RESUMO
Mycolic acids represent a major component of the unique cell wall of mycobacteria. Mycolic acid biosynthesis is inhibited by isoniazid, a key frontline antitubercular drug that is inactivated by mycobacterial and human arylamine N-acetyltransferase (NAT). We show that an in-frame deletion of Mycobacterium bovis BCG nat results in delayed entry into log phase, altered morphology, altered cell wall lipid composition, and increased intracellular killing by macrophages. In particular, deletion of nat perturbs biosynthesis of mycolic acids and their derivatives and increases susceptibility of M. bovis BCG to antibiotics that permeate the cell wall. Phenotypic traits are fully complemented by introduction of Mycobacterium tuberculosis nat. We infer from our findings that NAT is critical to normal mycolic acid synthesis and hence other derivative cell wall components and represents a novel target for antituberculosis therapy. In addition, this is the first report of an endogenous role for NAT in mycobacteria.
Assuntos
Antibacterianos/farmacologia , Antituberculosos/farmacologia , Arilamina N-Acetiltransferase/metabolismo , Isoniazida/farmacologia , Lipídeos/biossíntese , Mycobacterium bovis/enzimologia , Ácidos Micólicos/metabolismo , Arilamina N-Acetiltransferase/genética , Arilamina N-Acetiltransferase/ultraestrutura , Vacina BCG , Primers do DNA , Deleção de Genes , Genes Bacterianos/genética , Mycobacterium bovis/efeitos dos fármacos , Fases de Leitura Aberta , Proteínas Recombinantes/metabolismoRESUMO
Enteropathogenic Escherichia coli employs a filamentous type III secretion system, made by homopolymerization of the translocator protein EspA. In this study, we have shown that the N-terminal region of EspA has a role in EspA's protein stability, interaction with the CesAB chaperone, and filament biogenesis and function.
Assuntos
Aminoácidos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Aminoácidos/genética , Aminoácidos/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Imunofluorescência , Células HeLa , Humanos , Modelos Biológicos , Mutação , Estrutura Terciária de ProteínaRESUMO
Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains represent a major global health problem. Their virulence is mediated by the concerted activity of an array of virulence factors including toxins, a type III protein secretion system (TTSS), pili, and others. We previously showed that EPEC O127 forms a group 4 capsule (G4C), and in this report we show that EHEC O157 also produces a G4C, whose assembly is dependent on the etp, etk, and wzy genes. We further show that at early time points postinfection, these G4Cs appear to mask surface structures including intimin and the TTSS. This masking inhibited the attachment of EPEC and EHEC to tissue-cultured epithelial cells, diminished their capacity to induce the formation of actin pedestals, and attenuated TTSS-mediated protein translocation into host cells. Importantly, we found that Ler, a positive regulator of intimin and TTSS genes, represses the expression of the capsule-related genes, including etp and etk. Thus, the expression of TTSS and G4C is conversely regulated and capsule production is diminished upon TTSS expression. Indeed, at later time points postinfection, the diminishing capsule no longer interferes with the activities of intimin and the TTSS. Notably, by using the rabbit infant model, we found that the EHEC G4C is required for efficient colonization of the rabbit large intestine. Taken together, our results suggest that temporal expression of the capsule, which is coordinated with that of the TTSS, is required for optimal EHEC colonization of the host intestine.
Assuntos
Adesinas Bacterianas/metabolismo , Cápsulas Bacterianas/metabolismo , Escherichia coli Enteropatogênica/patogenicidade , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fatores de Virulência/metabolismo , Animais , Aderência Bacteriana , Cápsulas Bacterianas/ultraestrutura , Linhagem Celular , Escherichia coli Enteropatogênica/metabolismo , Escherichia coli Enteropatogênica/ultraestrutura , Células Epiteliais/microbiologia , Eritrócitos/microbiologia , Infecções por Escherichia coli , Escherichia coli O157/metabolismo , Escherichia coli O157/ultraestrutura , Proteínas de Escherichia coli/genética , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Humanos , Intestino Grosso/microbiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Mutagênese Insercional , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Coelhos , Transativadores/metabolismoRESUMO
Enterohemorrhagic Escherichia coli (EHEC) strains are important food-borne pathogens that use a filamentous type III secretion system (fT3SS) for colonization of the gut epithelium. In this study we have shown that EHEC O157 and O26 strains use the fT3SS apparatus for attachment to leaves. Leaf attachment was independent of effector protein translocation.
