Study On Function Of Two-Component System Response Regulator Gene RS11565 In Listeria Monocytogenes

Listeria monocytogenges, Lm, is a Gram-positive, food-borne and invasive pathogen that can cause listeriosis in both humans and animals, especially immunocompromised individuals, with a high mortality rate of 20%-30%. In recent years, the cases of listeriosiswere reported from 27 provinces in China. If the disease becomes prevalent in China, human health will be threatened and the development of livestock industry will be hampered. So study on the pathogenic mechanism in Lm should be carried out.Bacterial two-component signal transduction system(TCS) is responsible forresponding to external signals and can play a considerable role in regulating expressions of virulence factors in pathogenic bacteria. So the findings on TCS will contribute to uncover the pathogenic mechanism in Lm.Lm strain LM201 was isolated from food and stocked in our laboratory. The work of whole genome sequencing, and annotation of strain LM201 have been finished.The GenBank database accession number is AYPT00000000.In this study, by bioinformatics analysis, we found that RS11570/RS11565 was an unknown function TCS in LM201. In order to ascertain the function of RS11570/RS11565, a mutant strain DRS11565was constructed. The biological characteristics were detected in DRS11565.The findings of this study would be helpful to annotating the pathogenic mechanism in Lm.The following is the results in this study. 1. Bioinformatics analysis of TCSs in LM201Based on the whole genome sequence information of LM201, there were 14 histidine kinases(HKs) which included 11 structures, and 16 response regulator(RR) that are classified into 7 subfamilies in LM201. The analysis for the phylogenetic tree showed that the level of evolution of HKs and RRs was same. By function prediction, RS11570/RS11565 was found in LM201, which was an unknown function TCS. 2. Construction of mutant DRS11565According to the RS11565 sequence, primers were designed to amplify upstream and downstream fragments and the recombinant suicide plasmid p3007 was constructed. Plasmid p3007 was transformed into LM201 to obtain mutant DRS11565, name as S4070. 3. Biological characteristics of mutant DRS11565Growth curve and main biochemical properties of mutant DRS11565 were detected, there was no difference bentween mutant and parent strain.The experiments of infection of C. elegans N2 and mice were carried out to investigate the virulence of mutant strain. The results indicated that the average life spans of C. elegans N2 fed with E. coli OP50(as control group) was 15.5d. The average life spans of C. elegans N2 infected with S4070 and LM201 was 12 d.There was no significant difference between S4070 and LM201 group.The result of LD50 showed that the LD50 of LM201 and S4070 was 1.55×106CFU and 4.37×105CFU for Kunming mice, respectively. By statistical analysis, no difference was found between S4070 and LM201. However, S4070 had significantly increased liver bacterial loads at 72 hours after infection compared with LM201.Compared with LM201, the result displayed that the efficiencies of adhesion with cell line Caco-2 of S4070 was significantly descended and the invasion assay showed that deletion of RS11565 did not affect the ability of invasion. There were no distinct differences between LM201 and S4070 about the capability of anti-phagocytic with cell line RAW264.7.