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Authors
Advisor(s)
Abstract(s)
As bactérias do grupo Escherichia coli enteropatogénica (EPEC) são uma das maiores
causas de diarreia fatal em crianças com menos de cinco ano nos países em desenvolvimento.
A virulência de EPEC depende da presença do sistema de secreção do tipo III (SST3). O
SST3 é constituído por um corpo basal situado entre a membrana externa e interna da
bactéria, prolongado por uma agulha que é projetada a partir da superfície da bactéria. No
caso de EPEC, a agulha do SST3 é prolongada por um filamento formado pela proteína
hidrofílica, EspA. Depois de contactar com a célula hospedeira, o sistema ativa a secreção de
duas proteínas hidrofóbicas, EspD e EspB. Estas proteínas inserem-se na membrana
plasmática da célula hospedeira para formar o transclocão. O translocão é uma estrutura chave
para formar o poro na membrana da célula hospedeira pelo qual os efetores são inseridos na
célula.
O laboratório onde se realizou este trabalho estuda o papel da proteína EspC, que
pertence à família SPATE (serine protease autotransporter of Enterobacteriaceae), na
regulação e funcionamento do translocão. EspC é secretada pelo sistema de secreção do tipo
V, sendo independente do SST3. Foi demonstrado que EspC tem um papel importante no
controlo da formação do poro membranar e da virulência mediada pelo SST3. EspC tem
preferencialmente como alvo os complexos de EspA-EspD, envolvidos na formação do poro
membranar, presumivelmente após o destacamento do translocão do filamento da agulha.
Este trabalho de estágio utilizou procedimentos de purificação para isolar o complexo
EspA-EspD sensível à proteólise de EspC com o objetivo de realizar a caracterização
estrutural deste complexo por Microscopia Eletrónica. Implementamos várias fases de
purificação distintas para aumentar a pureza das amostras contendo os complexos desejados.
Conseguimos isolar complexos homogéneos contendo EspA que mostram um estrutura em
forma de anel com um diâmetro externo de 10 nm e um interno de 4 nm.
Enteropathogenic E. coli (EPEC) is a major cause of fatal diarrhea in childs under 5 years in developing countries. EPEC virulence is dependent on the presence of type III secretion system (T3SS). T3SSs are used by many pathogenic Gram-negative bacteria to inject bacterial effectors into host cells. T3SSs consist of a basal body embedded in the bacterial outer and inner membrane, prolonged by a needle protruding from the bacterial surface. In the case of EPEC, the T3SS needle is extended by a filament formed by hydrophilic EspA protomers. Upon cell contact, the system triggers the secretion of two hydrophobic proteins EspD and EspB, that insert in the host cell plasma membrane to form the translocon. The translocon is critical for the injection of type 3 effectors, presumably by forming a pore into host cell plasma membranes through which the effectors are channeled. The host lab studies the role of EspC, a serine protease autotransporter of Enterobacteriaceae (SPATE) family, on the regulation of the translocon function. EspC is a protein secreted by EPEC by a type V secretion system independently of the T3SS, although the translocation into epithelial cells requires active type III secretion. EspC has been shown to play a role in controlling pore formation and cytotoxicity mediated by the T3SS. EspC was shown to preferentially target an EspA-EspD complex, involved in the T3SS-dependent pore formation, supposedly following detachment of the translocon from the T3SS needle filament. This internship work took advantage of fractionation procedures worked out to isolate the EspA-EspD complex sensitive to EspC proteolysis, with the aim to perform a structural characterization of this complex by electron microscopy analysis. In this study, we increased the yield of EspA-EspD complex purification, that for the first time allowed the detection of EspB together with EspA-EspD complexes. We implemented additional purification steps to increase the purity of protein complexes. We were able to isolate homogenous EspAcontaining complexes showing a ring-link structure with an external diameter of 10 nm of and a 4 nm-inner diameter.
Enteropathogenic E. coli (EPEC) is a major cause of fatal diarrhea in childs under 5 years in developing countries. EPEC virulence is dependent on the presence of type III secretion system (T3SS). T3SSs are used by many pathogenic Gram-negative bacteria to inject bacterial effectors into host cells. T3SSs consist of a basal body embedded in the bacterial outer and inner membrane, prolonged by a needle protruding from the bacterial surface. In the case of EPEC, the T3SS needle is extended by a filament formed by hydrophilic EspA protomers. Upon cell contact, the system triggers the secretion of two hydrophobic proteins EspD and EspB, that insert in the host cell plasma membrane to form the translocon. The translocon is critical for the injection of type 3 effectors, presumably by forming a pore into host cell plasma membranes through which the effectors are channeled. The host lab studies the role of EspC, a serine protease autotransporter of Enterobacteriaceae (SPATE) family, on the regulation of the translocon function. EspC is a protein secreted by EPEC by a type V secretion system independently of the T3SS, although the translocation into epithelial cells requires active type III secretion. EspC has been shown to play a role in controlling pore formation and cytotoxicity mediated by the T3SS. EspC was shown to preferentially target an EspA-EspD complex, involved in the T3SS-dependent pore formation, supposedly following detachment of the translocon from the T3SS needle filament. This internship work took advantage of fractionation procedures worked out to isolate the EspA-EspD complex sensitive to EspC proteolysis, with the aim to perform a structural characterization of this complex by electron microscopy analysis. In this study, we increased the yield of EspA-EspD complex purification, that for the first time allowed the detection of EspB together with EspA-EspD complexes. We implemented additional purification steps to increase the purity of protein complexes. We were able to isolate homogenous EspAcontaining complexes showing a ring-link structure with an external diameter of 10 nm of and a 4 nm-inner diameter.