Assuntos
Aderência Bacteriana/fisiologia , Escherichia coli Êntero-Hemorrágica/fisiologia , Proteínas de Escherichia coli/metabolismo , Lactuca/microbiologia , Folhas de Planta/microbiologia , Contagem de Colônia Microbiana , Proteínas de Escherichia coli/genética , Deleção de GenesRESUMO
During the course of infection, enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) subvert the host cell signalling machinery and hijack the actin cytoskeleton to tighten their interaction with the gut epithelium, while avoiding phagocytosis by professional phagocytes. Much progress has been made recently in our understanding of how EPEC and EHEC regulate the pathways leading to local activation of two regulators of actin cytoskeleton dynamics, the Wiskott-Aldrich syndrome protein (N-WASP) and the Arp2/3 complex. A recent highlight is the unravelling of functions for effector proteins (particularly Tir, TccP, Map and EspG/EspG2) that are injected into the host cell by a type III secretion system.
Assuntos
Actinas/fisiologia , Aderência Bacteriana/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/patogenicidade , Mucosa Intestinal/microbiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/fisiologia , Animais , Citoesqueleto/fisiologia , Humanos , Receptores de Superfície Celular/fisiologia , Fatores de Virulência/fisiologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/fisiologiaRESUMO
The enteropathogenic Escherichia coli (EPEC) effector protein EspG, like the Shigella effector VirA, functions through disruption of the host cell microtubule network. Reports have differed as to whether the EspG homologue, EspG2, is also responsible for microtubule disruption. In this study we show that following translocation, EspG2 and VirA are localised under adherent bacteria and able to restore the microtubule disruption phenotype to an espG/espG2 double EPEC mutant. The espG/espG2 double mutant produced A/E lesions similar to wild-type EPEC on human intestinal in vitro organ cultures. Determining the distribution of espG and espG2 among clinical EPEC isolates revealed two different types of espG (espG alpha and espG beta) and espG2 (intact and pseudo genes), which were associated with specific EPEC serotypes and closely followed the EPEC lineage. This investigation has established a role for EspG2 in the disruption of the microtubule network and associated different espG and espG2 types with different groups of EPEC.
Assuntos
Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/fisiologia , Escherichia coli/química , Escherichia coli/patogenicidade , Fatores de Virulência/genética , Células CACO-2 , Citoplasma/química , DNA Bacteriano/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Deleção de Genes , Teste de Complementação Genética , Humanos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Microtúbulos/metabolismo , Técnicas de Cultura de ÓrgãosRESUMO
Like many Gram-negative pathogens, enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) use a macromolecular type III secretion system (TTSS) to inject effector proteins into eukaryotic host cells. The membrane-associated needle complex (NC) of the TTSS, which shows broad similarity to the flagellar basal body, is conserved amongst bacterial pathogens. However, the extracellular part of the TTSS of EPEC and EHEC is unique, in that it has a hollow, approximately 12 nm in diameter, filamentous extension to the NC. EspA filaments are homo-polymers made of the translocator protein EspA. The three-dimensional structure of EspA filaments is comparable to that of flagella; the helical symmetry and packing of the subunits forming both filamentous structures are very similar. Like flagella, EspA filaments show antigenic polymorphism as EspA from different EPEC and EHEC clones show no immunological cross-reactivity. In this study, we determined the molecular basis of the antigenic polymorphism of EspA filaments and identified a surface-exposed hypervariable domain that contains the immunodominant EspA epitope. By exchanging the hypervariable domains of EspA(EPEC) and EspA(EHEC) we swapped the antigenic specificity of the EspA filaments. As for the flagellin D3 domain, which is known to tolerate insertions of natural and artificial amino acid sequences, we have inserted short peptides into the surface-exposed, hypervariable domain of EspA. We demonstrated that the inserted peptides are presented on the surface of the recombinant EspA filaments forming a new immunodominant epitope. Accordingly, EspA filaments have a potential to be developed into a novel epitope display system.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Biblioteca de Peptídeos , Polimorfismo Genético/genética , Actinas/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Proteínas de Escherichia coli/química , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Alinhamento de SequênciaAssuntos
Membrana Eritrocítica/microbiologia , Eritrócitos/microbiologia , Escherichia coli/patogenicidade , Hemólise , Técnicas de Cultura de Células/métodos , Células Cultivadas , Membrana Eritrocítica/ultraestrutura , Eritrócitos/ultraestrutura , Escherichia coli/classificação , Imunofluorescência , Humanos , Microscopia Eletrônica de Varredura/métodosRESUMO
Enteropathogenic Escherichia coli (EPEC) is the single most important contributor to child diarrhoea in developing countries. Nevertheless, the mechanism responsible for EPEC diarrhoea remains elusive. Using the yeast two-hybrid system to determine the target host cell protein of the EPEC type III secretion system effector Map led to identification of ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50), also known as Na+/H+ exchanger regulatory factor 1 (NHERF1). Protein interaction is mediated by the carboxy-terminal Thr-Arg-Leu (TRL) motif of Map and the PSD-95/Disk-large/ZO-1 domain 1 (PDZ1) of EBP50/NHERF1. Although EBP50/NHERF1 is recruited to site of EPEC adhesion in a Map-independent mechanism, co-immunoprecipitation and immunostaining revealed that Map binds to, induces proteolysis of, and colocalizes with EBP50/NHERF1 during infection of cultured epithelial cells. The TRL motif of Map was involved in Map-induced filopodia formation and brush border elongation on infected HeLa and Caco-2 cells respectively. As EBP50/NHERF1 regulates ion channels in the intestine we assessed the involvement of Map in diarrhoea using the Citrobacter rodentium mouse model of EPEC. We report significantly greater diarrhoea following infections with wild-type C. rodentium compared with C. rodentiumDeltamap. These results provide new insights into the mechanisms of EPEC diarrhoea.
