RESUMO
We screened a total of 672 plant-tissue extracts to search for phytochemicals that inhibit the function of the type III secretion system (T3SS) of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among candidates examined, we found that an extract from the leaves of Psidium guajava (guava) inhibited the secretion of the EspB protein from EPEC and EHEC without affecting bacterial growth. The guava extract (GE) also inhibited EPEC and EHEC from adhering to and injecting EspB protein into HEp-2 cells. GE seemed to block the translocation of EspB from the bacterial cells to the culture medium. In addition to EPEC and EHEC, GE also inhibited the T3SS of Yersinia pseudotuberculosis and Salmonella enterica serovar Typhimurium. After exposure to GE, Y. pseudotuberculosis stopped the secretion of Yop proteins and lost its ability to induce the apoptosis of mouse bone marrow-derived macrophages. S. Typhimurium exposed to GE ceased the secretion of Sip proteins and lost its ability to invade HEp-2 cells. GE inhibited EspC secretion, the type V secretion protein of EPEC, but not Shiga toxin2 from EHEC. Thus, our results suggest that guava leaves contain a novel type of antimicrobial compound that could be used for the therapeutic treatment and prevention of gram-negative enteropathogenic bacterial infections.
RESUMO
Bacterial pathogens utilize pore-forming toxins or sophisticated secretion systems to establish infection in hosts. Recognition of these toxins or secretion system by nucleotide-binding oligomerization domain leucine-rich repeat proteins (NLRs) triggers the assembly of inflammasomes, the multiprotein complexes necessary for caspase-1 activation and the maturation of inflammatory cytokines such as IL-1ß or IL-18. Here we demonstrate that both the NLRP3 and NLRC4 inflammasomes are activated by thermostable direct hemolysins (TDHs) and type III secretion system 1 (T3SS1) in response to V. parahaemolyticus infection. Furthermore, we identify T3SS1 secreted effector proteins, VopQ and VopS, which induce autophagy and the inactivation of Cdc42, respectively, to prevent mainly NLRC4 inflammasome activation. VopQ and VopS interfere with the assembly of specks in infected macrophages. These data suggest that bacterial effectors interfere with inflammasome activation and contribute to bacterial evasion from the host inflammatory responses.
Assuntos
Autofagia/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Inflamassomos/imunologia , Vibrioses/imunologia , Vibrio parahaemolyticus/patogenicidade , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/imunologia , Toxinas Bacterianas/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Células Cultivadas , Citocinas/metabolismo , Inibidores Enzimáticos , Proteínas Hemolisinas/metabolismo , Evasão da Resposta Imune/imunologia , Inflamassomos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas Repressoras/imunologia , Proteínas Repressoras/metabolismo , Transdução de Sinais , Vibrioses/metabolismo , Vibrio parahaemolyticus/imunologiaRESUMO
Members of the nucleotide-binding, oligomerization domain (NOD)-like receptor (NLR) proteins assemble into a multiprotein platform, known as the inflammasome, to induce caspase-1 activation followed by the subsequent secretion of IL-1ß and IL-18. In this review, we focus on the role of NLRs in inflammasome activation as part of the host defence against bacterial pathogens. One of activators of the NLRC4 inflammasome is bacterial flagellin secreted through type III or IV secretion systems, which are important for the pathogenicity of many Gram-negative bacteria. The NLRP3 inflammasome is mainly activated by a large number of bacterial pore-forming toxins. Despite our knowledge of inflammasome activation upon bacterial infection, the function of antibacterial defence under in vivo conditions remains to be elucidated. Further understanding of NLR function should provide new insights into the mechanisms of host pro-inflammatory responses and the pathogenesis of bacterial infections.
