Pesquisa de polimorfismos nas proteínas EspB e EspD e seu efeito na aderência de escherichia coli enteropatogênica atípica a células HeLa
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Data
2018-03-29
Autores
Santos, Fernanda Fernandes dos [UNIFESP]
Orientadores
Amaral, Tania Aparecida Tardelli Gomes do [UNIFESP]
Tipo
Tese de doutorado
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Resumo
As cepas de Escherichia coli enteropatogênica (EPEC) são subdivididas em EPEC típicas
(tEPEC) e EPEC atípicas (aEPEC). Elas diferem, principalmente, em relação à produção
da fímbria bundleforming
pilus (BFP), que ocorre apenas em tEPEC. A proteína intimina
é a principal adesina envolvida na aderência íntima de EPEC a células epiteliais, porém,
enquanto BFP participa dos estágios iniciais de interação de tEPEC com essas células,
pouco se sabe sobre as estruturas de aEPEC envolvidas no início do processo de
colonização. Este estudo pretende investigar o papel do translocon do sistema de
secreção do tipo III (SST3) como uma estrutura de aderência envolvida nos estágios
iniciais do contato de aEPEC com células epiteliais e avaliar se polimorfismos em EspB
e EspD, proteínas que compõem o translocon, bem como a expressão mais elevada dos
genes correspondentes, além da presença de outras adesinas, poderiam resultar em
diferenças na eficiência dessa aderência. Foram construídos mutantes em intimina a
partir de sete cepas de aEPEC, utilizandose
o vetor suicida pJP5603. Esses mutantes
foram testados em células HeLa quanto a possíveis modificações na sua eficiência de
aderência. Cinco desses mutantes perderam a capacidade aderente, enquanto dois
permaneceram aderentes (20121
wt e 38813
wt) (p> 0,05). Esses últimos foram
utilizados para gerar duplos mutantes eae/escN (genes que codificam intimina e a
ATPase do SST3, respectivamente). A cepa 20121
wt, cujo duplo mutante em eae/escN
perdeu sua capacidade aderente, foi também utilizada para gerar o mutante simples em
espD e o duplo mutante eae/espD, empregandose
o sistema de recombinação Lambda
Red. Ambos os mutantes perderam a capacidade aderente, sugerindo que EspD contribui
para a aderência dessa cepa. Em seguida, as sequências dos genes espB e espD das
sete cepas de aEPEC foram obtidas e traduzidas em sequências de aminoácidos, as
quais foram, então, comparadas entre si e com as sequências correspondentes às da
cepa protótipo tEPEC E2348/69, utilizandose
o software Clustal Omega. As análises
preliminares dos domínios transmembrana de EspB e EspD e do domínio coiled coil da
região carboxiterminal
de EspD, que são importantes para ancoragem dessas proteínas
à membrana de células epiteliais, mostraram que existem diferentes aminoácidos entre
20121 wt e as outras cepas de aEPEC, bem como em relação à cepa E2348/69. Interessantemente, o aminoácido 352 do domínio coiled coil da cepa 20121
wt difere,
inclusive, quanto a sua carga, em relação aos aminoácidos encontrados nessa mesma
posição nas outras cepas. Comparandose
a expressão relativa de espB e espD por qRTPCR,
demonstrouse
que a cepa 20121
wt expressa 18 vezes mais esses genes do que
a cepa aEPEC BA4095, cujo mutante em intimina perde a capacidade de aderência. Além
disso, a pesquisa de adesinas potenciais, realizada com o software SPAAN, revelou que,
o genoma da cepa 20121
wt codifica uma proteína homóloga a YadA que poderia
contribuir para aumentar a eficiência da aderência dessa cepa por meio do translocon do
SST3. Nossos achados sugerem que o translocon do SST3 pode desempenhar um papel
adicional importante, como adesina, no início do processo de colonização. Além disso, a
alta expressão de espB e espD, polimorfismos específicos nesses genes e/ou a presença
de outras adesinas que atuem juntamente com o SST3 podem tornar cepas de aEPEC
colonizadores intestinais mais eficientes.