Assuntos
Diarreia/metabolismo , Infecções por Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Actinas/metabolismo , Motivos de Aminoácidos/genética , Células CACO-2 , Escherichia coli/metabolismo , Células HeLa , Humanos , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Modelos Biológicos , Fosfoproteínas/análise , Fosfoproteínas/genética , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína/genética , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/análise , Trocadores de Sódio-Hidrogênio/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
EspF of enteropathogenic Escherichia coli targets mitochondria and subverts a number of cellular functions. EspF consists of six putative Src homology 3 (SH3) domain binding motifs. In this study we identified sorting nexin 9 (SNX9) as a host cell EspF binding partner protein, which binds EspF via its amino-terminal SH3 region. Coimmunoprecipitation and confocal microscopy showed specific EspF-SNX9 interaction and non-mitochondrial protein colocalization in infected epithelial cells.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Motivos de Aminoácidos , Proteínas de Transporte/química , Células Epiteliais/química , Células Epiteliais/microbiologia , Proteínas de Escherichia coli/química , Células HeLa , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular , Microscopia Confocal , Ligação Proteica , Nexinas de Classificação , Proteínas de Transporte Vesicular , Domínios de Homologia de srcRESUMO
Type III secretion systems (TTSS) are sophisticated macromolecular structures that play an imperative role in bacterial infections and human disease. The TTSS needle complex is conserved among bacterial pathogens and shows broad similarity to the flagellar basal body. However, the TTSS of enteropathogenic and enterohemorrhagic Escherichia coli, two important human enteric pathogens, is unique in that it has an approximately 12-nm-diameter filamentous extension to the needle that is composed of the secreted translocator protein EspA. EspA filaments and flagellar structures have very similar helical symmetry parameters. In this study we investigated EspA filament assembly and the delivery of effector proteins across the bacterial cell wall. We show that EspA filaments are elongated by addition of EspA subunits to the tip of the growing filament. Moreover, EspA filament length is modulated by the availability of intracellular EspA subunits. Finally, we provide direct evidence that EspA filaments are hollow conduits through which effector proteins are delivered to the extremity of the bacterial cell (and subsequently into the host cell).
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Transporte Proteico/fisiologia , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas de Escherichia coli/química , Flagelos/química , Flagelos/metabolismoRESUMO
Enteropathogenic Escherichia coli (EPEC) strains deliver effector proteins Tir, EspB, Map, EspF, EspH, and EspG into host cells to induce brush border remodeling and produce attaching and effacing (A/E) lesions on small intestinal enterocytes. In this study, the role of individual EPEC effectors in brush border remodeling and A/E lesion formation was investigated with an in vitro human small intestinal organ culture model of EPEC infection and specific effector mutants. tir, map, espB, and espH mutants produced "footprint" phenotypes due to close bacterial adhesion but subsequent loss of bacteria; an espB mutant and other type III secretion system mutants induced a "noneffacing footprint" associated with intact brush border microvilli, whereas a tir mutant was able to efface microvilli resulting in an "effacing footprint"; map and espH mutants produced A/E lesions, but loss of bacteria resulted in a "pedestal footprint." An espF mutant produced typical A/E lesions without associated microvillous elongation. An espG mutant was indistinguishable from the wild type. These observations indicate that Tir, Map, EspF, and EspH effectors play a role in brush border remodeling and production of mature A/E lesions.
