[Association of fliR gene in Leptospira interrogans with adhesion and pathogenicity to host cells].
To investigate the pathogenicity of Leptospira interrogans fliR gene to J774A.1 cells.



fliR gene from L. interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 and kana gene from plasmid pET42a were amplified by PCR. Suicide plasmid of fliR gene was constructed; and specific siRNA for fliR gene was designed and synthesized. fliR gene mutants were constructed by gene knock-out with suicide plasmid (56601fliR-Kana) and gene silencing with siRNA (56601siRNA-R2). The mutants were identified by PCR, sequencing and semi-quantitative RT-PCR. Adhesion to mouse mononuclear-macrophage J774A.1 and induction of cell necrosis and apoptosis by 56601fliR-Kana and 56601siRNA-R2 were examined by adhesion test and flow cytometry, respectively.



The nucleotide and putative amino acid sequences of cloned fliR gene had 99.9% and 100% similarities to those of reported sequences in GenBank. The nucleotide sequence of the cloned kana gene was identical to the corresponding sequence in pET42a map. The results of PCR and sequencing confirmed that kana gene was inserted in the sequence of 56601fliR-Kana fliR gene. The mRNA level of fliR gene in 56601fliR-Kana was remarkably decreased (P<0.01) while the mRNA level of fliR gene in 56601siRNA-R2 was much lower than that in wild strain 56601 (P<0.05). 56601fliR-Kana and 56601siRNA-R2 lost the ability to adhere J774A.1 cells; and their ability to induce cell necrosis and apoptosis was markedly weakened (P<0.01).



fliR is a virulence-associated gene of L. interrogans and the function of the gene is closely related to adhesion, induction of cell necrosis and apoptosis of the microbe.
Ruan
Ping
P
Medical School of Shaoxing University, Shaoxing 312000, China.
Wang
Xin-ying
XY
Sun
Ai-hua
AH
Li
Shi-jun
SJ
Yan
Jie
J
D000818:Animals; D017209:Apoptosis; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D007921:Leptospira interrogans / Q000235:genetics* / Q000472:pathogenicity*; D008264:Macrophages / Q000382:microbiology* / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics
D001426:Bacterial Proteins; C085248:FliR protein, Bacteria; D008565:Membrane Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering