) strains had their complete genomes sequenced. All strains were submitted to polymerase chain reactions (PCR) for the detection of the ISAba1/blaOXA-51-like and ISAba1/blaOXA-23-like genes, while the expression of genes encoding the carO porin, AdeABC (adeB), AdeFGH (adeG), and AdeIJK (adeJ) efflux pumps was determined by real time PCR (qPCR). Most of the strains were characterized as extensively drug-resistant (XDR) with high minimal inhibitory concentrations (MICs) detected for tigecycline and carbapenems. Associations between ISAba1/OXA-51 and ISAba1/OXA-23 were observed in 91.3% and 52.2% of the strains, respectively. Only the adeB gene was considered hyper-expressed. Furthermore, most of the strains analyzed by the MuLtilocus Sequence-Typing (MLST) were found to belong to the clonal complex 113 (CC113). In addition, a new ST, ST1399, belonging to CC229, was also discovered herein. Strains analyzed by whole genome sequencing presented resistance genes linked to multidrug-resistance phenotypes and confirmed the presence of Tn2008, which provides high levels carbapenem-resistance.
Assuntos
Acinetobacter baumannii/enzimologia , Proteínas de Bactérias/metabolismo , beta-Lactamases/metabolismo , Acinetobacter baumannii/genética , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Porinas/química , Porinas/genética , Alinhamento de Sequência , Tigeciclina/farmacologia , Sequenciamento Completo do Genoma , beta-Lactamases/genéticaRESUMO
OmpD is the major Salmonella enterica serovar Typhimurium (S. Typhimurium) porin and mediates hydrogen peroxide (H2O2) influx. The results described herein extend this finding to hypochlorous acid (HOCl), another reactive oxygen species that is also part of the oxidative burst generated by the phagosome. S. Typhimurium cells lacking OmpD show decreased HOCl influx, and OmpD-reconstituted proteoliposomes show an increase in the uptake of the toxic compound. To understand this physiologically relevant process, we investigated the role of key OmpD residues in H2O2 and NaOCl transport. Using a theoretical approach, residue K16 was defined as a major contributor to the channel electrostatic properties, and E111 was shown to directly participate in the size-exclusion limit of the channel. Together, we provide theoretical, genetic, and biochemical evidence that OmpD mediates H2O2 and NaOCl uptake, and that key residues of the channel are implicated in this process.
Assuntos
Peróxido de Hidrogênio/metabolismo , Ácido Hipocloroso/metabolismo , Porinas/metabolismo , Salmonella typhimurium/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Porinas/química , Porinas/genética , Salmonella typhimurium/química , Salmonella typhimurium/genética , Alinhamento de SequênciaRESUMO
The main goal of this work was to identify the mechanisms responsible for carbapenem resistance in 61 Chilean clinical isolates of Enterobacteriaceae (Enterobacter spp., Serratia marcescens, Morganella morganii, Escherichia coli and Klebsiella pneumoniae) with reduced susceptibility to at least one carbapenem (ertapenem, imipenem or meropenem). All of the isolates were analysed for the presence of carbapenemases, extended spectrum ß-lactamases (ESBLs), AmpC enzymes and outer-membrane proteins. None of the isolates exhibited carbapenemase activity nor did they have any of the carbapenemase genes that were screened for. Most of the 61 strains produced at least one ESBL and/or one AmpC enzyme and either lost their porins or had altered porins according to sequence analysis. The distribution of ESBLs and AmpC enzymes was different among the species studied. Resistance in K. pneumoniae and E. coli isolates was associated with ESBLs; in M. morganii isolates, resistance was attributed to overexpression of an AmpC enzyme; and in Enterobacter spp. isolates, resistance was associated with both types of enzymes. In K. pneumoniae isolates, porin integrity was more a determinant of carbapenem resistance than the presence of ESBLs, whereas in isolates of Enterobacter spp., M. morganii and S. marcescens, the presence of an overexpressed AmpC enzyme was associated with higher imipenem and meropenem MIC values. Therefore, carbapenem resistance in Chilean isolates is not due to true carbapenemases but rather to a combination of porin loss/alteration and ß-lactamase activity. The fact that carbapenemases were not detected in this study is unique, given that many countries in the region have already reported the presence of these enzymes.