Assuntos
Escherichia coli/metabolismo , Mucosa Intestinal/metabolismo , Microvilosidades/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Duodeno/ultraestrutura , Escherichia coli/genética , Escherichia coli/patogenicidade , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/ultraestrutura , Microscopia Eletrônica de Varredura , Microvilosidades/microbiologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
Enteropathogenic Escherichia coli infection of intestinal epithelial cells leads to localized depletion of the microtubule cytoskeleton, an effect that is dependent on delivery of type III translocated effector proteins EspG and Orf3 (designated EspG2) to the site of depletion. Microtubule depletion involved disruption rather than displacement of microtubules.
Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/patogenicidade , Mucosa Intestinal/microbiologia , Microtúbulos/fisiologia , Actinas/química , Células CACO-2 , Humanos , Transporte ProteicoRESUMO
The type III secretion system (TTSS) is a macromolecular structure that spans the cell wall of Gram-negative bacterial pathogens, enabling delivery of virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. TTSS consists of a conserved needle complex (NC) that is composed of sets of inner and outer membranes rings connected by a periplasmic rod. Enteropathogenic Escherichia coli (EPEC) is an extracellular diarrhoeagenic pathogen that uses TTSS to induce actin polymerization and colonizes the intestinal epithelium. In EPEC, EscJ is predicted to be targeted to the periplasm, in a sec-dependent manner, and to bridge the TTSS membrane-associated rings. In this study we determined the global fold of EscJ using Nuclear Magnetic Resonance spectroscopy. We show that EscJ comprises two subdomains (D1 - amino acid residues 1-55 in the mature protein, and D2 - amino acid residues 90-170), each comprising a three-stranded beta-sheet flanked by two alpha-helices. A flexible region (residues 60-85) couples the structured regions D1 and D2. Periplasmic overexpression of EscJ(D1) and EscJ(D2) in a single escJ mutant bacterium failed to restore protein secretion activity, suggesting that the flexible linker is essential for the rod function. In contrast, periplasmic overexpression of EscJ(D1) and EscJ(D2) in the same wild-type bacterium had a dominant-negative phenotype suggesting defective assembly of the TTSS and protein translocation.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Substâncias Macromoleculares/metabolismo , Sequência de Aminoácidos , Membrana Celular/química , Membrana Celular/ultraestrutura , Sequência Conservada , Escherichia coli/química , Escherichia coli/metabolismo , Deleção de Genes , Genes Bacterianos , Teste de Complementação Genética , Substâncias Macromoleculares/química , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Alinhamento de Sequência , Deleção de Sequência/genética , Deleção de Sequência/fisiologia , Homologia de Sequência de AminoácidosRESUMO
Enterohemorrhagic Escherichia coli, enteropathogenic E. coli, and Citrobacter rodentium are highly adapted enteropathogens that successfully colonize their host's gastrointestinal tract via the formation of attaching and effacing (A/E) lesions. These pathogens utilize a type III secretion system (TTSS) apparatus, encoded by the locus of enterocyte effacement, to translocate bacterial effector proteins into epithelial cells. Here, we report the identification of EspJ (E. coli-secreted protein J), a translocated TTSS effector that is carried on the 5' end of the cryptic prophage CP-933U. Infection of epithelial cells in culture revealed that EspJ is not required for A/E lesion activity in vivo and ex vivo. However, in vivo studies performed with mice demonstrated that EspJ possesses properties that influence the dynamics of clearance of the pathogen from the host's intestinal tract, suggesting a role in host survival and pathogen transmission.
Assuntos
Infecções Bacterianas/imunologia , Proteínas de Escherichia coli/metabolismo , Prófagos/imunologia , Animais , Aderência Bacteriana/imunologia , Citrobacter rodentium/imunologia , Infecções por Enterobacteriaceae/imunologia , Células Epiteliais/imunologia , Escherichia coli/imunologia , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Camundongos , Virulência/imunologiaRESUMO
Escherichia coli produces polysaccharide capsules that, based on their mechanisms of synthesis and assembly, have been classified into four groups. The group 4 capsule (G4C) polysaccharide is frequently identical to that of the cognate lipopolysaccharide O side chain and has, therefore, also been termed the O-antigen capsule. The genes involved in the assembly of the group 1, 2, and 3 capsules have been described, but those required for G4C assembly remained obscure. We found that enteropathogenic E. coli (EPEC) produces G4C, and we identified an operon containing seven genes, ymcD, ymcC, ymcB, ymcA, yccZ, etp, and etk, which are required for formation of the capsule. The encoded proteins appear to constitute a polysaccharide secretion system. The G4C operon is absent from the genomes of enteroaggregative E. coli and uropathogenic E. coli. E. coli K-12 contains the G4C operon but does not express it, because of the presence of IS1 at its promoter region. In contrast, EPEC, enterohemorrhagic E. coli, and Shigella species possess an intact G4C operon.
