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1.
Infect Immun ; 85(12)2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28893912

RESUMO

Locus of enterocyte effacement (LEE)-negative Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are human pathogens that lack the LEE locus, a pathogenicity island (PAI) involved in the intimate adhesion of LEE-positive strains to the host gut epithelium. The mechanism used by LEE-negative STEC strains to colonize the host intestinal mucosa is still not clear. The cell invasion determinant tia, previously described in enterotoxigenic E. coli strains, has been identified in LEE-negative STEC strains that possess the subtilase-encoding pathogenicity island (SE-PAI). We evaluated the role of the gene tia, present in these LEE-negative STEC strains, in the invasion of monolayers of cultured cells. We observed that these strains were able to invade Caco-2 and HEp-2 cell monolayers and compared their invasion ability with that of a mutant strain in which the gene tia had been inactivated. Mutation of the gene tia resulted in a strong reduction of the invasive phenotype, and complementation of the tia mutation with a functional copy of the gene restored the invasion activity. Moreover, we show that the gene tia is overexpressed in bacteria actively invading cell monolayers, demonstrating that tia is involved in the ability to invade cultured monolayers of epithelial cells shown by SE-PAI-positive E. coli, including STEC, strains. However, the expression of the tia gene in the E. coli K-12 strain JM109 was not sufficient, in its own right, to confer to this strain the ability to invade cell monolayers, suggesting that at least another factor must be involved in the invasion ability displayed by the SE-PAI-positive strains.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Endocitose , Células Epiteliais/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli Shiga Toxigênica/patogenicidade , Fatores de Virulência/metabolismo , Linhagem Celular , Escherichia coli K12/genética , Escherichia coli K12/patogenicidade , Técnicas de Inativação de Genes , Teste de Complementação Genética , Ilhas Genômicas , Humanos , Escherichia coli Shiga Toxigênica/genética
2.
Front Microbiol ; 7: 942, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27379071

RESUMO

Monoclonal antibodies (MAbs) specific for the lipopolysaccharide (LPS) of Escherichia coli O104:H4 were produced by fusion of Sp2/O-Ag-14 mouse myeloma cells with spleen cells of Balb/c mice, immunized with heat-inactivated and sonicated E. coli O104:H4 bacterial cells. Four MAbs specific for the E. coli O104:H4 LPS (1E6G6, 1F4C9, 3G6G7, and 4G10D2) were characterized and evaluated for the use in a method for the detection of E. coli O104:H4 in milk samples that involves antibody conjugation to magnetic microbeads to reduce time and increase the efficiency of isolation. MAb 1E6G6 was selected and coupled to microbeads, then used for immuno-magnetic separation (IMS); the efficiency of the IMS method for E. coli O104:H4 isolation from milk was evaluated and compared to that of the EU RL VTEC conventional culture-based isolation procedure. Milk suspensions also containing other pathogenic bacteria that could potentially be found in milk (Campylobacter jejuni, Listeria monocytogenes, and Staphylococcus aureus) were also tested to evaluate the specificity of MAb-coated beads. Beads coated with MAb 1E6G6 showed a good ability to capture the E. coli O104:H4, even in milk samples contaminated with other bacteria, with a higher number of E. coli O104:H4 CFU reisolated in comparison with the official method (121 and 41 CFU, respectively, at 10(3) E. coli O104:H4 initial load; 19 and 6 CFU, respectively, at 10(2) E. coli O104:H4 initial load; 1 and 0 CFU, respectively, at 10(1) E. coli O104:H4 initial load). The specificity was 100%.

3.
J Leukoc Biol ; 84(4): 1019-27, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18625912

RESUMO

Human intestinal infections by Shiga toxin (Stx)-producing Escherichia coli cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), which represents the main cause of acute renal failure in early childhood. In HUS, Stx released in the gut enter the bloodstream and are targeted to renal endothelium. The mechanism of toxin delivery is still a matter of debate, although the role of polymorphonuclear leukocytes (PMN) as a Stx carrier has been indicated. The aim of this paper was to better define the interactions between Stx and human PMN. Direct and indirect flow cytometric analysis and binding experiments with radiolabeled toxins demonstrated that Stx bind to the surface of human mature PMN but not to immature PMN from G-CSF-treated donors. The use of the human myeloid leukemia cell (HL-60) model for inducible cell differentiation confirmed that the toxin binding occurs only after granulocytic differentiation. Stx binding caused a delay of the spontaneous apoptosis of PMN, as shown by the delayed appearance of apoptotic nuclei and activation of caspase 3 and by the higher number of cells negative to the annexin V-binding assay after 48 h. Moreover, flow cytometric analysis of mixed Stx-positive and Stx-negative PMN populations showed that the toxins were transferred from positive to negative PMN. The delayed, spontaneous apoptosis and the passage of the toxic ligand from older PMN to new, mature cells entering the circulation from the bone marrow may explain the previously reported persistence of Stx in the blood of children with HUS.


Assuntos
Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Toxinas Shiga/toxicidade , Apoptose/efeitos dos fármacos , Transporte Biológico , Caspase 3/sangue , Caspase 3/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Pré-Escolar , Escherichia coli/patogenicidade , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células HL-60/patologia , Síndrome Hemolítico-Urêmica/induzido quimicamente , Humanos , Cinética , Neutrófilos/patologia , Toxinas Shiga/farmacocinética
4.
Infect Immun ; 75(2): 604-12, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17101643

RESUMO

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) trigger actin polymerization at the site of bacterial adhesion by inducing different signaling pathways. Actin assembly by EPEC requires tyrosine phosphorylation of Tir, which subsequently binds the host adaptor protein Nck. In contrast, Tir(EHEC O157) is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspF(U), which mimics the function of Nck. tccP is carried on prophage CP-933U/Sp14 (TccP). Typical isolates of EHEC O157:H7 harbor a pseudo-tccP gene that is carried on prophage CP-933 M/Sp4 (tccP2). Here we report that atypical, beta-glucuronidase-positive and sorbitol-fermenting, strains of EHEC O157 harbor intact tccP and tccP2 genes, both of which are secreted by the LEE-encoded type III secretion system. Non-O157 EHEC strains, including O26, O103, O111, and O145, are typically tccP negative and translocate a Tir protein that encompasses an Nck binding site. Unexpectedly, we found that most clinical non-O157 EHEC isolates carry a functional tccP2 gene that encodes a secreted protein that can complement an EHEC O157:H7 DeltatccP mutant. Using discriminatory, allele-specific PCR, we have demonstrated that over 90% of tccP2-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated. These results suggest that the TccP pathway can be used by both O157 and non-O157 EHEC and that non-O157 EHEC can also trigger actin polymerization via the Nck pathway.


Assuntos
Actinas/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Escherichia coli O157/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fatores de Virulência/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Proteínas de Transporte/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Células Epiteliais/química , Células Epiteliais/microbiologia , Escherichia coli/metabolismo , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Teste de Complementação Genética , Células HeLa , Humanos , Imuno-Histoquímica , Microscopia de Fluorescência , Dados de Sequência Molecular , Fosforilação , Reação em Cadeia da Polimerase , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Análise de Sequência de DNA
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