lrp、stm0551 與 fimY 基因在鼠傷寒沙門氏菌第
一型線毛表現調控所扮演的角色

在感染過程中，細菌的吸附能力是個重要的致病因子。鼠傷寒沙門氏菌可以表現出第一型線毛，其具有甘露醣專一性的結合能力可以使細菌吸附於宿主細胞。過去的研究指出鼠傷寒沙門氏菌第一型線毛的表現受到 fimZ、fimY、fimW 與 lrp 基因的調控。在我們的研究中，我們首先將已經表現第一型線毛的鼠傷寒沙門氏菌 LB5010 感染於巨噬細胞株 RAW 264.7。實驗結果顯示當細菌感染巨噬細胞後其線毛的主要結構基因 fimA、主要調控基因 fimZ 與 lrp 的表現會隨時間逐漸減少。另外在模擬感染的環境中，我們發現當培養環境由pH 7 降低至pH 4 時，fimA、 fimZ 與 lrp 的表現也會同樣地下降。此外，fimA 的突變株在感染巨噬細胞後反而比正常可表現第一型線毛的菌株有更好的生長率。而帶有 lrp 質體的菌株難以於 pH 4 的培養條件下生長。接下來的研究，我們了分析了一個位在 fimY 與 fimW 基因之間一個尚未研究的 stm0551 基因，stm0551 會產生一個 11.4-kDa 具有磷酸二酯酶活性的蛋白質，可以參與二級訊息分子 c-di-GMP 的降解。我們利用了對偶基因交換的模式構築了 stm0551 的突變株並發現了此突變株無論在 LB broth 或是 LB agar 的培養環境皆會持續性地表現第一型線毛。透過定量 RT-PCR 的分析則發現在 stm0551 突變株中 fimA 與 fimZ 的表現程度皆會增加。接著我們純化了 STM0551 的重組蛋白並發現該蛋白具有磷酸二酯酶的活性。此外，將 STM0551 蛋白上 EAL 區域的 glutamic acid (E)置換為 alanine (A)時，STM0551 蛋白會失去磷酸二酯酶的活性。最後一個部分，我們分析了 FimY 蛋白的序列並發現了 FimY 蛋白的 C 端序列類似於 LuxR 蛋白。接著利用電泳遷移試驗的模式發現了 FimY 具有與 DNA 結合的能力，其可以與 fimY 與 fimZ 之間約 605-bp 的啟動子區域結合。接下來將 FimY 蛋白的 C 端具有 helix-turn-helix 的區域進行突變時，當第 189 胺基酸位置的 glycine (G)置換為 aspartate (D)以及第 195 胺基酸位置的 isoleucine (I)置換為 lysine (K)的時候，FimY 蛋白會失去與 DNA 結合的能力。此外 fimZ 的啟動子發現有個類似於 lux box 的區域(5’-TCTGTTATTACATAACAAATACT-3’) II 與 FimY 的結合有關。而 FimY 並無法與其他基因包括 fimA、fimY 與 fimW 的啟動子區域結合。統整上述三個階段的研究可以發現模擬巨噬細胞感染的酸性環境會透過調控 fimZ 與 lrp 來抑制鼠傷寒沙門氏菌第一型線毛的表現。而 STM0551 可以藉由與 c-di-GMP 交互作用來控制第一型線毛的表現。最後我們發現 FimY 可以結合於 fimZ 的啟動子區域並調控其表現。

關鍵字

鼠傷寒沙門氏菌；第一型線毛

並列摘要

Adherence is one of the important steps in the pathogenesis. Salmonella enterica serovar Typhimurium (S. Typhimurium) produces type 1 fimbriae mediating mannose-specific binding to host cells. The genetic regulations of type 1 fimbriae are controlled by fimZ, fimY, fimW and global regulatory gene lrp. In the first part of the studies, we used a type 1 fimbrial phase-variable strain S. Typhimurium LB5010 and its derivatives to infect RAW 264.7 macrophages. Following entry into macrophages, S. Typhimurium LB5010 gradually decreased the transcript levels of fimbrial subunit gene fimA, positive regulatory gene fimZ, and global regulatory gene lrp. A similar decrease in transcript levels was detected by RT-PCR when the pH of medium was shifted from pH 7 to pH 4. The proliferation rate of fimA-deleted strain was superior to that of the parental strain after infection. An lrp deletion strain was unimpaired for in vitro growth at pH 7 or pH 4, while a strain harboring an lrp-containing plasmid exhibited impaired in vitro growth at pH 4. The next part of these studies, we identified a novel gene, stm0551, located between fimY and fimW that encodes an 11.4-kDa putative phosphodiesterase specific for the bacterial second messenger cyclic-diguanylate monophosphate (c-di-GMP). A stm0551-deleted stain constructed by allelic exchange constitutively produced type 1 fimbriae in both static-broth and solid-agar medium conditions. Quantative RT-PCR revealed that expression of fimA and fimZ were comparably increased in the stm0551-deleted strain compared with the parental strain when grown on the solid-agar medium, a condition that normally inhibits expression of type 1 fimbriae. A purified STM0551 protein exhibited a phosphodiesterase activity in vitro while a point mutation in the putative EAL domain, substituting glutamic acid (E) with alanine (A), of STM0551 protein abolished this activity. The third part of the studies, we aligned the amino acid sequence of FimY. IV The amino acid sequences of the C-terminal portion of FimY revealed similarity with those of LuxR-like proteins. Electrophoretic mobility shift assays indicated that FimY possessed DNA-binding capacity and bound a 605-bp DNA fragment spanning the intergenic region between fimY and fimZ, while a FimY protein harboring a double mutation in the C-terminal helix-turn-helix region containing a glycine (G) to aspartate (D) substitution at residue 189 and isoleucine (I) to lysine (K) substitution at residue 195 lost its ability to bind this DNA fragment. A lux box sequence (5’-TCTGTTATTACATAACAAATACT-3’) within the fimZ promoter was required for binding. None of the DNA fragments derived from the promoters for fimA, fimY, or fimW was shifted by FimY. According to the above results, we propose that acidic medium, which resembles one aspect of the intracellular environment in a macrophage, inhibits type 1 fimbrial production by down-regulation of the expression of lrp, fimZ and fimA. In addition, the stm0551 gene plays a negative regulatory role in the regulation of type 1 fimbriae by modulating the concentration of c-di-GMP, and FimY functions as an activator by binding to the fimZ promoter.

並列關鍵字

Salmonella Typhimurium ； type 1 fimbriae

參考文獻

Angulo. 1997. Emerging quinolone-resistant Salmonella in the United States.

Emerg. Infect. Dis. 3:371-372.

K. Smith, F. J. Angulo. 2004. Invasive Salmonella infections in the United

Clin. Infect. Dis. 38 Suppl 3:S149-156.

invertible element of DNA controls phase variation of type 1 fimbriae of

國際替代計量

lrp、stm0551 與 fimY 基因在鼠傷寒沙門氏菌第一型線毛表現調控所扮演的角色

全文下載

主題瀏覽