Assuntos
Bactérias/imunologia , Inflamassomos/imunologia , Inflamassomos/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Animais , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Flagelina/imunologia , Flagelina/metabolismo , HumanosRESUMO
Vibrio vulnificus and Vibrio cholerae are Gram-negative pathogens that cause serious infectious disease in humans. The beta form of pro-IL-1 is thought to be involved in inflammatory responses and disease development during infection with these pathogens, but the mechanism of beta form of pro-IL-1 production remains poorly defined. In this study, we demonstrate that infection of mouse macrophages with two pathogenic Vibrio triggers the activation of caspase-1 via the NLRP3 inflammasome. Activation of the NLRP3 inflammasome was mediated by hemolysins and multifunctional repeat-in-toxins produced by the pathogenic bacteria. NLRP3 activation in response to V. vulnificus infection required NF-kappaB activation, which was mediated via TLR signaling. V. cholerae-induced NLRP3 activation also required NF-kappaB activation but was independent of TLR stimulation. Studies with purified V. cholerae hemolysin revealed that toxin-stimulated NLRP3 activation was induced by TLR and nucleotide-binding oligomerization domain 1/2 ligand-mediated NF-kappaB activation. Our results identify the NLRP3 inflammasome as a sensor of Vibrio infections through the action of bacterial cytotoxins and differential activation of innate signaling pathways acting upstream of NF-kappaB.
Assuntos
Toxinas Bacterianas/farmacologia , Proteínas de Transporte/metabolismo , NF-kappa B/fisiologia , Proteína Adaptadora de Sinalização NOD1/fisiologia , Proteína Adaptadora de Sinalização NOD2/fisiologia , Transdução de Sinais/imunologia , Receptores Toll-Like/fisiologia , Vibrio cholerae/patogenicidade , Vibrio vulnificus/patogenicidade , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/microbiologia , Células da Medula Óssea/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Caspase 1/metabolismo , Imunidade Inata/genética , Inflamação/enzimologia , Inflamação/imunologia , Inflamação/microbiologia , Interleucina-1beta/metabolismo , Ligantes , Macrófagos/imunologia , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transdução de Sinais/genética , Vibrio cholerae/imunologia , Vibrio vulnificus/imunologiaRESUMO
The majority of Shiga toxigenic Escherichia coli (STEC) strains isolated from severe STEC disease are those harboring the locus of enterocyte effacement (LEE), which encodes factors involved in adherence to epithelial cells. However, LEE-negative STEC are increasingly isolated from clinical cases. STEC autoagglutinating adhesin (Saa) is widely used as a marker of adhesin in the absence of LEE. In the present study, we compared the adherence of 32 saa-harboring STEC strains to cultured epithelial cells in the absence or presence of d-mannose. In the absence of d-mannose, 19 strains were adherent to HEp-2 and Caco-2 cells, while 12 were non-adherent. One strain showed detachment of epithelial cells. The adherence of 13 strains was sensitive to the presence of d-mannose. The saa mutant of strain T141, in which adherence was mannose resistant, did not show a significant decrease in adherence compared to the wild type, suggesting a Saa-independent mechanism of adherence. saa-harboring STEC exhibited differential binding properties to epithelial cells, which could not be attributed to the number of C-terminal repeats of Saa, or to the expression of Saa as detected by Western blotting. Our results suggest that multiple adherence mechanisms are present in saa-harboring STEC, implying a high degree of diversity in this group of STEC.
Assuntos
Adesinas Bacterianas/fisiologia , Aderência Bacteriana , Células Epiteliais/microbiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli Shiga Toxigênica/fisiologia , Adesinas Bacterianas/genética , Linhagem Celular Tumoral , Proteínas de Escherichia coli/genética , Humanos , Manose/metabolismo , Escherichia coli Shiga Toxigênica/genéticaRESUMO
A rapid immunochromatographic (IC) test to identify enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) was developed to detect EspB secreted by the type III secretion system of these bacteria. The detection limit of the test system was 4 ng/mL. All 33 of 34 strains harboring the eae gene encoding intimin were positive in the IC test, and all 40 of the eae-negative strains were negative. The results showed that the sensitivity was 96.9% and specificity was 100%. The IC test also detected EspB in a stool sample artificially supplemented with 60 ng EspB/mL. The IC test for the detection of EspB may be a practical method to define EPEC or EHEC both in clinical laboratories and the field.
