| Summary: | 碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 94 === L0033 is encoded by the 4th open-reading frame of LEE3 that is located in the pathogenic island of Escherichia coli O157:H7 named locus of enterocyte effacement (LEE). This protein comprises 125 amino acids, and its function remains unclear. Biochemically, the purified protein prepared from a lab Escherichia coli strain forms dimeric structure, as evidenced by SDS PAGE and gel-filtration. However, in a natural setting or when the expression system was switched to E. coli O157:H7, L0033 was below the detectable level of Western blotting, a fact indicating a stringent control. When the first open reading frame of LEE3, mpc, was silenced by mutation at the start ATG codon, L0033 was successfully detected, presumably due to a release of the stringent control. To localize L0033 in the bacteria, protein fractionation was carried out and analyzed for the presence of L0033 by anti-L0033. It was found that L0033 was mainly in the outer membrane fraction. When the coding gene l0033 was deleted from the LEE in E. coli O157:H7, the type III secretion of the bacteria was completely abolished while detection of representative type III proteins in the bacterial lysate varied differently. These changes in the deletion mutant could be completely restored after complementation with L0033. Thus, L0033 is a tightly regulated outer membrane protein that may assist the assembly of the type three secretion apparatus by forming a dimeric structure. Furthermore, we found that L0033 could form complex with EspA under a native condition. Thus we hypothesized that during the working of the type III secretion system, L0033 may assist EspA to pass through the channel. Another topic in this thesis is L0038, may act as one of the accessory components for the type III secretion system. We identified that L0038 is mainly located to the inner membrane of the EHEC and general protein transport system is sufficient to transit it to the favorable site.
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