Virulence Factors Identification And FliL Genes Characteristic Study Of Proteus Vulgaris In Alligator Sinensis

Proteus vulgaris is a kind of gram-negative condition pathogen.It widely exsits in the natural environment and exists in the intestines of humans,wild animals and livestock.Under given conditions,it can cause humans and animals getting infection,such as urinary tract infections,bacteremia,diseases of the central nervous system,wound infections,rheumatoid arthritis and other related diseases.In recent years,there are many reports about the death of shrimp,soft shelled turtle and groupers caused by Proteus vulgaris.At present,researches into the pathogenicity of Proteus genus bacteria mainly focus on Proteus mirabilis and its urinary tract infection,but few researches focus on Proteus vulgaris.It is rare for the research on Proteus infection of Alligator sinensis.In the past researches of our laboratory,Proteus vulgaris was found in the diseased alligator and alligator eggs.Those Alligator sinensis suffered from loss of appetite,swelling of limbs and joints.By the method of pathological anatomy,those alligator sinensis were found severe kidney damageand deposition of urate in the joints of the kidneys and limbs.Proteus is a common urinary tract infection bacteria.It is proved by studies that Proteus can cause rheumatic diseases.Furthermore,urinary tract damage will affect the normal discharge of urate.For the purpose of exploring the relationship between Proteus vulgaris and the Alligator sinensis,we study the virulence factors of Proteus vulgaris from the Alligator sinensis.The results of the study are significant for prevention and control of Proteus vulgaris infection,and for healthy artificial breeding and species protection of Alligator sinensis.In this experiment,17 strains of Proteus vulgaris which isolated from alligators in Anhui Research Center for Alligator sinensis Reproduction from 2012 to 2013 were tested for virulence and common virulence genes of Proteus.Get the results of 17 strains of FliL,ZapA,HlyA,HpmA,HpmB,and RopA genes,and LD50 of 8 pathogenic strains.Among them,the detection rate of FliL was the highest(17/17,100%),and other genes were ZapA(9/17,52.9%),HlyA(0/17,0),HpmA(4/17,23.5%),HpmB(3/17,17.6%),RopA(10/17,58.8%).The experimental results showed that there was no clear linear relationship between the virulence of strains and individual virulence factors.However,17 strains were found to contain the common virulence factor FliL through this testing.FliL is a major gene in the class Ⅱ flagellar operon,and flagella is directly related to the pathogenicity of bacteria.One of the virulent strains was selected as the object and the bacteria were knocked out of the FliL gene for further exploration.Using the Red-mediated system,FliL targeting fragments were first constructed using the Kan-resistant pKD4 plasmid with as a template.The Amp-resistant pKD46 plasmid was electroporated into Proteus vulgaris XX2 to obtain the recombinant strain XX2/pKD46.XX2/pKD46 competent cells were then prepared by induction of L-arabinose and the targeting fragment was transferred to XX2/pKD46 competent cells.Under the action of the Red-mediated system,the bacterial gene and the targeting fragment were recombined to form kanami-resistant XX2△FliL::Kan.Then,introduct the pCP20 plasmid into XX2△FliL::Kan.By using the FLP enzyme the resistance gene on XX2△FliL::Kan was removed,the temperature-sensitive pCD20 plasmid was removed by culture at 42℃,and the deletion strain XX2△FliL was obtained around the mouth.By constructing complementary plasmids,and then electrotransferred into XX2dFliL competent cells,a complementary complement strain XX2△FliL/pFliL was constructed.The growth rate of FliL wild-type strains,deletion strains and complement strains was not significantly different in the first 6 hours.After 6 hours,the wild-type strains had faster growth rates than the FilL-deficient strains and the FilL-complemented strains.The wild-type strains and the deletion strains were compared in terms of their movements,group motility and virulence in order to investigate the effect of flagellin FilL gene on the virulence of Proteus vulgaris in Alligator sinensis,.The related influence of biological characteristics and the influence of the FilL gene on the virulence of the common proteobacteria of the Alligator sinensis.FliL knockout strain was unable to swarm but was able to swim.FliL is indispensable to the common Proteus migrating group,but does not work on its motility.For the virulence of the mice,the gap in LD50 between the knockout strain and the wild strain was about 100 times,indicating that flagella affected the virulence of Proteus.The result of this study lays a foundation for further exploration of relationship between Proteus vulgaris and alligator in proteus infection and treatmentfor proteus infection in alligator.