Assuntos
Proteínas da Membrana Bacteriana Externa/análise , Cromatografia/métodos , Infecções por Escherichia coli/diagnóstico , Escherichia coli O157/classificação , Proteínas de Escherichia coli/análise , Escherichia coli/classificação , Testes Sorológicos/métodos , Adesinas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Proteínas de Escherichia coli/genética , Humanos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Epigallocatechin gallate (EGCG), a major polyphenol in green tea, inhibits the type III secretion system (T3SS) of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), Salmonella enterica serovar Typhimurium, and Yersinia pseudotuberculosis. The inhibitory effect causes the inhibition of hemolysis, cell invasion, cell adhesion and apoptosis, which are functions of the type III secretion device. In the case of EPEC, EspB accumulates in the cells. RT-PCR showed that the translation of EspB was not blocked. The transcription of escN, which supplies energy for the injection of the effector factor into the host cells, was also not inhibited. EGCG does not suppress the transcription and translation of T3SS constitutive protein in bacterial cells, but it seems to suppress the normal construction or secretion of T3SS. When Luria-Bertani (LB) medium was used to visualize the EGCG-induced inhibition of T3SS, the inhibitory effect disappeared. The inhibition of T3SS was partially canceled when the T3SS inhibitory potency of EGCG was examined by adding yeast extract, which is a component of LB medium, to DMEM. These results suggest that EGCG probably inhibits secretion by suppressing some metabolic mechanisms of T3SS.
Assuntos
Catequina/análogos & derivados , Escherichia coli Êntero-Hemorrágica/efeitos dos fármacos , Escherichia coli Êntero-Hemorrágica/patogenicidade , Escherichia coli Enteropatogênica/efeitos dos fármacos , Salmonella typhi/efeitos dos fármacos , Sistemas de Secreção Tipo III/efeitos dos fármacos , Yersinia pseudotuberculosis/efeitos dos fármacos , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Catequina/farmacologia , Linhagem Celular , Meios de Cultura/farmacologia , Escherichia coli Enteropatogênica/patogenicidade , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Microbiologia de Alimentos , Humanos , Salmonella typhi/patogenicidade , Fatores de Virulência/metabolismo , Yersinia pseudotuberculosis/patogenicidadeRESUMO
Enterohemorrhagic Escherichia coli O157:H7 was isolated for the first time in Vietnam. Shiga toxin-producing E. coli were isolated from 8 of 100 cows examined. The two strains showing serotype O157:H7 carried the eae, ehxA, and stx2c genes, but the other six were negative for the eae gene.
Assuntos
Escherichia coli O157/isolamento & purificação , Fezes/microbiologia , Animais , Bovinos , Escherichia coli O157/genética , Toxinas Shiga/genética , VietnãRESUMO
Here we report a loop-mediated isothermal amplification (LAMP) method for detecting Shigella and enteroinvasive Escherichia coli. The target for this LAMP method is the ipaH gene which is carried by both of the pathogens. The LAMP method efficiently detected the gene within 2 h at a minimal amount of bacteria (8 CFU) per reaction.
Assuntos
Escherichia coli/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Shigella/isolamento & purificação , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Sondas de DNA , Diarreia/microbiologia , Disenteria Bacilar/microbiologia , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Humanos , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Shigella/genética , Fatores de TempoRESUMO
To evaluate the serogrouping-based diagnosis of diarrheagenic Escherichia coli, a total of 1,130 strains of E. coli isolated in several countries were studied. The strains were regarded as enterovirulent on the basis of their O-antigens determined using a commercially available kit containing 43 antisera, and the presence of diarrhea-associated genes (eae, stx, aggR, est, elt, ipaH) was evaluated by PCR. Two hundred sixty-three strains of 1,130 (23.3%) were identified as diarrheagenic based on the presence of at least one pathogenic gene. The probability that E. coli identified as diarrheagenic on the basis of serogrouping actually possessed some pathogenic gene was highest for serogroup O119 (78.4%); other serogroups with a positive rate for pathogenic genes higher than 60% were O111 and O126. No target genes were detected among the strains belonging to serogroups O1, O29, O112ac, O143, O158 and O168. Our results suggest that, in practice, serogrouping is useful for the identification of diarrheagenic E. coli in a very limited number of serogroups.
