Functional activities of the Tsh protein from avian pathogenic Escherichia coli (APEC) strains

The temperature-sensitive hemagglutinin (Tsh) expressed by strains of avian pathogenic Escherichia (E.) coli (APEC) has both agglutinin and protease activities. Tsh is synthesized as a 140 kDa precursor protein, whose processing results in a 106 kDa passenger domain (Tsh(s)) and a 33 kDa beta-domain (Tsh(beta)). In this study, both recombinant Tsh (rTsh) and supernatants from APEC, which contain Tsh(s) (106 kDa), caused proteolysis of chicken tracheal mucin. Both rTsh (140 kDa) and pellets from wild-type APEC, which contain Tsh(beta) (33 kDa), agglutinated chicken erythrocytes. On Western blots, the anti-rTsh antibody recognized the rTsh and 106 kDa proteins in recombinant E. coli BL21/pET 101-Tsh and in the supernatants from APEC grown at either 37degrees C or 42degrees C. Anti-rTsh also recognized a 33 kDa protein in the pellets from APEC13 cultures grown in either Luria-Bertani agar, colonization factor antigen agar, or mucin agar at either 26degrees C, 37degrees C, or 42degrees C, and in the extracts of outer membrane proteins of APEC. The 106 kDa protein was more evident when the bacteria were grown at 37degrees C in mucin agar, and it was not detected when the bacteria were grown at 26degrees C in any of the culture media used in this study. Chicken anti-Tsh serum inhibited hemagglutinating and mucinolytic activities of strain APEC13 and recombinant E. coli BL21/pET101-Tsh. This work suggests that the mucinolytic activity of Tsh might be important for the colonization of the avian tracheal mucous environment by APEC.

Virulence factors of avian pathogenic Escherichia coli (APEC) / Fatores de virulência de Escherichia coli aviária patogênica (APEC)

Avian pathogenic Escherichia coli (APEC) strains cause a great diversity of diseases in birds and are responsible for great economic losses in the avian industry. To date, several studies have been carried out to better understand the APEC pathogenesis for a possible development of tools which could prevent the economics losses caused by these strains. This review discusses the virulence factors described do date to be expressed by these strains and the advances made to understand and identify virulence determinants present in APEC.

Linhagens de Escherichia coli patogênicas para aves (APEC) causam uma grande diversidade de doenças em aves e são responsáveis por grandes prejuízos na indústria aviária. Nos últimos anos, vários estudos foram realizados para melhor entender a patogênese de linhagens APEC e para desenvolver ferramentas que podem prevenir as perdas econômicas causadas por estas linhagens. Esta revisão discute os fatores de virulência descritos nestas linhagens e os avanços realizados para entender e identificar os determinantes de virulência presentes em APEC.

Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli

The aim of this work was to isolate, clone and characterize the iron uptake gene iutA from avian pathogenic E. coli (APEC). The iutA gene was isolated from the strain APEC 9, serotype O2:H9, which was cloned in the expression vector pET101/D-TOPO. The gene of 2.2 Kb was sequenced (AY602767, which showed high similarity to the iutA gene from three plasmids, two from APEC, pAPEC-02-ColV (AY545598.4) and pTJ100 (AY553855.1), and one from a human invasive E. coli strain, the pColV K30. The recombinant protein IutA was over expressed in E. coli BL21(DE-3) and was solubilized with urea and purified by Ni-NTA column. This method produced a relatively high yield of r-IutA of approximately 74kDa, which was used to produce the antibody anti-IutA. This anti-IutA reacted with the protein r-IutA and native IutA of APEC 9, as demonstrated by Western blot, showing that the r-IutA conserved epitopes and its antigenicity was preserved. The anti-IutA IgY was able to inhibit the IutA biological activity, inhibiting the sensitivity to cloacin DF13 of APEC9. However, it did not inhibit the growth of APEC9 in M9 and did not protect the chickens inoculated with the APEC, suggesting that the APEC possessed another iron acquisition mechanism distinct of aerobactin.

