RESUMO
PURPOSE: This study aims to comprehensively review the multifaceted factors underlying the successful colonization and infection process of Helicobacter pylori (H. pylori), a prominent Gram-negative pathogen in humans. The focus is on elucidating the functions, mechanisms, genetic regulation, and potential cross-interactions of these elements. METHODS: Employing a literature review approach, this study examines the intricate interactions between H. pylori and its host. It delves into virulence factors like VacA, CagA, DupA, Urease, along with phase variable genes, such as babA, babC, hopZ, etc., giving insights about the bacterial perspective of the infection The association of these factors with the infection has also been added in the form of statistical data via Funnel and Forest plots, citing the potential of the virulence and also adding an aspect of geographical biasness to the virulence factors. The biochemical characteristics and clinical relevance of these factors and their effects on host cells are individually examined, both comprehensively and statistically. RESULTS: H. pylori is a Gram-negative, spiral bacterium that successfully colonises the stomach of more than half of the world's population, causing peptic ulcers, gastric cancer, MALT lymphoma, and other gastro-duodenal disorders. The clinical outcomes of H. pylori infection are influenced by a complex interplay between virulence factors and phase variable genes produced by the infecting strain and the host genetic background. A meta-analysis of the prevalence of all the major virulence factors has also been appended. CONCLUSION: This study illuminates the diverse elements contributing to H. pylori's colonization and infection. The interplay between virulence factors, phase variable genes, and host genetics determines the outcome of the infection. Despite biochemical insights into many factors, their comprehensive regulation remains an understudied area. By offering a panoramic view of these factors and their functions, this study enhances understanding of the bacterium's perspective, i.e. H. pylori's journey from infiltration to successful establishment within the host's stomach.
Assuntos
Helicobacter pylori , Úlcera Péptica , Neoplasias Gástricas , Humanos , Virulência/genética , Helicobacter pylori/genética , Úlcera Péptica/microbiologia , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Antígenos de Bactérias/genéticaRESUMO
BACKGROUND AND AIMS: Diabetes and periodontal disease are chronic disorders with complex interplay. Periodontal microbiota may play a major role in the development of periodontal disease (PD). The study was framed to identify oral microorganisms and assess oral biofilm in children & adolescents with T1DM and PD. METHODS: In this cross-sectional study we recruited a total of 60 subjects aged 10-18 years (in 3 groups of 20 each). Group 1: Diabetes with periodontal disease (DMPD), Group 2: Diabetes without periodontal disease (DM), Group 3: Periodontal disease without Diabetes (PD).Gingival plaque samples were collected and processed for culture based microbial identification and biofilm assay. RESULTS: The microbial diversity in the DMPD group was higher. Staphylococcus warneri was the only organism specifically isolated from DMPD group. Staphylococcus vitulinus, Streptococcus sanguinis, Pseudomonas aeruginosa, was commonly isolated from both DMPD and PD group especially higher incidence in DMPD group (P ≤ 0.001).There was a strong positive correlation between poor glycaemic control and biofilm formation in both Groups 1 & 2 (DMPD and DM) patients (Spearman's Rho: 0.868, P < 0.001). CONCLUSION: Children & adolescents with T1DM with worse glycaemic control, associated with higher abundance of biofilm formation and greater microbial diversity, especially in those with T1DM with PD.