Assuntos
Diarreia/microbiologia , Escherichia coli/genética , Escherichia coli/patogenicidade , Antígenos O/fisiologia , Antígenos de Bactérias , Escherichia coli/classificação , Escherichia coli/imunologia , Fezes/microbiologia , Humanos , Kit de Reagentes para Diagnóstico , Sorotipagem , VirulênciaRESUMO
A novel protease produced by Aeromonas caviae was purified and characterized. The molecular weight of the protease (AP19) was estimated as 19 kDa on SDS-polyacrylamide gel electrophoresis. The protease activity was not inhibited completely by heating at 100 degrees C for 15 min. The proteolytic activities were inhibited by metalloprotease inhibitor. The N-terminal amino acid sequence of AP19 was VTASVSFSGRCTN. AP19 did not activate Aeromonas proaerolysin, and did not show fluid accumulation in the rabbit intestinal loop test. A high concentration of the protease showed cytotoxic activity against Vero cells.
Assuntos
Aeromonas/enzimologia , Metaloproteases/isolamento & purificação , Metaloproteases/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Chlorocebus aethiops , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Temperatura Alta , Intestino Delgado/microbiologia , Intestino Delgado/patologia , Metaloproteases/química , Peso Molecular , Proteínas Citotóxicas Formadoras de Poros , Coelhos , Análise de Sequência de Proteína , Células VeroRESUMO
Aeromonas veronii is an opportunistic human pathogen that causes diarrhoea and extraintestinal infections, such as wound infection and septicaemia. An A. veronii protease (AVP) from a biovar sobria strain, AeG1, was partially purified and characterized. Mature AVP hydrolysed casein but not elastin, and protease activity was inhibited by metalloprotease inhibitors. Nucleotide sequence analysis showed that AVP belongs to the thermolysin family of proteases. An AVP-deficient mutant was constructed to investigate the role of AVP in aerolysin activation. Western blot analysis using anti-aerolysin antisera revealed that proaerolysin (52 kDa) in the AVP-deficient mutant was not completely activated to mature aerolysin (47 kDa) as seen in the wild-type strain. The AVP-deficient mutant showed lower cytotoxic and haemolytic activities than wild type. AVP and proaerolysin had no haemolytic activity; however, activity appeared after incubating both proteins. Taken together, these results suggested that AVP plays an indirect role in virulence through activating aerolysin, which is an essential step for cytotoxic activity.
Assuntos
Aeromonas/metabolismo , Toxinas Bacterianas/metabolismo , Metaloproteases/metabolismo , Fatores de Virulência/metabolismo , Aeromonas/patogenicidade , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Western Blotting , Linhagem Celular , Clonagem Molecular , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Metaloproteases/antagonistas & inibidores , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas Citotóxicas Formadoras de PorosRESUMO
The nucleotide sequence of an ORF (vcfQ) within the type IV pilus gene cluster of Vibrio cholerae O34 strain NAGV14 was determined, thereby completing the sequence analysis of the structural operon. The vcfQ gene showed homology to the mshQ gene of the mannose-sensitive haemagglutinin pilus gene cluster. The vcfQ was 651 bp larger than mshQ, and the G+C content of the extra 651 bp portion (35.6 mol%) was lower than that of the overall vcfQ gene (42.5 mol%). Except for the first 270 aa residues, the deduced amino acid sequence of VcfQ showed high homology to the MshQ protein. There was immunological cross-reaction between VcfQ and MshQ by Western blotting. Cell fractionation studies showed that VcfQ is located in both the inner and the outer membranes. Mutational analysis showed that vcfQ-deficient mutant expressed detectable levels of major pilin (VcfA), but failed to assemble them into pili, indicating that VcfQ is essential for pilus assembly. Colony-blotting analyses showed that the N-terminal region of vcfQ is variable in V. cholerae strains.
