Study On The Molecular Mechanism Of Vibrio Cholerae Colonization In The Intestinal Of The Adult Mouse

Vibrio cholerae is a curved gram-negative bacteria that can cause the acute diarrheal disease cholera.When V.cholerae enters an animal or human body,it needs to overcome the stomach acid barrier and colonize the surface of epithelium and produced virulence,through the host's excretion,V.cholerae returns to the in vitro environment,thus completing its infection cycle.The effective colonization of V.cholerae in the intestine is a necessary condition for its pathogenic effect on the host.The main animal models to study the colonization mechanism of V.cholerae are mouse and rabbit models,and mice are divided into two categories,the infant mouse and the adult mouse.The molecular mechanism of V.cholerae colonization in the infant mouse is relatively clear,and the expression of virulence gene is necessary for the colonization of V.cholerae in infant mouse.But the molecular mechanism of the colonization of V.cholerae in the adult mouse isn't clear.Previous studies found that the cysteine residue in the structure domain of virulence regulatory protein TcpP is mutated,the colonization ability of the mutant strain decreased significantly in the adult mouse.Studies has shown that Reactive oxygen species(ROS)had an important effect on the colonization of V.cholerae in the adult mouse,and the regulatory function of TcpP is inhibited under ROS conditions.TcpP cysteine mutants are insensitive to the inhibitory effect of ROS.Despite the existence of ROS,it also can activate the expression of downstream virulence genes in a large amount.It is speculated that V.cholerae regulates the function of TcpP by sensing ROS in the intestinal tract of adult mice to inhibit the expression of virulence genes,it makes V.cholerae better colonize in the adult mouse.Therefore,in order to clarify the molecular mechanism of V.cholerae colonization in the adult mice,this thesis carried out the following research.First of all,this paper studies the effect of virulence regulatory protein TcpP on the colonization of V.cholerae in adult mice.TcpP has an N-terminal intracellular domain with DNA activation function,and its intracellular cysteine residues affect the formation of disulfide bonds.AMS experiment proved that TcpP intracellular cysteine residues can sense the presence of extracellular ROS.When using fluorescent quantitative PCR to detect the transcription efficiency of the target gene downstream of TcpP,it shous that under the condition of ROS,the ability of TcpP to activate downstream gene expression was greatly reduced.When the cysteine residue in the intracellular domain of TcpP is mutated,it can still activate the expression of virulence genes normally.It shous that TcpP can use the cysteine residues in the intracellular domain to sense extracellular ROS,and change its own function of activating the expression of downstream virulence genes.Study the colonization ability of TcpP cysteine mutants in the colonization model of adult mice treated with streptomycin,it was found that in the intestines of adult mouse treated with streptomycin,the colonization ability of TcpP cysteine mutant strain was significantly weaker than that of wild-type strains.However,after infusion of N-acetylcysteine that can eliminate ROS,the colonization ability of TcpP cysteine mutant strain is equivalent to that of wild-type strains.When the tcp P gene was deleted,the colonization ability of V.cholerae in the adult mouse was significantly enhanced.This shows that V.cholerae can sense ROS in the intestine through TcpP,thereby inhibiting the expression of downstream virulence genes,and this inhibition plays an important role in the colonization of V.cholerae in the adult mice.It is known that omp T,vca0536,toxT can be regulated by TcpP,in order to determine which downstream gene is inhibited by TcpP to promote the colonization,the omp T,vca0536,and toxT gene deletion mutant strains were constructed.By analyzing the colonization ability of mutant strains,it was found that after toxT was deleted,the colonization ability of V.cholerae in the adult mouse is significantly enhanced.These results all indicate that inhibiting the expression of virulence genes can promote the colonization of V.cholerae in the adult mouse.ToxT is a key virulence gene expression regulatory protein of V.cholerae.In order to study the effect of ToxT on the colonization of V.cholerae in the adult mouse,the ToxT,downstream gene cluster(vc(0819-0825),vc(0828-0837),vc(0840-0845))gene deletion mutant strains and toxT overexpression strain were constructed.Through the analysis results,it was found that the colonization ability of these downstream gene cluster deletion strains in the adult mouse was significantly enhanced compared with that of wild-type bacteria.But the colonization ability of toxT overexpression strain was significantly weakened.This shows that the expression of virulence genes is not conducive to the colonization of V.cholerae in the adult mouse.In order to prove whether ToxT affects the colonization by regulating the expression of downstream virulence genes.This experiment knocked out gene clusters on the basis of toxT overexpression strain.However,the colonization ability of this mutant strain in the adult mouse is the same as that of the toxT overexpression strain,and its colonization ability is significantly weaker than that of the wild-type strain.This shows that in addition to the virulence genes regulated by ToxT,there are some unknown factors regulated by ToxT have an important influence on the colonization of V.cholerae in the adult mouse.In V.cholerae,the transcription of toxT is regulated by the transcriptional regulators TcpP and Tox R.In order to study the effect of Tox R on the colonization of V.cholerae in the adult mouse.When studying the colonization effect of Tox R on V.cholerae in the intestinal tract of adult mice,it was found tox R was deleted,the colonization ability of V.cholerae was significantly weakened,this had the opposite effect to TcpP.This shows that Tox R is essential for the colonization of V.cholerae in the adult mouse.The Tox R regulator is known to regulates a variety of virulence genes,the outer membrane porins Omp U and Omp T,and other downstream genes,etc.Studies have found that the colonization ability of ?omp T was not significantly different from that of wild-type,while ?omp U could hardly colonize in the adult mouse.This indicates that Tox R may enhance the colonization ability of V.cholerae in the adult mouse by activating the outer membrane porin Omp U.Above,this study found that V.cholerae senses the ROS through the virulence regulation protein TcpP intracellular cysteine residues,it inhibits the expression of downstream toxT genes,and assists the colonization of V.cholerae in the adult mouse.ToxT enhances the colonization of V.cholerae in the adult mouse by inhibiting the expression of virulence genes and other unknown factors.In addition,the regulatory protein Tox R enhances the competitive colonization ability of V.cholerae in the adult mouse by activating the expression of the outer membrane porin Omp U.Based on these experimental results,we proposed a model for the survival and competition of V.cholerae in the adult mouse.This study could provide a theoretical basis for revealing the molecular mechanism of V.cholerae colonization in the adult mice and could have a great significance to provide prevention and treatment ideas for further prevention and treatment of V.cholerae infection.