Assuntos
Biofilmes/crescimento & desenvolvimento , Diabetes Mellitus Tipo 1/epidemiologia , Diabetes Mellitus Tipo 1/microbiologia , Gengiva/microbiologia , Doenças Periodontais/epidemiologia , Doenças Periodontais/microbiologia , Adolescente , Criança , Estudos Transversais , Diabetes Mellitus Tipo 1/diagnóstico , Feminino , Humanos , Masculino , Doenças Periodontais/diagnósticoRESUMO
BACKGROUND: To analyze the molecular epidemiology and to compare between the major methicillin resistant Staphylococcus aureus biotypes for association with patient characteristics who had an implant for closed fracture and developed early post-operative wound infections (POWI) in a tertiary care hospital of India. METHODS: Pulsed-field gel electrophoresis (PFGE), antimicrobial resistance, accessory gene regulator (agr) and staphylococcal cassette chromosome mec (SCCmec) types, Paton-Valentine leukocidin (PVL) gene, toxin gene profiling, biofilm formation and patient demographics were correlated with MLST clonal complexes (CC). FINDINGS: Overall eight different sequence types (STs) were detected with a predominance of ST239 (66%), ST22 (18%) and some minor types ST772, ST30 (4% each) ST1, ST642, ST6, ST107 (2% each). All ST239 isolates belong to CC239 and SCCmec III whereas ST22 isolates belong to CC22 and SCCmec IV. The isolates varied in the distribution of various toxin genes. With 63.63% biofilm formers ST239 were all multidrug resistant with frequent resistance to erythromycin, clindamycin, gentamicin, cefuroxime, amoxyclav and ciprofloxacin indicating doxycycline, amikacin, vancomycin and linezolid can be the drug of choice. CONCLUSION: This study shows that ST239 MRSA is still most prevalent strain with new emergence of ST642 and ST107 isolates in association with orthopedic implant based POWI. As compare to other ST types ST239 strain was associated with adverse treatment outcomes. This highlights the importance of improving nosocomial infection control measures in this unit.
Assuntos
Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Próteses e Implantes , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Adulto , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Infecção Hospitalar , Eletroforese em Gel de Campo Pulsado/métodos , Exotoxinas/genética , Feminino , Fixação de Fratura , Genes Bacterianos , Humanos , Índia/epidemiologia , Leucocidinas/genética , Masculino , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem Molecular , Tipagem de Sequências Multilocus , Ortopedia , Atenção Terciária à Saúde , Transativadores/genéticaRESUMO
Leishmania donovani possesses a complex heteroxenic life cycle where infective metacyclic promastigotes are pre-adapted to infect their host and cope up with intracellular stress. Exploiting the similarities between cultured and sandfly derived promastigotes, we used early and late passage cultured promastigotes to show specific changes at genome level which compromise pathogen fitness reflected in gene expression and infection studies. The pathogen loses virulence mostly via transcriptional and translational regulations and long-time cultivation makes them struggle to convert to virulent metacyclics. At the genomic level very subtle plasticity was observed between the early and the late passages mostly in defense-related, nutrient acquisition and signal transduction genes. Chromosome Copy number variation is seen in the early and late passages involving several genes that may be playing a role in pathogenicity. Our study highlights the importance of ABC transporters and calpain like cysteine proteases in parasite virulence in cultured promastigotes. Interestingly, these proteins are emerging as important patho-adaptive factors in clinical isolates of Leishmania. We found that the currently available genome of Leishmania in the NCBI database are from late passages. Our early passage genome can act as a reference for future studies on virulent isolates of Leishmania. The annotated leads from this study can be used for virulence surveillance and therapeutic studies in the Indian subcontinent.
RESUMO
Vibrio cholerae converts glucose into either acid or the neutral end product acetoin and its survival in carbohydrate enriched media is linked to the nature of the byproducts produced. It has been demonstrated in this study that Escherichia coli strain isolated from the gut of healthy human volunteers and the commonly used probiotic E. coli Nissle strain that metabolize glucose to acidic byproducts drastically reduce the survival of V. cholerae strains irrespective of their glucose sensitivity and acetoin production status. Accordingly, E. coli glucose transport mutants that produce lower amounts of acidic metabolites had little effect on the survival of V. cholerae in cocultures. Thus, cross feeding of byproducts of glucose metabolism by heterologous bacteria modulates the survival of V. cholerae in glucose rich medium suggesting that composition of the gut microbiota could influence the outcome of V. cholerae infection especially when glucose based ORS is administered.
