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.
Date of Award | 22 Sept 2016 |
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Original language | English |
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Awarding Institution | - Universidade Católica Portuguesa
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Supervisor | Guy Tran Van Nhieu (Supervisor) |
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- Mestrado em Microbiologia Aplicada
Regulation of the pore-forming translocon of the type III secretion system by the EspC Serine Protease in Enteopathogenic Escherichia Coli
Gomes, L. T. (Student). 22 Sept 2016
Student thesis: Master's Thesis