Assuntos
Proteínas de Bactérias/biossíntese , Carbapenêmicos/farmacologia , Infecções por Enterobacteriaceae/epidemiologia , Enterobacteriaceae/efeitos dos fármacos , Porinas/química , Resistência beta-Lactâmica/genética , beta-Lactamases/biossíntese , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Chile/epidemiologia , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Porinas/genética , Porinas/metabolismo , beta-Lactamases/genéticaRESUMO
Crithidia deanei is a trypanosomatid protozoan that harbours a symbiotic bacterium. The partners maintain a mutualistic relationship, thus constituting an excellent model for studying metabolic exchanges between the host and the symbiont, the origin of organelles and cellular evolution. According to molecular analysis, symbionts of different trypanosomatid species share high identity and descend from a common ancestor, a ß-proteobacterium of the genus Bordetella. The endosymbiont is surrounded by two membranes, like Gram-negative bacteria, but its envelope presents special features, since phosphatidylcholine is a major membrane component and the peptidoglycan layer is highly reduced, as described in other obligate intracellular bacteria. Like the process that generated mitochondria and plastids, the endosymbiosis in trypanosomatids depends on pathways that facilitate the intensive metabolic exchanges between the bacterium and the host protozoan. A search of the annotated symbiont genome database identified one sequence with identity to porin-encoding genes of the genus Bordetella. Considering that the symbiont outer membrane has a great accessibility to cytoplasm host factors, it was important to characterize this single porin-like protein using biochemical, molecular, computational and ultrastructural approaches. Antiserum against the recombinant porin-like molecule revealed that it is mainly located in the symbiont envelope. Secondary structure analysis and comparative modelling predicted the protein 3D structure as an 18-domain ß-barrel, which is consistent with porin channels. Electrophysiological measurements showed that the porin displays a slight preference for cations over anions. Taken together, the data presented herein suggest that the C. deanei endosymbiont porin is phylogenetically and structurally similar to those described in Gram-negative bacteria, representing a diffusion channel that might contribute to the exchange of nutrients and metabolic precursors between the symbiont and its host cell.
Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/química , Crithidia/microbiologia , Porinas/química , Simbiose , Sequência de Aminoácidos , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Crithidia/fisiologia , Dados de Sequência Molecular , Filogenia , Porinas/genética , Porinas/metabolismo , Alinhamento de SequênciaRESUMO
The PhoB/PhoR-dependent response to inorganic phosphate (Pi)-starvation in Vibrio cholerae O1 includes the expression of vc0719 for the response regulator PhoB, vca0033 for an alkaline phosphatase and vca1008 for an outer membrane protein (OMP). Sequences with high identity to these genes have been found in the genome of clinical and environmental strains, suggesting that the Pi-starvation response in V. cholerae is well conserved. VCA1008, an uncharacterized OMP involved in V. cholerae pathogenicity, presents sequence similarity to porins of Gram-negative bacteria such as phosphoporin PhoE from Escherichia coli. A three-dimensional model shows that VCA1008 is a 16-stranded pore-forming beta-barrel protein that shares three of the four conserved lysine residues responsible for PhoE anionic specificity with PhoE. VCA1008 beta-barrel apparently forms trimers that collapse into monomers by heating. Properties such as heat modifiability and resistance to denaturation by sodium dodecyl sulfate at lower temperatures permitted us to suggest that VCA1008 is a classical porin, more precisely, a phosphoporin due to its Pi starvation-induced PhoB-dependent expression, demonstrated by electrophoretic mobility shift assay and promoter fusion-lacZ assays.