RESUMO
Vibrio cholerae, the etiological agent of cholera, is known to form biofilms to persist in the environment. It is demonstrated here that even during infection, biofilm genes are upregulated, and microscopic observation indicated that biofilm formation is initiated almost immediately after adherence of V. cholerae to intestinal cells. About 7-fold upregulation of the biofilm regulatory gene vpsT was observed within 30 minutes of adherence of V. cholerae to the intestinal cell line INT 407, and a massive induction of about 700-fold was observed in rabbit ileal loops. The upregulation was observed in the classical and El Tor biotype strains of serogroup O1 that is most frequently associated with epidemic cholera. vpsT upregulation was primarily dependent on the virulence master regulator AphA. Of possible clinical relevance was the observation that V. cholerae in the INT 407-associated biofilms was significantly more resistant to antibiotics than unadhered planktonic cells.
Assuntos
Aderência Bacteriana , Biofilmes/crescimento & desenvolvimento , Células Epiteliais/microbiologia , Vibrio cholerae O1/fisiologia , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Genes Bacterianos , Humanos , Íleo/microbiologia , Coelhos , Fatores de TempoRESUMO
Contact with host cells is recognized as a signal capable of triggering expression of bacterial genes important for host pathogen interaction. Adherence of Helicobacter pylori to the gastric epithelial cell line AGS strongly upregulated expression of a gene, HP0102, in the adhered bacteria in all strains examined, including several Indian clinical isolates. The gene is highly conserved and ubiquitously present in all 69 sequenced H. pylori genomes at the same genomic locus, as well as in 15 Indian clinical isolates. The gene is associated with 2 distinct phenotypes related to pathogenicity. In AGS cell-adhered H. pylori, it has a role in upregulation of cagA expression from a specific σ(28)-RNAP promoter and consequent induction of the hummingbird phenotype in the infected AGS cells. Furthermore, HP0102 has a role in chemotaxis and a ΔHP0102 mutant exhibited low acid-escape response that might account for the poor colonization efficiency of the mutant.
Assuntos
Aderência Bacteriana , Células Epiteliais/microbiologia , Helicobacter pylori/patogenicidade , Ativação Transcricional , Fatores de Virulência/biossíntese , Linhagem Celular , Quimiotaxia , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Helicobacter pylori/fisiologia , Humanos , Fatores de Virulência/genéticaRESUMO
Adherence of Helicobacter pylori to the gastric epithelial cell line AGS strongly induces expression of fliK encoding a flagellar hook-length control protein. FliK has a role in triggering dissociation of the alternate sigma factor, σ(28), from a nonfunctional σ(28)-FlgM complex, releasing free, functional σ(28). The σ(28)-RNA polymerase initiates transcription of cagA, the major virulence gene, from a promoter identified in this study. Consequently, significant up-regulation of cagA was observed in AGS-adhered H. pylori. Direct binding of σ(28) to the cagA promoter was demonstrated by chromatin immunoprecipitation and the transcription start site was identified by 5' RACE (rapid amplification of complementary DNA ends). The σ(28)-dependent cagA promoter was active specifically in AGS-adhered H. pylori, and this motif might be associated with high cagA expression and severity of disease. These results also indicate that H. pylori has evolved to integrate expression of the major virulence gene cagA with the flagellar regulatory circuit, essential for colonization of the human host.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Fator sigma/metabolismo , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Sequência de Bases , Linhagem Celular Tumoral , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Fator sigma/genética , Estômago/citologiaRESUMO
This manuscript has been withdrawn by the author.
RESUMO
After entering the gastrointestinal (GI) tract on the way to their physiological site of infection, enteric bacteria encounter a remarkable diversity in environmental conditions. There are gross differences in the physico-chemical parameters in different sections of the GI tract e.g. between the stomach, small intestine and large intestine. Furthermore, even within a certain anatomical site, there are subtle differences in the microenvironment e.g. between the lumen, mucous layer and epithelial surface. Enteric pathogens must not only survive passage through the rapidly changing environments encountered at different niches of the GI tract but must also appropriately coordinate expression of virulence determinants in response to environmental cues at different stages of infection. There are some common themes in the responses of enteric pathogens to environmental cues, there are also distinct differences that may reflect differences in basic pathogenesis mechanisms. The role of bile and oxygen concentration in spatiotemporal regulation of virulence genes in selected enteric pathogens has been reviewed.