Assuntos
Proteínas de Bactérias/genética , Fímbrias Bacterianas/genética , Vibrio cholerae/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Sequência de Bases , Western Blotting , Clonagem Molecular , Reações Cruzadas , Análise Mutacional de DNA , DNA Bacteriano/química , Fímbrias Bacterianas/fisiologia , Humanos , Dados de Sequência Molecular , Família Multigênica/genética , Mutagênese Insercional , Fases de Leitura Aberta/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Vibrio cholerae/fisiologiaRESUMO
The nucleotide sequences of five open reading frames within the Vibrio cholerae NAGV14 type IV pilus gene cluster were determined. The genes showed high homology to the mannose-sensitive hemagglutinin (MSHA) pilus genes mshB, mshC, mshD, mshO and mshP. PCR analysis showed that a MSHA-like gene cluster is highly conserved among different V. cholerae strains, with the exception of the previously reported major pilin subunit. Recombinant MshB and MshO proteins were purified and specific antiserum was raised to each of them. Western blotting analyses showed that these antisera reacted with purified NAGV14 and MSHA pili. The results suggested that MshB and MshO are minor components of the pilus fiber. Although there was no cross-reaction between the major pilin subunits of NAGV14 and MSHA pili, minor components seemed to be highly homologous and immunologically cross-reactive.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Fímbrias , Fímbrias Bacterianas/genética , Hemaglutininas/genética , Vibrio cholerae/genética , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Reações Cruzadas , DNA Bacteriano/genética , Fímbrias Bacterianas/química , Fímbrias Bacterianas/imunologia , Genes Bacterianos , Hemaglutininas/imunologia , Lectina de Ligação a Manose , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Sorotipagem , Especificidade da Espécie , Vibrio cholerae/classificação , Vibrio cholerae/imunologia , Vibrio cholerae/isolamento & purificaçãoRESUMO
Staphylococcus aureus isolates in 2001 from the nose and the throat of an adult population were characterized for their incidence and type. The incidence was 51%, present in 80 out of 157 individuals examined, consisting of 34 nasal carriers, 24 throat carriers, and 22 who carried the isolates in both the nose and throat. Among these isolates, 2 and 5 from the nose and the throat, respectively, were identified as methicillin-resistant S. aureus. S. aureus from the nose and throat of the same individuals were characterized for identification. Examination of their phenotypes revealed that in 11 individuals the clone of S. aureus in the throat was different from the nasal clone. These results suggested that staphylococcal flora in the nose and the throat were independently formed, and that attention should also be directed to the carriers of S. aureus in the throat for the control of nosocomial infection.
Assuntos
Portador Sadio/epidemiologia , Cavidade Nasal/microbiologia , Faringe/microbiologia , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/isolamento & purificação , Adulto , Tipagem de Bacteriófagos , Portador Sadio/microbiologia , Infecção Hospitalar/prevenção & controle , Farmacorresistência Bacteriana , Feminino , Humanos , Incidência , Masculino , Resistência a Meticilina , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacosRESUMO
The incidence of Escherichia coli having pathogenic genes for diarrhea was studied in Laos in 2002. A total of 525 E. coli strains from 278 patients (basically, two E. coli isolates from each patient) were examined by PCR to detect the known pathogenic genes (stx, eae, elt, est, ipaH, and aggR). These genes were detected in 23 strains from 16 patients (16/278: 5.8%). In 10 cases of the 16, one of the two isolates from each individual was negative for the gene, and in the other six cases, both isolates had the gene (same gene in four cases). E. coli having eae but no stx (enteropathogenic E. coli [EPEC]) was found in two cases out of 278 (0.7%). Nevertheless, Class I classical EPEC (serogroup-based) was found in 77 cases (28%). Enterotoxigenic E. coli, enteroaggregative E. coli, and enterohemorrhagic E. coli were found in 9, 4, and 1 cases, respectively. Enteroinvasive E. coli was not detected. This study suggested that the incidence of diarrhea due to E. coli is not as high as has been previously thought.
