Role Of Virulence Plasmid In The Proteome Ofyersiniapestis And Shigellaflexneri

Shigella spp. is a kind of nonspore-forming, nonmotile, Gram-negative bacteria, which is the main cause of human shigellosis. Yersinia pestis is a genus of capsule-forming, nonspore-forming, nonmotile, Gram-negative short rod-shaped bacillus, which is the causative agent of plague. Both of them have a special cellular structure(T3SS,type III secretion system) cross the bacterial inner membrane, the outer membrane and the host cell membrane, secretingthe protein inside the bacteria directly into the host cell,and playing an important role in the invasion of pathogens and pathogenic process.Shigella and Yersinia pestis are pathogens with serious harm. In both of these bacteria, the structure proteins and effectors of T3SS are encoded by an evolutional acquired virulence plasmid. In order to better understand the common and specific influences of exogenous virulence plasmid on bacterial protein expression profiling, this study will compare the results obtained from two different pathogenic bacteria.As we all known,most proteins could not perform their accurate functions in the cell independently, without bingding to other proteins or subunits to forma functional protein complex. Blue Native Polyacrylamide Gel Electrophoresis(BN-PAGE)is able to maintain the biological activity of protein complexes at the time of separation.It was commonly used in the protein complex analysis, detecting the activity of proteins in gel and protein-protein interactions and other fields.Through separation and comparison of the whole-cell protein complexes and membrane protein complexes between the deletion mutant strains without virulence plasmid and wild-type strains using BN-PAGE, we found that lost of virulence plasmid of Shigella will change the abundance of glutamic acid decarboxylase and ATP synthase complex.But we did not observe this phenomenon in Yersinia pestis.Then we compare the proteomic changes ofShigella and Y. pestis when their virulence plasmids were cured, to evaluate the similarity and difference in the gene regulations in these bacteria.We did a comparative proteomics analysis between the deletion mutant strains and wild-type strain of Yersinia pestis by using two-dimensional electrophoresis coupled with mass spectrometry in this study.The proteomes of wild-type and virulence plasmid cured strain of Yersinia pestis grown in 26 ℃ and 37℃were separated.A total of 24 differently expressed proteins were successfully identified from the samples of bacteria grown at 26 ℃, while 25 proteins at 37℃. Among those,7 proteins were encoded by virulence plasmid.But we did not found protein expression changes of glutamate decarboxylase and ATP synthase.Only two of these proteins were reported in the similar research performed in Shigella, suggesting the significant difference of gene regulation between the two pathogens.Further more,we use the λ-Red recombination system to knock out the encoded genes of the glutamic acid decarboxylase, whose expression had been proved to be related with the virulence plasmid of Shigella flexneri. There are two different isoforms of glutamic acid decarboxylase encoded by two paralogous genes.The glutamic acid decarboxylase single gene and double gene deletion mutant strains were successfully constructed. DNA binding proteins with affinity for the regulatory region of the GadA/B genes were also purifiedby magnetic beads, and then identified by mass spectrometry.The results revealed that protein StpA, RuvA might also control the expression of the GadA/B genes besides the known regulator H-NS.In summary, Shigella and Yersinia pestis both have T3SS, which is the most important virulence factor of intestinal and respiratory pathogens.We opened up new ideas screening virulence-related proteins in this study by comparison of protein and proteomes in deletion mutant and wild-type strains.The technical improvements and the experience gained inthis study made a foundation for depth study.