以基因體抑制相減雜交法分析病原性鉤端螺旋體特異性基因暨病原性鉤端螺旋體台灣分離株omp52外膜蛋白質基因之選殖、表現與分析

Abstract

鉤端螺旋體症在台灣的主要流行血清型為Leptospira santarosai serovar Shermani。利用抑制相減雜交法 (suppression subtractive hybridization; SSH) 分析病原性L. santarosai serovar Shermani及非病原性L. biflexa serovar Patoc基因體的差異。選殖23個DNA片段含有25個基因片段只存在於病原性L. santarosai serovar Shermani而不存在於非病原性L. biflexa serovar Patoc。以BLASTX程式分析有8個選殖DNA片段與轉位酶 (transposase) 基因相似，3個與轉錄及代謝有關基因相似。4個選殖的片段分別為外膜蛋白質 (outer membrane protein；OMP)、Penicillin結合蛋白質 (Penicillin binding protein；PBP)、CreD-like蛋白質及Two-component訊息傳導系統蛋白質。另外還有1個含有TPR (Tetratrico peptide repeat) repeat domain的蛋白質與5個為未知功能的假設性蛋白質 (hypothetical protein)。其餘的選殖片段經比對沒有與任何已知基因相似。實驗的結果顯示抑制相減雜交法可以成功的確認存在於病原性鉤端螺旋體而不存在於非病原性鉤端螺旋體的基因，而提供病原性與非病原性鉤端螺旋體差異基因研究的開始。2000年本實驗室由病人血清分離得到鉤端螺旋體CCF株，以顯微凝集試驗 (microscopic agglutination test；MAT) 方式證明為血清型Shermani。為了進一步確認CCF株的基因種別，利用flaB-RFLP typing的方法及以抑制相減雜交法所得的鉤端螺旋體Shermani DNA片段為探針進行沙忍氏墨點轉漬分析 (Southern blot analysis )，實驗結果顯示CCF株的基因種別為L. santarosai，確認CCF株為L. santarosai serovar Shermani strain CCF。以抑制相減雜交法所得到C67片段經BLASTX程式比對可能為一個外膜蛋白質，為了解整個基因的序列利用Inverted PCR (IPCR) 技術選殖已知序列鄰近的DNA片段，約6 kb的DNA片段被選殖定序，整段DNA序列中含有二個開放讀碼區(open reading frame；ORF)為omp52及orf1。Omp52有1371 bp可轉譯成456個胺基酸，分子量爲52.6 kD，為原C67 DNA片段的基因，其C端含有OmpA的Domain，為OmpA familiy外膜蛋白質，而ORF1含有1128 bp，位於omp52的下游約547 bp，可能為一個Lysophospholipase。以西方氏墨點分析法(Western blot analysis)分析各個時期生長之L. santarosai serovar Shermani strain CCF的Omp52蛋白質表現，發現Omp52蛋白質在對數期表現量少，進入Stationary phase時表現量才大增。利用Triton X-114萃取CCF株的外膜蛋白質，於清潔劑層 (detergent phase) 偵測到Omp52蛋白質，以Proteinase K切割外膜蛋白質，發現Omp52被切割消化。免疫螢光抗體分析可見Omp52被Anti-Omp52的抗體標示，這三方面的實驗結果驗證Omp52為外膜蛋白質且有部份的蛋白質是曝露於細菌外表。以感染鉤端螺旋體Shermani血清型病人的血清對Recombinant Omp52蛋白質進行西方氏墨點分析法，可發現陽性病人血清可偵測到該蛋白質，而陰性血清則不能，顯示該蛋白質在鉤端螺旋體感染人類時會被表現。鉤端螺旋體所引起的腎臟疾病主要是腎小管間質性腎炎，且常聚集於近端腎小管間，引起腎小管的損傷及炎症反應。以鉤端螺旋體Shermani血清型之外膜蛋白質及LipL32重組蛋白質處理小鼠近端腎小管細胞可引起細胞之MCP-1 (Monocyte chemoattractant protein–1) 及iNOs (Inducible nitrite oxide synthase) mRNA表現上升數倍，而以Omp52重組蛋白質處理小鼠近端腎小管細胞也可正向調控MCP-1及iNOs mRNA的表現量。綜合以上的結果，顯示Omp52蛋白質在鉤端螺旋體的致病機制上扮演相當重要的角色。

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.