A proteína de membrane externa IutA (iron uptake transport) é o receptor para aerobactina férrica, um fator de virulência encontrado mais frequentemente entre as amostras de E. coli pathogênicas para aves (APEC) do que entre os isolados fecais de aves saudáveis. O gene iutA da amostra APEC 9, sorotipo O2:H9, foi amplificado e clonado no vetor pET101/D-TOPO. O gene iutA 2.2 Kb foi sequenciado (AY602767) e mostrou alta similaridade para gene iutA de três plasmidios, dois da APEC, pAPEC-02-ColV (AY545598.4) e pTJ100 (AY553855.1), e um da amostra E. coli invasiva humana, pColV K30. A proteína IutA recombinante (r-IutA) foi produzida em Escherichia coli BL21(DE-3), solubilizada com uréia e purificada em coluna de níquel Ni-NTA. A r-IutA tem aproximadamente 74kDa e foi utilizada para produzir anticorpos anti-IutA. Este anticorpo reagiu com a r- IutA e com IutA da APEC13, como demonstrado por Western blot, mostrando que a r-IutA tem epitopos conservados e sua antigenicidade foi preservada. O anticorpo anti-IutA foi capaz de inibir a atividade biológica da IutA, inibindo o teste positivo de sensibilidade à cloacina DF13 apresentada pela APEC 9, contudo não inibiu o crescimento da APEC9 crescida em M9 e não protegeu os pintinhos inoculados com APEC 9, sugerindo que a APEC possui outro mecanismo de captação de íons ferro distinto da aerobactina.

Clonagem, sequenciamento, expressão e caracterização do gene tsh de uma cepa de Escherichia coli patogênica de aves / Cloning, sequencing, expression, and characterization of the tsh gene from an avian pathogenic Escherichia coli strain

A hemaglutinina temperatura sensível (Tsh) pertence à família das serino-proteases autotransporte de Enterobacteriacea (SPATE), as quais são capazes de clivar diferentes substratos. Nós isolamos e caracterizamos o gene de Escherichia coli patogênica aviária (APEC) amostra APEC 13, sorotipo O2:H9, clonado em pET 101. A região de 4.2 kb do DNA clonado codificou uma proteína de aproximadamente 140 kDa (r-Tsh). O plasmídio recombinante pET 101-tsh conferiu um fenótipo de hemaglutinação positivo para a linhagem BL21 (tsh) para eritrócitos de galinha. A proteína r-Tsh foi purificada em coluna de níquel e utilizada na produção de anticorpos anti-Tsh. Um fragmento de 1.6 kb foi amplificado e subclonado em pCR4, e a seqüência parcial mostrou alta homologia com outras seqüências analisadas. O anti-Tsh reagiu com as proteínas r-Tsh e Tsh nativa da amostra APEC13, como demonstrado pela técnica de Western blot, mostrando que a r-Tsh tem epitopos conservados e que sua antigenicidade foi preservada. O anti-Tsh também inibiu a atividade hemaglutinante das amostras APEC 13 e BL12/pET 101-tsh

The temperature-sensitive hemagglutinin (Tsh) belongs to a family of high-molecular-weight serineprotease autotransporters of Enterobacteriaceae (SPATEs), which can cleave different substrates. Weisolated and characterised the tsh gene from an avian pathogenic Escherichia coli (APEC) strain, APEC13serotype O2:H9, which was cloned in pET101. The 4.2 kb region of cloned DNA coded one protein ofapproximately 140 kDa (r-Tsh). The recombinant plasmid pET101-tsh conferred to E. coli BL21 strain(tsh) the hemagglutination-positive phenotype against chicken erythrocytes. The r-Tsh was purified byNi-NTA column and used to produce antibody anti-Tsh. A 1.6 kb fragment of the tsh sequence was alsoamplified and cloned in pCR4, and a partial sequence showed high homology with other sequenceanalysed. The anti-Tsh reacted with the protein r-Tsh and native Tsh of APEC13, as demonstrated byWestern blot, showing that r-Tsh has conserved epitopes and that its antigenicity was preserved. Theanti-Tsh also inhibited the hemagglutinating activity of strains APEC13 and BL21/pET101-tsh