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Master's Dissertation
DOI
https://doi.org/10.11606/D.87.2009.tde-08052009-094943
Document
Author
Full name
Renato de Mello Ruiz
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2009
Supervisor
Committee
Elias Junior, Waldir Pereira (President)
Oliveira, Maria Leonor Sarno de
Salvador, Fábia Andréia
Title in Portuguese
Caracterização genotípica e fenotípica de mutantes não aderentes de Escherichia coli enteropatogênica atípica do sorotipo O125ac:H6.
Keywords in Portuguese
Escherichia coli
Diarréia
Proteínas de membrana
Sistemas de secreção
Abstract in Portuguese
O sorotipo O125ac:H6 compreende amostras de Escherichia coli enteropatogênica atípicas que apresentam o padrão de adesão agregativa (AA) em células HEp-2. A construção de um banco de mutantes da amostra protótipo Ec292/84 com o transposon TnphoA gerou quatro mutantes não aderentes. O objetivo deste estudo foi a caracterização genotípica e fenotípica desses mutantes. As regiões adjacentes à inserção do TnphoA no mutante Ec2921/84::01 foram amplificadas, clonadas e seqüenciadas, revelando que a inserção do TnphoA ocorreu no gene secD, parte do sistema de secreção de proteínas do tipo 2 (SST2). O perfil de proteínas de membrana externa (OMP) dos mutantes, em comparação com a amostra selvagem, revelou a ausência de proteínas de 21 e 30 kDa nos mutantes. Um antissoro obtido contra o extrato de OMP da amostra protótipo inibiu o padrão AA e reconheceu a proteína de 30 kDa em immunoblottings com extratos de OMP. Esses dados indicam que esta proteína está envolvida no estabelecimento do padrão AA de E. coli O125ac:H6 e que essa proteína é transportada através do SST2.
Title in English
Genotypic and phenotypic characterization of nonadherent mutants of atypical enteropathogenic Escherichia coli of serotype 0125ac:H6.
Keywords in English
Escherichia coli
Diarrhea
Membrane protein
Secretion system
Abstract in English
The serotype O125ac:H6 comprises atypical Enteropathogenic Escherichia coli strains that express the aggregative adherence (AA) pattern to HEp-2 cells. We obtained four nonadherent mutants using TnphoA insertion in the Ec292/84 strain. The aim of this study was the genetic and phenotypic characterization of these mutants. The genetic analysis of the mutants revealed that the insertion of TnphoA ocurred in the secD gene, part of the bacterial type 2 secretion system (T2SS). The mutant outer membrane proteins (OMP) profile, in comparison to the prototype strain, demonstrated the lack of expression of proteins of 21 and 30 kDa in the mutant profile. An antiserum raised against the OMP extract of the prototype strain, in addition to inhibit the AA pattern, recognized the 30 kDa protein in immunoblotting assays with OMP extract. These data indicate this OMP is involved in the establishment of the AA pattern presented by the atypical EPEC strains of the O125ac:H6 serotype, and that this protein is transported via the T2SS.
 
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Publishing Date
2009-05-14
 
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