Analysis of pathogenic leptospires-specific genes by genomic suppression subtractive hybridization and cloning, expression and characterization of the outer membrane protein gene “omp52” from one Taiwanese leptospiral isolate
Date Issued
2005
Date
2005
Author(s)
Hsieh, Wang-Ju
DOI
zh-TW
Abstract
Abstract
In Taiwan, leptospirosis is caused mainly by Leptospira santarosai serovar shermani. Suppression subtractive hybridization was employed to isolate DNA fragments present in pathogenic Leptopsira santarosai serovar shermani but absent in non-pathogenic Leptospira biflexa serovar patoc. Analysis of 23 subtracted DNA clones revealed 25 gene fragments by BLAST program. Eight clones showed similarity to transposase genes and three clones displayed homology with either translation or metabolism related genes. Four clones were similar to outer membrane protein, penicillin-binding protein, CreD-like protein and the protein of two-component signal transduction system, respectively. One clone had TPR repeat domain and 5 clones had significant similarity with hypothetical proteins of unknown functions. The remaining 4 clones exhibited no homology with any known genes. These results indicate that subtractive hybridization can successfully identify genes that are absent from the non-pathogenic leptospira and provide with a starting point for clarifying the differential genes expression between pathogenic and non-pathogenic Leptospira species. We isolated a leptospira "strain CCF" from patient serum at our laboratory in 2000 and proved that its serovar type was Shermani by MAT (microscopic agglutination test). In order to identify the genotype of strain CCF, we used the flaB-RFLP typing method and confirmed the result by Southern blot with the probes of SSH clones to prove that strain CCF belongs to Leptospira santarosai. We used the method of inverted PCR to amplify the gene in L. santarosai serovar Shermani strain CCF isolated in Taiwan. A DNA fragment about 6 kb with 2 open reading frames was cloned and sequenced. 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. Another open reading frame with 1128 bp named ORF1 is located 547 bp downstream of omp52. ORF1 is a putative lysophospholipase gene. Omp52 was shown to be an outer membrane protein containing a C-terminal OmpA consensus domain and exposed on the cell surface. Furthermore, Omp52 increases dramatically during the stationary phase, indicating that the expression of Omp52 is environmentally regulated. By using immunoblotting analysis, we proved that Omp52 was expressed in patients infected with leptospires. Tubulointerstitial nephritis is a main renal manifestation caused by pathogenic leptospira that accumulates mostly in the proximal tubules, thereby inducing tubular injury and tubulointerstitial nephritis. To elucidate the role of leptospira outer membrane proteins in tubulointerstitial nephritis, outer membrane proteins from pathogenic Leptospira shermani by Triton X-114 and recombinant LipL32 protein were administered to cultured mouse proximal tubule cells and they significantly increased the mRNA expression of monocyte chemoattractant protein–1 (MCP-1) and inducible nitrite oxide synthase (iNOS). Recombinant Omp52 protein was added into the culture medium of mouse proximal tubule cells and increases both the mRNA expression of MCP-1 and iNOS, revealing the same result as treating mouse proximal tubule cells with LipL32. These observations suggest that Omp52 may play roles in the leptospiral pathogenesis.
Subjects
基因體抑制相減雜交法
鉤端螺旋體
Omp52外膜蛋白
genomic suppression subtractive hybridization
Omp52 outer membrane protein
leptospires
Type
thesis
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