Enteropathogenic Escherichia coli strains (EPEC) are subdivided into typical EPEC (tEPEC) and atypical EPEC (aEPEC). They differ mainly regarding the production of the bundleforming pilus (BFP), which occurs only in tEPEC. The intimin protein is the major adhesin involved in the intimate adherence of EPEC to epithelial cells, but while BFP participates in the early stages of the interaction of tEPEC with these cells, little is known about the structures involved in the early colonization process of aEPEC. This study intends to investigate the role of the type III secretion system (SST3) translocon as an adhesin in the initial stages of the aEPEC contact with epithelial cells. Furthermore, the study aims to evaluate whether polymorphisms in EspB and EspD, proteins that make up the translocon, as well as the higher differential expression of their corresponding genes, besides the presence of other adhesins, could result in differences in their adherence efficiency. Intimin insertion mutants were constructed with seven aEPEC strains, using the suicide vector pJP5603, and tested in HeLa cells regarding any modification in their adherence efficiency. Five intimin mutants were no longer adherent while two intimin mutants remained adherent (20121 wt and 38813 wt) (p>0.05). Each of the later mutants were then used to generate an eae/escN (encoding intimin and the T3SS ATPase, respectively) double mutant. The 20121 wt strain, whose derivative eae/escN double mutant lost its adherent capacity, was used to generate the espD single mutant and the eae/espD double mutant, employing the Lambda Red recombinant system. Both espD and eae/espD aEPEC 20121 mutants were nonadherent, thus suggesting that EspD contributes to the adherence of this strain. Next, the espB and espD sequences of the seven aEPEC strains were obtained and translated into amino acid sequences, which were compared with each other and with the corresponding sequences of tEPEC prototype E2348/69 strain, using Clustal Omega. Preliminary analyses of the EspB and EspD transmembrane domains, and the EspD carboxiterminal coiled coil domain, which are important to anchor these proteins into the epithelial cell membrane, showed that there are different amino acids among the 20121 wt, the other aEPEC strains, and the E2348/69 strain. Interestingly, the charge of the amino acid 352 of the coiled coil domain also differed from the amino acids on the same position in the other strains. By comparing the relative expression of espB and espD by qRTPCR, it was shown that aEPEC 20121 wt expresses 18 times more these genes than the aEPEC BA4095 strain, whose mutant was no longer adherent. Moreover, the search for potential adhesins using the SPAAN software revealed that there is a YadA homologue protein, encoded by the 20121 genome, that could contribute with the T3SStranslocon to a more efficient adherence. Our findings suggest that the T3SStranslocon could play an additional important role as an adhesin at the beginning of the colonization process. Furthermore, the higher expression of espB and espD, specific polymorphisms in these genes and/or the occurrence of other adhesins acting in conjunction with the SST3 could render certain aEPEC strains more efficient intestinal colonizers.
Enteropathogenic Escherichia coli strains (EPEC) are subdivided into typical EPEC (tEPEC) and atypical EPEC (aEPEC). They differ mainly regarding the production of the bundleforming pilus (BFP), which occurs only in tEPEC. The intimin protein is the major adhesin involved in the intimate adherence of EPEC to epithelial cells, but while BFP participates in the early stages of the interaction of tEPEC with these cells, little is known about the structures involved in the early colonization process of aEPEC. This study intends to investigate the role of the type III secretion system (SST3) translocon as an adhesin in the initial stages of the aEPEC contact with epithelial cells. Furthermore, the study aims to evaluate whether polymorphisms in EspB and EspD, proteins that make up the translocon, as well as the higher differential expression of their corresponding genes, besides the presence of other adhesins, could result in differences in their adherence efficiency. Intimin insertion mutants were constructed with seven aEPEC strains, using the suicide vector pJP5603, and tested in HeLa cells regarding any modification in their adherence efficiency. Five intimin mutants were no longer adherent while two intimin mutants remained adherent (20121 wt and 38813 wt) (p>0.05). Each of the later mutants were then used to generate an eae/escN (encoding intimin and the T3SS ATPase, respectively) double mutant. The 20121 wt strain, whose derivative eae/escN double mutant lost its adherent capacity, was used to generate the espD single mutant and the eae/espD double mutant, employing the Lambda Red recombinant system. Both espD and eae/espD aEPEC 20121 mutants were nonadherent, thus suggesting that EspD contributes to the adherence of this strain. Next, the espB and espD sequences of the seven aEPEC strains were obtained and translated into amino acid sequences, which were compared with each other and with the corresponding sequences of tEPEC prototype E2348/69 strain, using Clustal Omega. Preliminary analyses of the EspB and EspD transmembrane domains, and the EspD carboxiterminal coiled coil domain, which are important to anchor these proteins into the epithelial cell membrane, showed that there are different amino acids among the 20121 wt, the other aEPEC strains, and the E2348/69 strain. Interestingly, the charge of the amino acid 352 of the coiled coil domain also differed from the amino acids on the same position in the other strains. By comparing the relative expression of espB and espD by qRTPCR, it was shown that aEPEC 20121 wt expresses 18 times more these genes than the aEPEC BA4095 strain, whose mutant was no longer adherent. Moreover, the search for potential adhesins using the SPAAN software revealed that there is a YadA homologue protein, encoded by the 20121 genome, that could contribute with the T3SStranslocon to a more efficient adherence. Our findings suggest that the T3SStranslocon could play an additional important role as an adhesin at the beginning of the colonization process. Furthermore, the higher expression of espB and espD, specific polymorphisms in these genes and/or the occurrence of other adhesins acting in conjunction with the SST3 could render certain aEPEC strains more efficient intestinal colonizers.
Descrição
Citação
SANTOS, Fernanda Fernandes dos. Pesquisa de polimorfismos nas proteínas EspB e EspD e
seu efeito na aderência de Escherichia coli enteropatogênica atípica a células HeLa. 2018. 94 f. Tese (Doutorado em Microbiologia e Imunologia) – Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2018.