探討在促進或抑制鼠傷寒沙門氏菌第一型線毛表現時fim基因mRNA的差異並選殖其他mRNA有差異的基因

Determination of the fim related gene expression and cloning the genes that express differently in mRNA level from the culture conditions that favor and inhibit type 1 fimbrial expression in Salmonella enterica serotype Typhimurium

范嘉純(Chia-Chun Fan)

指導教授 ： 葉光勝

臺北醫學大學/醫學院/醫學科學研究所/碩士(2007年)

摘要

鼠傷寒沙門氏菌在其菌體的表面擁有第一型線毛的構造，帶有吸附素可與具有甘露糖受體的宿主專一結合，與表現第一型線毛相關的fim基因群包含九個開放讀碼區和一個tRNA基因。過去的研究發現，鼠傷寒沙門氏菌經過一連串的繼代培養在靜置的液態培養基後，可以篩選出大量表現第一型線毛的細菌，而在固態培養基上的繼代培養則會抑制細菌表現第一型線毛。本研究一方面偵測在靜置液態培養基中或固態培養上細菌的fim相關基因mRNA的表現量，另一方面則是想找出在這兩種不同的培養條件下有表現不同的基因，進而研究此基因與第一型線毛表現的相關性。反轉錄聚合酶鏈鎖反應顯示鼠傷寒沙門氏菌的fimA、fimI、fimD、fimZ和fimW基因在固態培養基不表現但會表現在液態培養基中生長的細菌。fimC、fimF、fimY在靜置的液態培養基中則是有比在固態培養基中有較多的mRNA表現。利用GeneFishingTM聚合酶鏈鎖反應試劑組選殖出在靜置液態培養與固態培養基表現不同的基因，結果發現在靜置的液態培養下有較多ompC, gph, STM1128, modE, upp, dut, cheY的mRNA表現，相反的，在固態培養基則有較多ytfK, yfgB, uup, ycgB, ndk, aroC的mRNA表現。以上基因與fim基因之間的關聯性，將會持續研究並闡明其控制鼠傷寒沙門氏菌第一型線毛表現的機制。

關鍵字

鼠傷寒沙門氏菌 ； 第一型線毛

並列摘要

Salmonella enterica serovar Typhimurium possesses surface appendages called type 1 fimbriae that carry adhesins specific for mannosylated host glycoconjugates. The expression of type 1 fimbriae is associated with the fim gene cluster within which 9 open reading frames and one tRNA gene are present. Previous observation indicated that serial subculturing of S. Typhimurium in static liquid broth selected for highly type 1 fimbriate bacteria, while solid agar media inhibited the bacteria to produce type 1 fimbriae. The purpose of the present study is on one hand to detect the mRNA expression level of individual fim related gene from static broth and solid agar culture conditions. In addition, those genes that may express differently at static broth and solid agar were identified. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that the mRNA of fimA, fimI, fimD, fimZ, and fimW were present when S. Typhimurium was grown in static broth but were absent when cultured on solid agar. However, S. Typhimurium had more fimC, fimF, and fimY mRNA when grown in static broth than grown on solid agar. GeneFishingTM PCR kit was used to clone the genes that express differently in static broth and solid agar culture conditions. The mRNA of ompC, gph, STM1128, modE, upp, dut, cheY were found to express more in static broth than on the solid agar medium, on the contrary, the mRNA of ytfK, yfgB, uup, ycgB, ndk, aroC were more on solid agar than in the static broth culture. The association between the above identified genes and the fim related genes will be further investigated to elucidate the mechanism that may control the expression of the type 1 fimbriae in S. Typhimurium in response to static broth and solid agar culture conditions.

並列關鍵字

Salmonella Typhimurium ； type 1 fimbriae

參考文獻

58. Poppe, C., N. Smart, R. Khakhria, W. Johnson, J. Spika, and J. Prescott. 1998. Salmonella typhimurium DT104: a virulent and drug-resistant pathogen. Can. Vet. J. 39:559-65.

1. Ackman, D. M., P. Drabkin, G. Birkhead, and P. Cieslak. 1995. Reptile-associated salmonellosis in New York State. Pediatr. Infect. Dis. J. 14:955-9.

2. Allen, R. M., R. Chatterjee, M. S. Madden, P. W. Ludden, and V. K. Shah. 1994. Biosynthesis of the iron-molybdenum cofactor of nitrogenase. Crit. Rev. Biotechnol. 14:225-49.

3. Andersen, P. S., J. M. Smith, and B. Mygind. 1992. Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12. Eur. J. Biochem. 204:51-6.

4. Bäumler, A. J., and F. Heffron. 1995. Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium. J. Bacteriol. 177:2087-97.