RESUMO
Synthetic N-acetyllactosamine (LacNAc) glycoside sequences coupled to BSA competitively inhibit enteropathogenic Escherichia coli (EPEC) localized adherence (LA) to human intestinal biopsy specimens and tissue culture cell monolayers. The LacNAc-specific adhesin appears to be associated with the bundle-forming pili (BFP) expressed by EPEC during the early stages of colonization. Herein, we report that recombinant bundlin inhibits EPEC LA to HEp-2 cells and binds to HEp-2 cells. Recombinant bundlin also binds, with millimolar association constants (K(assoc)), to synthetic LacNAc-Benzene and LacNAc-O(CH(2))(8)CONH(2) glycosides as assessed in the gas phase by nanoelectrospray ionization mass spectrometry. Furthermore, LacNAc-BSA inhibits LA only of EPEC strains that express alpha bundlin alleles, suggesting putative locations for the LacNAc-binding pocket in the alpha bundlin monomer. Collectively, these results suggest that alpha bundlin possesses lectin-like properties that are responsible for LacNAc-specific initial adherence of alpha bundlin-expressing EPEC strains to host intestinal epithelial cells.
Assuntos
Amino Açúcares/metabolismo , Aderência Bacteriana/fisiologia , Escherichia coli Enteropatogênica/fisiologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Lectinas/metabolismo , Linhagem Celular , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/isolamento & purificação , Humanos , Cinética , Lectinas/genética , Lectinas/isolamento & purificação , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
In a previous article, the authors reported that exposing wild-type enteropathogenic Escherichia coli (EPEC) to chemically synthesized N-acetyllactosamine glycosides covalently coupled to BSA (LacNAc-BSA) inhibited localized adherence (LA) by these organisms and also caused them to lose their bundle-forming pili (BFP), the filamentous surface appendages responsible for their LA phenotype. This effect has now been further investigated by screening a panel of LacNAc-BSA-related glycosides for their ability to inhibit EPEC LA, which revealed that LacNAc-BSA retained its status as the most effective inhibitor of EPEC LA. It was also shown that LacNAc-BSA did not cause the loss of BFP in an EPEC strain containing a non-polar mutation in the bfpF gene and, as a consequence, unable to retract its BFP. LacNAc-BSA also effectively inhibited LA of the bfpF mutant EPEC. Taken together, these observations suggest that, as well as triggering BfpF-mediated BFP retraction, LacNAc-BSA likely functions as a competitive inhibitor of EPEC binding to LacNAc-related receptors on host cells. Moreover, transmission electron microscopy revealed that LacNAc conjugated to gold nanoparticles bound specifically to BFP. This observation indicated that either the major BFP structural subunit (BfpA) itself or, possibly, an accessory protein co-assembled with BfpA into the BFP filaments, contains a LacNAc-specific EPEC adhesin. The results suggest a mechanism whereby the initial binding of EPEC to LacNAc-like receptors on host cells triggers BfpF-mediated BFP retraction. This could then expedite the intimate adherence phase of the multi-step EPEC colonization process by drawing the organisms closer to the host-cell plasma membrane.
Assuntos
Amino Açúcares/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/metabolismo , Animais , Bovinos , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Escherichia coli/genética , Escherichia coli/patogenicidade , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/efeitos dos fármacos , Fímbrias Bacterianas/ultraestrutura , Genes Bacterianos , Glicoconjugados/farmacologia , Humanos , Técnicas In Vitro , Intestinos/efeitos dos fármacos , Intestinos/microbiologia , Microscopia Eletrônica , Soroalbumina BovinaRESUMO
Evaluation of: Ferreira GM, Spira B: The pst operon of enteropathogenic Escherichia coli enhances bacterial adherence to epithelial cells. Microbiology 154(7), 2025-2036 (2008). Enteropathogenic Escherichia coli (EPEC) colonization of the human intestinal epithelium constitutes a complex, two-stage process, which involves multiple virulence factors that are expressed in response to numerous environmental signals. Ferreira and Spira present evidence that the phosphate transport system is involved in regulating EPEC colonization in a novel manner that does not appear to involve inorganic phosphate sensing.
RESUMO
We previously reported that the bundle-forming pilus-mediated localized adherence of enteropathogenic Escherichia coli to HEp-2, T84, and Caco-2 cells is inhibited by N-acetyllactosamine neoglycoconjugates. The results presented here extend this observation to the epithelium of biopsy specimens obtained from the human adult duodenum, terminal ileum, and colon.