The survival of E. coli expressing leptospiral outer membrane protein Omp52 within macrophages
Date Issued
2006
Date
2006
Author(s)
Chen, Wei-Ting
DOI
zh-TW
Abstract
Leptospirosis is a worldwide zooanthroponosis casused by bacteria belonging to the Leptospira. In Taiwan, leptospirosis is caused mainly by Leptospira santarosai serovar Shermani. We isolated 23 DNA fragments present in pathogenic Leptopsira santarosai serovar shermani but absent in non-pathogenic Leptospira biflexa serovar patoc. An open reading frame of 1371 bp encoding a protein of 456 amino acids (designated omp52) with a predicted molecular mass of 52.6 kD is matched to the clone C67. Omp52 gene identified among pathogenic L. santarosai serovar Shermani strain CCF but absent in non-pathogenic L. biflexa serovar Patoc. It was shown to be an outer membrane protein containing a C-terminal OmpA consensus domain and exposed on the cell surface. OmpA expression is necessary for effective binding to and subsequent uptake of E. coli by human and murine macrophages, and OmpA+ E. coli can replicate in the phagocytic cells. Infected with OmpA+ E. coli macrophages will disrupt after postinfecion. Pathogenic leptospires survive by evading phagocytosis and induct the apoptosis of macrophages. This phenomenon is similar to OmpA+ E. coli. The function of Omp52 is still unknown. Because there is a OmpA consensus domain on Omp52 structure, and Omp52 only present in pathogenic Leptopsira. We used invasion assays to investigate the interactions of transformed E. coli with macrophage cell lines. The amount of survived bacterium will reveal whether evading phagocytosis is related to Omp52 in pathogenic leptospires. This study is divided into two parts, E. coli BL21 (OmpA+) and E98 (OmpA- E. coli). Using BL21 (pWJN1) which can express leptospiral outer membrane protein Omp52 to perform invasion assays. The results of invasion assay which were carried out with 10-fold less ( MOI=0.1) and MOI of 1 showed BL21 (pWJN1) survival better than E. coli without Omp52 expression ( p < 0.05 ). In 10-fold more ( MOI=0.1), BL21 (pWJN1) also survival better than E. coli without Omp52 expression ( p < 0.001）. E98 （OmpA- E. coli） was transformed with pWJN1 containing the Omp52 gene. In MOI= 1and MOI= 10, the amount of E98（pWJN1）in macrophages are lager than E. coli without Omp52 expression （p < 0.05）. The results reveal one of Omp52 functions is to help evade phagocytosis, and E. coli which express Omp52 can still replicate in macrophage cell lines. Furthermore, E98 was transformed with pRSETc-LipL32 to express the other leptospiral outer membrane protein LipL32. The invasion assays results showed LipL32 can’t help bacterium survival in macrophages.
Subjects
鉤端螺旋體
外膜蛋白質
leptospira
Omp52
outer membrane protein
Type
thesis