Flexneri 2a, Shigella The 2457t Argt Gene Function Of The Study

Shigella spp.,commonly called Bacillus dysenteriae,is a group of Gram-negative,non-spore forming,facultative pathogens.The bacteria cause a disease called shigellosis in humans,an infection of colonic epithelial cells characterized by abdominal cramps,diarrhea,and fever. According to statistics of WHO,there're approximately 164.7 million cases every year,of which 11 thousand are dead and of which majority are children under 5 years old.Serum type 2a of Shigella spp.is responsible for the majority of cases of endemic dysentery prevalent in developing countries where sanitation is poor.While in China,there're about 20 million cases every year,and Shigella flexneri 2a is responsible for 50-70%of these.Thus it is important to research on Shigella flexneri for our sanitary and antiepidemic work.It was reported that the invasion into HeLa cells is available when Shigella flexneri 2a cultivated at 37℃but not at 30℃.We previously did proteomic research of S.flexneri.2457T under different temperatures and found that the abundance of ArgT is very low when cultured to stationary phase at 37℃while high at 30℃.The periplasmic binding protein ArgT,also called lysinearginine-ornithine-binding protein(LAO),participates in the uptake of arginine as a nitrogen source. In order to make clear the function of ArgT in invasion,the deletion mutant 2457TΔargT::Kan was constructed withλ-Red recombination system and the over-expression mutant 2457T/pAK-ArgT_Y225Dby site-specific mutagenesis.Comparing mutants with the wild strain,we have the conclusions as follows:When grown in LB medium,the mutants grow slower than the wild stain at the beginning,but no difference in the end.The ability to ultilize mannitol of mutants are higher than the wild strain, but lower to glucose.The HeLa cell invasion test and infected lung model in mice showed that argT deletion has no effects on virulence,while over-expression decreases the invasion.So,we infer that argT is an anti-virulunce gene.By means of two-dimensional gel electrophoresis combined with MALDI-TOF MS,we compare the protein expression profiles between mutants and the wild strain. The data showed that expressions of 8 proteins were significantly altered in mutants.OmpR was down-regulated in ArgT over-expressed mutant.The Heat Shock Protein HtrA was significantly altered in ArgT different mutants.The outermembrane protein regulator OmpR interacting with ArgT by GST-pulldown and Western-blotting was identified.OmpR is a part of osmotic response regulator OmpR-EnvZ and also a virulence factor,which regulates the expression of vir and OmpF/C.The results show that,argT has little effects on the metabolism of Shigella flexneri 2a 2457T, but over-expression ArgT can decrease the virulence,and change the permeability of the membrane.