RESUMO
BACKGROUND: Helicobacter pylori, a gram negative bacterium, colonizes the stomach in a majority of the world population. The two major virulence factors of H. pylori VacA and CagA, thought to be associated with chronic inflammation and disease, have been extensively studied, but the regulation of the expression of these virulence genes in H. pylori remains poorly understood. METHODS: qRT-PCR was performed to quantify gene expression in unadhered and AGS-adhered H. pylori. Δfur mutant was constructed by splicing by overlap extension PCR and allelic exchange. RESULTS: Adherence of H. pylori to the gastric epithelial cell line AGS strongly induces the expression of both cagA and vacA. Induction of cagA and vacA in the AGS cell-adhered H. pylori Δfur mutant strain was consistently lower than in the adhered parent strain. However, expression of the genes was similar between the wild-type and Δfur mutant strains in the unadhered state, suggesting that Fur has a role in the upregulation of cagA and vacA expression, especially in AGS-adhered H. pylori. Consistent with these results, microscopic observations revealed that infection of AGS cells with H. pylori Δfur mutant strain produced much less damage as compared to that produced by the wild-type H. pylori strain. CONCLUSIONS: These results suggested that cagA and vacA gene expression is upregulated in H. pylori, especially by host cell contact, and Fur has a role in the upregulation.
Assuntos
Antígenos de Bactérias/biossíntese , Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica , Helicobacter pylori/fisiologia , Interações Hospedeiro-Patógeno , Fatores de Virulência/biossíntese , Antígenos de Bactérias/genética , Aderência Bacteriana , Proteínas de Bactérias/genética , Linhagem Celular , Células Epiteliais/microbiologia , Deleção de Genes , Helicobacter pylori/genética , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Virulência/genéticaRESUMO
Exposure to arsenic (As) causes serious health hazards. Therefore, there is a sustained effort to understand the molecular basis of the risk posed by the toxicant. It has been reported that apoptotic changes ensue on exposure to As. To investigate the molecular basis of such changes, we sequenced the entire mitochondrial (mt) genome from PBMC of a subset of these individuals (As-exposed=16 and unexposed=18) using Affymetrix platform. Our analysis revealed that As exposure does not induce large-scale mt-DNA variations, but that specific deleterious changes could induce mt dysfunction. A Glu115Ter mutation as well as 17 other in silico predicted deleterious variants were identified exclusively in exposed individuals. The number of variants in mt Complex I in As-exposed individuals was positively correlated with their respective intracellular ROS level. In addition, the extent of potentially damaging variants in As-exposed individuals had significant positive correlation to the degree of G0 /G1 cell cycle arrest.
Assuntos
Apoptose/genética , Arsênio/toxicidade , Variação Genética , Genoma Mitocondrial/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Estudos de Casos e Controles , Humanos , Mutação , Espécies Reativas de Oxigênio/sangueRESUMO
Vibrio cholerae, an important human intestinal pathogen, is responsible for the diarrheal disease cholera. The pathogenesis of V. cholerae is a highly coordinated process that involves diverse regulatory factors. It has recently been demonstrated that disruption of the V. cholerae fadD gene, encoding a long-chain fatty acyl coenzyme A (acyl-CoA) ligase, drastically reduces expression of the major virulence genes and in vivo lethality of this important human pathogen. This effect was due to reduced membrane localization of the central virulence regulator TcpP. In this study, the reason for the impaired membrane localization of TcpP in the fadD mutant was investigated. We demonstrate that extracytoplasmic stress is induced in the V. cholerae ΔfadD strain. In response to the extracytoplasmic stress, the integral membrane protease RseP is activated and degrades the membrane-localized TcpP in the fadD mutant strain. Indeed, disruption of the rseP gene in a fadD mutant background restored membrane localization of TcpP and expression of the downstream virulence genes toxT, ctxA, and tcpA. Increased expression of the σ(E) regulon genes in ethanol-treated wild-type V. cholerae indicated that ethanol exposure could induce an extracytoplasmic stress response in V. cholerae. Ethanol treatment also led to activation of the RseP protease activity and resulted in degradation of membrane-localized TcpP and subsequent reduction in expression of the virulence genes. Taken together, these results suggest that extracytoplasmic stress response per se reduces virulence of V. cholerae by impairing membrane localization of TcpP.