Assuntos
Cápsulas Bacterianas/genética , Proteínas de Bactérias/genética , Escherichia coli/genética , Genes Bacterianos , Óperon , Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Mutação , Antígenos O/metabolismo , Proteínas Tirosina Quinases/genéticaRESUMO
OBJECTIVES: In developed countries, small bowel histology in coeliac disease is a spectrum, ranging from normal with increased intraepithelial lymphocytes to the classic flat mucosa. In developing countries, mild to moderate enteropathies in children with chronic diarrhea and growth failure are assumed to be caused by tropical sprue, persistent infections, or malnutrition with bacterial overgrowth. We report the prevalence and histology of coeliac disease in children with chronic diarrhea at a tertiary referral hospital in North India. METHODS: Two hundred fifty-nine children with symptoms indicating coeliac disease attended the All India Institute of Medical Sciences. Histology was graded after a modified Marsh classification. Serum immunoglobulin A anti-endomysial antibodies (AEA) were assayed using indirect immunofluorescence. Subjects with abnormal histology and positive AEA were put on a gluten free diet (GFD). Coeliac disease was diagnosed on small intestinal biopsy changes and a clinical response to a GFD. RESULTS: Severe enteropathies were present in 63 (24%) subjects, and 58 (92%) responded to a GFD. Sixty-six (25%) had moderate histologic changes, 61 responding to a GFD. AEA was positive in 56 of 63 patients with severe and 65 of 66 with moderate enteropathies. Fifty-seven children had mild enteropathies, and 19 of 20 with positive AEA responded clinically to a GFD. CONCLUSIONS: Coeliac disease is more common than previously believed. It presents a variable histology, and diagnoses may be missed or delayed if based only on severe enteropathies. Serology is a useful adjunct to diagnosis, and diagnostic criteria need to be developed appropriately for coeliac disease in developing countries despite limited facilities.
Assuntos
Autoanticorpos/sangue , Doença Celíaca/complicações , Diarreia/etiologia , Glutens/efeitos adversos , Adolescente , Doença Celíaca/diagnóstico , Doença Celíaca/dietoterapia , Doença Celíaca/patologia , Criança , Pré-Escolar , Doença Crônica , Diarreia/diagnóstico , Diarreia/dietoterapia , Diarreia/patologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Glutens/administração & dosagem , Humanos , Índia/epidemiologia , Lactente , Mucosa Intestinal/patologia , Masculino , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
Type III secretion allows bacteria to inject effector proteins into host cells. In enteropathogenic Escherichia coli (EPEC) the type III secreted protein, Tir, is translocated to the host-cell plasma membrane where it functions as a receptor for the bacterial adhesin intimin, leading to intimate bacterial attachment and "attaching and effacing" (A/E) lesion formation. To study EPEC type III secretion the interaction of EPEC with monolayers of red blood cells (RBCs) has been exploited and in a recent study [Shaw, R. K., Daniell, S., Ebel, F., Frankel, G. & Knutton, S. (2001 ). Cell Microbiol 3, 213-222] it was shown that EPEC induced haemolysis of RBCs and translocation of EspD, a putative pore-forming type III secreted protein in the RBC membrane. Here it is demonstrated that EPEC are able to translocate and correctly insert Tir into the RBC membrane and produce an intimin-Tir intimate bacterial attachment, identical to that seen in A/E lesions. Following translocation Tir did not undergo any change in apparent molecular mass or become tyrosine-phosphorylated and there was no focusing of RBC cytoskeletal actin beneath intimately adherent bacteria, and no pedestal formation. This study, employing an RBC model of infection, has demonstrated that Tir translocation can be separated from host-cell-mediated Tir modifications; the data show that the EPEC type III protein translocation apparatus is sufficient to deliver and correctly insert Tir into host-cell membranes independent of eukaryotic cell functions.
Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Proteínas de Transporte/metabolismo , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/microbiologia , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Receptores de Superfície Celular/metabolismo , Membrana Eritrocítica/ultraestrutura , Escherichia coli/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Biológicos , Ligação Proteica , Transporte ProteicoRESUMO
Using a DNA microarray, we determined changes in enterohemorrhagic Escherichia coli O157:H7 gene expression during binding to plasma membranes. Analysis of the complete transcriptomes of the bound bacteria revealed increased levels of stress-associated mRNAs and decreased levels of mRNA encoding proteins involved in translation and type III secretion.