fimW 基因在鼠傷寒沙門氏菌第一型線毛調控系統所扮演的角色

Abstract

鼠傷寒沙門氏菌是造成人和動物沙門氏菌感染症的最重要致病原之一。鼠傷寒沙門氏菌的傳播是藉由糞口途徑進行的，並且此微生物是透過菌體外膜上的附屬物來吸附於腸道上皮細胞，其中此附屬物大部分是線毛結構。第一型線毛或所謂的普通線毛常見於鼠傷寒沙門氏菌，並且與鼠傷寒沙門氏菌的致病機制有關。第一型線毛的表現是由fim基因組所編碼，該基因組是由六個結構基因fimA、fimI、fimC、fimD、fimH和fimF以及五個調控基因fimZ、fimY、stm0551、fimW和fimU所組成的。其中FimW的胺基酸序列與具有螺旋-轉角-螺旋DNA結合結構組元的大多數轉錄調控子之胺基酸序列相似。為了進一步探討fimW基因在線毛調控方面所扮演的角色，藉由等位基因置換的方法構築了一株fimW突變菌株。當fimW突變菌株培養在有利於第一型線毛表現的靜置液態的培養液中，或者培養於不利第一型線毛表現的固態培養基環境，此菌株都會持續表現第一型線毛。由RT-PCR分析顯示，在固態環境培養的fimW突變菌株，其線毛主結構基因fimA的mRNA表現量比wild type菌株高。而在固態培養環境的fimW突變菌株，其線毛的調控基因fimZ、fimY、fimU和stm0551的mRNA表現量比wild type菌株來得高。本研究也使用pET-30a系統構築FimW融合蛋白。進行電泳凝膠遷移實驗以研究FimW蛋白是否與特定DNA片段相互作用。實驗使用FimW蛋白和含有大小200至300鹼基對的fimA、fimU、fimW、fimY、fimZ和stm0551之上游區域片段以及鼠傷寒沙門氏菌中另外10種線毛的主要次單位的DNA片段進行。在本研究中未觀察到FimW蛋白與DNA有相互作用的情形。由於fimW突變菌株持續性地產生第一型菌毛，因此我們意圖進一步研究這種突變菌株是否適合做為減毒基因表現（AGE）疫苗開發的候選菌株。目前初步結果顯示，抗生素polyimox B和紅黴素的最小抑制濃度在fimW突變菌株中會降低，這說明了fimW基因的缺失，會降低細菌細胞膜的穩定性。使用fimW突變菌株做為減毒沙門氏菌疫苗株的適用性值得進一步的闡明。總結上述說明，FimW做為抑制因子來調節鼠傷寒沙門氏菌第一型線毛的表現。目前的結果，EMSA未觀察到FimW與fim專一性的DNA或其他10種線毛次單位的啟動子DNA之間有相互作用。缺乏fimW基因會影響細菌細胞膜的完整性，fimW突變菌株可能可以成為AGE疫苗的候選菌株。

Salmonella enterica serovar Typhimurium is one of the most important causative agents responsible for salmonellosis in both human and animals. Transmission of S. Typhimurium is by oral-fecal route and this microorganism adheres epithelial cells of intestinal tract by the appendages present on the outer membrane, most of which are fimbrial structures. Type 1 fimbriae or so-called common fimbriae are frequently found and have been implicated in pathogenesis of S. Typhimurium. Phenotypic expression of type 1 fimbriae is encoded by the fim gene cluster which is composed of six structural genes fimA, fimI, fimC, fimD, fimH and fimF, and five regulatory genes fimZ, fimY, stm0551, fimW and fimU. The amino acid sequence of FimW shares similarities to those of most transcriptional regulators possessing helix-turn-helix DNA binding motif. To further explore the role that fimW plays in terms of fimbrial regulation, a fimW mutant strain was constructed by allelic exchange. The fimW mutant constitutively produced type 1 fimbriae when cultured in static broth, a medium favorable for fimbrial production, or on solid agar medium, which is a condition usually impedes fimbrial expression. RT-PCR analysis revealed that the mRNA level of fimbrial subunit gene fimA was more prominent in the fimW mutant strain cultured on solid agar than that of the parental strain. The mRNA level of the fimbrial regulator gene fimZ, fimY, fimU and stm0551 were increased in the fimW mutant strain grown on solid agar than the parental strain. A FimW fusion protein was constructed using a pET-30a system. An electrophoretic mobility shift assay (EMSA) was performed to investigate if FimW protein interacted with the specific DNA fragments. It was performed using FimW and the DNA fragments containing the 200-300 base pairs of the upstream regions of the fimA, fimU, fimW, fimY, fimZ, and stm0551 and also of the other 10 major fimbrial subunit in S. Typhimurium. No FimW protein /DNA interaction was observed in the present study. Since the fimW mutant strain constitutively produced type 1 fimbriae, it was our intention to further explore if this mutant strain would be suitable to serve as a candidate strain for attenuating gene expression (AGE) vaccine development. Initial results suggested that the minimal inhibitory concentrations of polymxin B and erythromycin were decreased in the fimW mutant, indicating that the absence of fimW reduced the stability of the bacterial membrane. The suitability of using the fimW mutant strain as an attenuated Salmonella vaccine strain warrants further elucidation. In summary, FimW functions as a repressor to modulate type 1 fimbrial expression in S. Typhimurium. Currently, no interaction between FimW and fim-specific DNA, or other 10 fimbrial subunit promoter DNA was observed by EMSA. Deletion of fimW compromises the integrity of the bacterial membrane, making the fimW mutant a possible AGE vaccine candidate.