Assuntos
Proteínas de Bactérias/metabolismo , Vibrio cholerae/metabolismo , Vibrio cholerae/patogenicidade , Animais , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Íleo/microbiologia , Mutação , Transporte Proteico , Coelhos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , VirulênciaRESUMO
In view of the world wide prevalence of Helicobacter pylori infection, its potentially serious consequences, and the increasing emergence of antibiotic resistant H. pylori strains there is an urgent need for the development of alternative strategies to combat the infection. In this study it has been demonstrated that polyethyleneimine (PEI) functionalized zinc oxide (ZnO) nanoparticles (NPs) inhibit the growth of a metronidazole-resistant strain of H. pylori and the molecular basis of the anti-bacterial activity of ZnO-PEI NP has been investigated. The ZnO-PEI NP was synthesized using a wet chemical method with a core size of approximately 3-7 nm. Internalization and distribution of ZnO-PEI NP without agglomeration was observed in H. pylori cytosol by electron microscopy. Several lines of evidence including scanning electron microscopy, propidium iodide uptake and ATP assay indicate severe membrane damage in ZnO-PEI NP treated H. pylori. Intracellular ROS generation increased rapidly following the treatment of H. pylori with ZnO-PEI NP and extensive degradation of 16S and 23S rRNA was observed by quantitative reverse-transcriptase PCR. Finally, considerable synergy between ZnO-PEI NP and antibiotics was observed and it has been demonstrated that the concentration of ZnO-PEI NP (20 µg/ml) that is non-toxic to human cells could be used in combination with sub-inhibitory concentrations of antibiotics for the inhibition of H. pylori growth.
Assuntos
Antibacterianos/farmacologia , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Metronidazol/farmacologia , Nanopartículas/química , Polietilenoimina/farmacologia , Óxido de Zinco/farmacologia , Farmacorresistência Bacteriana , Helicobacter pylori/citologia , Helicobacter pylori/crescimento & desenvolvimento , Humanos , Polietilenoimina/química , Óxido de Zinco/químicaRESUMO
Vibrio cholerae, a noninvasive bacterium, colonizes the intestinal epithelium and secretes cholera toxin (CT), a potent enterotoxin that causes the severe fluid loss characteristic of the disease cholera. In this study, we demonstrate that adherence of V. cholerae to the intestinal epithelial cell line INT 407 strongly induces the expression of the major virulence genes ctxAB and tcpA and the virulence regulatory gene toxT. No induction of toxR and tcpP, which encode transcriptional activators of toxT, was observed in adhered bacteria, and the adherence-dependent upregulation of toxT expression was independent of ToxR and TcpP. A sharp increase in the expression of the vieA gene, which encodes a cyclic di-GMP (c-di-GMP) phosphodiesterase, was observed in INT 407-adhered V. cholerae immediately after infection. Induction of toxT, ctxAB, and tcpA in INT 407-adhered vieA mutant strain O395 ΔvieA was consistently lower than in the parent strain, although no effect was observed in unadhered bacteria, suggesting that VieA has a role in the upregulation of toxT expression specifically in host cell-adhered V. cholerae. Furthermore, though VieA has both a DNA binding helix-turn-helix domain and an EAL domain conferring c-di-GMP phosphodiesterase activity, the c-di-GMP phosphodiesterase activity of VieA is necessary and sufficient for the upregulation of toxT expression.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , Proteínas de Bactérias/genética , Cólera/microbiologia , Regulação Enzimológica da Expressão Gênica , Interações Hospedeiro-Patógeno , Vibrio cholerae/enzimologia , Fatores de Virulência/genética , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Regulação Bacteriana da Expressão Gênica , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade , Vibrio cholerae/fisiologia , Fatores de Virulência/metabolismoRESUMO