Assuntos
Porinas/genética , Porinas/metabolismo , Vibrio cholerae O1/genética , Vibrio cholerae O1/metabolismo , Sequência de Aminoácidos , Fusão Gênica Artificial , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Temperatura Alta , Modelos Moleculares , Dados de Sequência Molecular , Fosfatos/metabolismo , Porinas/química , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Microcins are a family of low-molecular weight bacteriocins produced and secreted by Gram-negative bacteria. This review is focused on microcin E492, a pore-forming bacteriocin produced by Klebsiella pneumoniae RYC492 that exerts its antibacterial action on related strains. The steps necessary for the production of active microcin E492 involve post-translational modification with a catechol-type siderophore at the C-terminal and proteolytic processing during export to the extracellular space. This bacteriocin has a modular structure, with a toxic domain at the N-terminal and an uptake domain at the C-terminal of the mature protein. The mechanism by which the C-terminal of microcin E492 is recognized by catecholate siderophore receptors is called the "Trojan horse" strategy, because the C-terminal structure mimics essential bacterial elements, which are recognized by the respective receptors and translocated across the outer membrane to exert antibacterial action. The C-terminal uptake module can be exchanged and used with other toxic domains. Microcin E492 also has a cytotoxic effect on malignant human cell lines. The cytotoxic mechanism is through apoptosis, a desired mechanism for cancer therapy. The ability of microcin E492 to form amyloid-like fibrils constitutes a property that can be exploited in the formulation of this bacteriocin as an antitumoral agent, because these fibrils can behave as stable depots to ensure the sustained release of a biologically active molecule. Alternatively, live bacteria can be used as a continuous source of microcin E492 production in specific tumors.
Assuntos
Antibacterianos/química , Antineoplásicos/química , Bacteriocinas/química , Porinas/química , Porinas/fisiologia , Animais , Bacteriocinas/genética , Bacteriocinas/farmacologia , Bacteriocinas/uso terapêutico , Humanos , Porinas/genéticaRESUMO
Identification of Neisseria meningitidis PorA types remains important, as the PorA protein is a major immunogenic component of several meningococcal vaccines under development. In this study, 191 N. meningitidis serogroup B isolates collected in Argentina through active laboratory-based surveillance from 2001 to 2003 were serosubtyped. Nucleotide sequences of the porA variable region 1 (VR1) and VR2 regions were determined in 52 non-serosubtypeable isolates. A substantial number of distinct VR types were identified, and a new VR2 variant from the P1.16 family was described. This is the first report describing PorA types in N. meningitidis serogroup B isolates in Argentina. Furthermore, the wide diversity of subtypes detected by serosubtyping and genosubtyping reveals the difficulty in designing a useful outer-membrane vaccine applicable in this country. A possible mechanism responsible for altered PorA expression was analysed in two PorA types.
Assuntos
Desenho de Fármacos , Variação Genética , Vacinas Meningocócicas , Neisseria meningitidis Sorogrupo B/classificação , Porinas/classificação , Argentina/epidemiologia , Sequência de Bases , Genótipo , Humanos , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/microbiologia , Vacinas Meningocócicas/química , Dados de Sequência Molecular , Neisseria meningitidis Sorogrupo B/genética , Neisseria meningitidis Sorogrupo B/isolamento & purificação , Neisseria meningitidis Sorogrupo B/metabolismo , Vigilância da População , Porinas/química , Porinas/genética , Porinas/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de DNA , SorotipagemRESUMO
The high genetic diversity found among the PorA regions VR1 and VR2 of 101 Neisseria meningitidis isolates from patients with meningococcal disease and healthy carriers in Brazil contrasts with the stability found in the PorA VR3 of these isolates. The presence of VR3 epitope variant 35 or 36 on the surfaces of 87% of the strains analyzed suggests that these antigens should be considered for inclusion in new formulations of vaccines against serogroup B meningococci in Brazil.