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

Abstract

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.