A unique event in bacterial epidemiology was the emergence of the El Tor biotype of Vibrio cholerae O1 and the subsequent rapid displacement of the existing classical biotype as the predominant cause of epidemic cholera. We demonstrate that when the El Tor and classical biotypes were cocultured in standard laboratory medium a precipitous decline in colony forming units (CFU) of the classical biotype occurred in a contact dependent manner. Several lines of evidence including DNA release, microscopy and flow cytometric analysis indicated that the drastic reduction in CFU of the classical biotype in cocultures was not accompanied by lysis, although when the classical biotype was grown individually in monocultures, lysis of the cells occurred concomitant with decrease in CFU starting from late stationary phase. Furthermore, uptake of a membrane potential sensitive dye and protection of genomic DNA from extracellular DNase strongly suggested that the classical biotype cells in cocultures retained viability in spite of loss of culturability. These results suggest that coculturing the classical biotype with the El Tor biotype protects the former from lysis allowing the cells to remain viable in spite of the loss of culturability. The stationary phase sigma factor RpoS may have a role in the loss of culturability of the classical biotype in cocultures. Although competitive exclusion of closely related strains has been reported for several bacterial species, conversion of the target bacterial population to the viable non-culturable state has not been demonstrated previously and may have important implications in the evolution of bacterial strains.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Viabilidade Microbiana , Vibrio cholerae/classificação , Vibrio cholerae/crescimento & desenvolvimento , Bacteriólise , Técnicas de Cocultura , Contagem de Colônia Microbiana , Desoxirribonucleases/metabolismo , Citometria de Fluxo , Concentração de Íons de Hidrogênio , Ensaios de Proteção de Nucleases , Vibrio cholerae/citologiaRESUMO
Earlier we had reported that irrespective of the source cigarette smoke (CS) contains substantial amounts of p-benzosemiquinone, which is readily converted to p-benzoquinone (p-BQ) by disproportionation and oxidation by transition metal containing proteins. Here we show that after CS-exposure, p-BQ-protein adducts are formed in the lungs as well as serum albumin of guinea pigs. We also show that serum of human smokers contains p-BQ-albumin adduct. It is known that human serum albumin (HSA) plays a very important role in binding and transport of a variety of ligands, including fatty acids and drugs. We show in vitro that p-BQ forms covalent adducts with free amino groups of all twenty amino acids as well as É-amino groups of lysine residues of HSA in a concentration dependent manner. When HSA is incubated with p-BQ in the molar ratio of 1:1, the number of p-BQ incorporated is 1. At the molar ratio of 1:60, the number of p-BQ incorporated is 40. The formation of HSA-p-BQ adduct has been demonstrated by absorption spectroscopy, MALDI-MS and MALDI-TOF-TOF-MS analyses. Upon complexation with p-BQ, the secondary structure and conformation of HSA are altered, as evidenced by steady state and time-resolved fluorescence, circular dichroism, 8-anilino-1-napthalenesulfonic acid binding and differential scanning calorimetry. Alteration of the structure and conformation of HSA results in impairment of its ligand binding properties with respect to myristic acid, quercitin and paracetamol. This might be one of the reasons why transport and distribution of lipids and drugs are impaired in smokers.