Assuntos
Diarreia/etiologia , Escherichia coli/genética , Escherichia coli/patogenicidade , Genes Bacterianos , Adolescente , Adulto , Criança , Humanos , Reação em Cadeia da PolimeraseRESUMO
The inflammasome is composed of nucleotide-binding, oligomerization domain (NOD)-like receptor (NLR) proteins, and leads to caspase-1 activation and subsequent secretion of the proinflammatory cytokines interleukin 1ß (IL-1ß) and interleukin-18 (IL-18). After certain pathogenic bacteria infect host cells, such as macrophages, NLR-mediated inflammasome activation is triggered to form part of the host defenses against the invading pathogens. However, recent evidence has shown that bacteria have strategies for evading inflammasome activation in host cells. In this review, we focus on NLR-mediated inflammasome activation and bacterial evasion of the inflammasome as part of the battle between the host defenses and pathogens.
Assuntos
Bactérias/imunologia , Bactérias/patogenicidade , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Inflamassomos/imunologia , Animais , Humanos , Modelos BiológicosRESUMO
The effects of detergents (cholic acid, deoxycholic acid, Triton X-100, and Nonidet P-40) on the secretion of EspB from the locus for enterocyte effacement (LEE) gene-positive Escherichia coli strains were examined. Clinical isolates of eight EPEC strains and seven STEC strains were used to detect EspB after they had been cultivated in Luria-Bertani (LB) broth containing one of the detergents. When the bacteria were cultured in LB broth supplemented with one of the detergents, the amount of EspB produced was increased by 2-32-fold depending on the detergent and the strain used. EspB was detected in all strains when they were cultured in LB broth containing all of the detergents. The results obtained in this study can be applied to immunological diagnostic methods for detecting EspB and also to the production of EspB for research purposes.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Detergentes/metabolismo , Escherichia coli Enteropatogênica/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Ativação Transcricional , Meios de Cultura/química , Escherichia coli Enteropatogênica/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Humanos , Escherichia coli Shiga Toxigênica/isolamento & purificaçãoRESUMO
Two outer membrane proteins (Omps) of Vibrio cholerae O1, expressed in the intestine (in vivo) but not in culture media (in vitro), were investigated. The molecular masses of those proteins were 116 kDa and 15 kDa, and they were not associated with iron-regulated proteins. Convalescent cholera patients' sera reacted with the 15 kDa protein but not with the 116 kDa protein. The N-terminal amino acid sequence of the 15 kDa protein was homologous to V. cholerae OmpT. Anti-serum to the 15 kDa protein caused agglutination of the organisms grown in the intestine, but not the organisms in in vitro culture. The anti-serum was bactericidal, but it did not inhibit the adhesion of the organisms to the intestine and HEp-2 cells. These findings suggest the possibility that the 15 kDa protein could be involved in post-infection immunity.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Vibrio cholerae/crescimento & desenvolvimento , Vibrio cholerae/metabolismo , Sequência de Aminoácidos , Animais , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Atividade Bactericida do Sangue , Western Blotting , Linhagem Celular , Meios de Cultura , Humanos , Intestino Delgado/microbiologia , Dados de Sequência Molecular , Coelhos , Análise de Sequência de Proteína , Vibrio cholerae/fisiologiaRESUMO
The usefulness of the clump-formation test described by Albert et al. for identifying enteroaggregative Escherichia coli (EAggEC) and the incidence of EAggEC in Japan were studied. One hundred and seventy strains of E. coli agglutinated with enteropathogenic E. coli diagnostic antisera were collected from a variety of districts in Japan. All isolates were from diarrheal stools. EAggEC was identified on the basis of the presence of the aggR gene accompanied by aggregative adhesion to HEp-2 cells. After 24 strains carrying eaeA, elt, est, stx-1, stx-2, or ipaH genes were eliminated, the remaining 145 strains were examined for adhesion to Hep-2 cells, the presence of the aggRgene, and clump formation on the surface of Muller-Hinton broth. aggR was detected in 10 strains, and 9 of them displayed aggregative adhesion to HEp-2 cells. Seven strains produced marked clumps and 22 showed moderate clump formation. The sensitivity and specificity of the clump-formation test for detecting EAggEC were each about 90%, and they varied slightly depending on the stringency of evaluation for the degree of clump formation. From these results, we conclude that the incidence of EAggEC cannot be ignored as a possible cause of diarrheal disease in Japan, and we strongly recommend the clump-formation test for detecting EAggEC.