Assuntos
Benzoquinonas/sangue , Albumina Sérica/metabolismo , Fumar/sangue , Acetaminofen/metabolismo , Adulto , Animais , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Cobaias , Humanos , Pulmão/metabolismo , Masculino , Ligação Proteica , Conformação Proteica , Quercetina/metabolismo , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria UltravioletaRESUMO
In the enteric pathogen Vibrio cholerae, expression of the major virulence factors is controlled by the hierarchical expression of several regulatory proteins comprising the ToxR regulon. In this study, we demonstrate that disruption of the fadD gene encoding a long-chain fatty acyl coenzyme A ligase has marked effects on expression of the ToxR virulence regulon, motility, and in vivo lethality of V. cholerae. In the V. cholerae fadD mutant, expression of the major virulence genes ctxAB and tcpA, encoding cholera toxin (CT), and the major subunit of the toxin-coregulated pilus (TCP) was drastically repressed and a growth-phase-dependent reduction in the expression of toxT, encoding the transcriptional activator of ctxAB and tcpA, was observed. Expression of toxT from an inducible promoter completely restored CT to wild-type levels in the V. cholerae fadD mutant, suggesting that FadD probably acts upstream of toxT expression. Expression of toxT is activated by the synergistic effect of two transcriptional regulators, TcpP and ToxR. Reverse transcription-PCR and Western blot analysis indicated that although gene expression and production of both TcpP and ToxR are unaffected in the fadD mutant strain, membrane localization of TcpP, but not ToxR, is severely impaired in the fadD mutant strain from the mid-logarithmic phase of growth. Since the decrease in toxT expression occurred concomitantly with the reduction in membrane localization of TcpP, a direct correlation between the defect in membrane localization of TcpP and reduced toxT expression in the fadD mutant strain is suggested.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Virulência/metabolismo , Animais , Animais Lactentes , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ácidos Graxos/metabolismo , Camundongos , Mutação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Virulência/genéticaRESUMO
Vibrio cholerae strains of the O1 serogroup that typically cause epidemic cholera can be classified into two biotypes, classical and El Tor. The El Tor biotype emerged in 1961 and subsequently displaced the classical biotype as a cause of cholera throughout the world. In this study we demonstrate that when strains of the El Tor and classical biotypes were cocultured in standard LB medium, the El Tor strains clearly had a competitive growth advantage over the classical biotype starting from the late stationary phase and could eventually take over the population. The classical biotype produces extracellular protease(s) in the stationary phase, and the amounts of amino acids and small peptides in the late stationary and death phase culture filtrates of the classical biotype were higher than those in the corresponding culture filtrates of the El Tor biotype. The El Tor biotype cells could utilize the amino acids more efficiently than the classical biotype under the alkaline pH of the stationary phase cultures but not in medium buffered to neutral pH. The growth advantage of the El Tor biotype was also observed in vivo using the ligated rabbit ileal loop and infant mouse animal models.
Assuntos
Vibrio cholerae O1/crescimento & desenvolvimento , Aminoácidos/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Cólera/microbiologia , Técnicas de Cocultura , Contagem de Colônia Microbiana , Meios de Cultura/química , Modelos Animais de Doenças , Concentração de Íons de Hidrogênio , Íleo/microbiologia , Camundongos , Peptídeo Hidrolases/metabolismo , Coelhos , Vibrio cholerae O1/patogenicidade , VirulênciaRESUMO
Cholera toxin (CT) is an archetypal bacterial toxin that binds with a high affinity to the receptor ganglioside GM1 on the intestinal epithelial surface and that causes the severe watery diarrhea characteristic of the disease cholera. Blockage of the interaction of CT with the GM1 receptor is an attractive approach for therapeutic intervention. We report here that crude bile prevents the interaction of CT with GM1 and reduces CT-mediated fluid accumulation in the rabbit intestine. The unsaturated fatty acids detected in crude bile, arachidonic, linoleic, and oleic acids, were found to be the most effective. Crude bile and the unsaturated fatty acids interacted with CT but not GM1 to prevent CT-GM1 binding. Neither crude bile nor the unsaturated fatty acids had any effect on the subunit structure of CT. The binding of CT to unsaturated fatty acids resulted in a shift of the apparent pI of CT from 6.8 to 8.2 and a marked decrease in intrinsic fluorescence. The Kd was calculated from fluorescence quenching assays. It was demonstrated by the rabbit ileal loop model that practically no fluid accumulated in the intestinal loops when CT was administered together with inhibitory concentrations of linoleic acid. The bile present in the intestine was sufficient to inhibit the activity of up to 300 ng CT. Bile and unsaturated fatty acids also inhibited the binding of Escherichia coli heat-labile enterotoxin (LT) to GM1, and no fluid accumulation was observed in rabbit ileal loops when LT was administered together with linoleic acid.