| Yersinia pestis, the causative agent of plague, belongs to the family Enterobacteriaceae, genus Yersinia. It is a Gram-negative intracellular bacterium, mainly spreads among reservoir hosts bited by infected flea and sometimes transmits to humans contact with skin tissue of infected animals or subject to bite by flea carrying the bacterium. Patients infected Y.pestis can display symptoms of bubonic, septicemic or pneumonic plague. Many virulent factors contribute to pathogenesis of Y.pestis, and one of the most important is T3SS(Type III Secretion System), a protein delivery apparatus which plays pivotal roles during infection in alternating the cytoskeleton structure of, disrupting the phagocytosis by, inhibiting cytokines production by, and inducing apoptosis of host cells. Through T3SS, Y.pestis injects into host cells at least six effectors, including YpkA, YopH, YopJ, YopM, YopE and YopT, which function as different biochemical features and act on their own targets to promote the infection progress. One of them, YpkA, is the first eukaryotic-like Serine/Threonine kinase identified from pathogenic bacteria. It consists of 732 amino acids and the molecular weight is 82kD. It has several functional domains, including the N-terminal signal peptide, the Ser/Thr kinase domain, and the Rho GDI domain followed by an actin-binding domain. When delivered into host cells, YpkA partly localizes at the cytoplasmic face of the plasma membrane and could induce a rounding up of host cells without detachment. The GDI domain of YpkA mimics the host guanidine nucleotide dissociation inhibitor(GDI), to bind to RhoA/Rac1 and inhibit their nucleotide exchange, resulting in inactivation of RhoA/Rac1 and disruption of actin cytoskeleton. Although there are some researches about the function of GDI domain YpkA, the targets and mechanisms of its kinase remain relatively unknown.In this study, applying a Yeast Two Hybrid(Y2H) system, we utilize YpkA as the bait to screening human kidney cDNA library and acquire three prey clones targeting to VASP, indicating the interaction between YpkA and VASP. VASP(Vasodilator-Stimulated Phosphoprotein), a member of Ena/VASP family, is a regulator of actin dynamics, playing key roles in actin ploymerization, modulating formation of lamellipodia and filopodia and maintaining morphology and behavior of cells. Functions of VASP are regulated by phosphorylation and it has three phosphorylating sites, S157, S239 and S278, which could be phospholylated in vivo by PKA, PKG and AMPK, respetively. The phosphorylation of VASP regulates its interactions with partners, and further influences the function of itself and downstream moleculars, resulting in affecting actin assembly, stress fiber formation and phagocytic ability of cells.Based on all of these, from the biochemical activity, localization features and known fucntions, I think the interaction between YpkA and VASP indeed exist, not only physically, but also functionally. Therefore, we apply several methods, such as GST Pull down, co-immunoprecipiation, and fluoresecent co-localization, to indenifying their interation in vitro and in vivo. Then we analyze the domain mediating binding of YpkA to VASP, and found that the kinase domain is necessary for its binding to VASP, and either the GDI domain or actin binding domain is also required. In order to investigating the function of kinase domain, we constuct a mutant lacking the GDI and retaining all other domains, and call it YpkA-1908. Next, we study the effect of YpkA on VASP phosphorylation and find that YpkA could induce VASP phosphorylation at S157 and the kinase activity is essential for this ability. Together with experiments about Reporter Gene Activity Assay, stress fiber formation and the phagocytosis ability of host cells, we conclude that the kinase domain of YpkA, through binding to and phosphorylating VASP, inhibit in vivo actin polymerization, actin stress fiber formation and the phagocytic ability of cells. In summery, this study presents a novel molecular target of YpkA and elucidates the important roles of their interaction on disrupting the function of host cells, which promotes our understanding of molecular mechanisms of Y.pestis pathogenesis and indicates a new therapeutic target for our combating with plague.Another, our lab has apply Y2H to screen the Protein-Protein Interaction(PPI) between Y.pestis and Homo sapienis and obtained 208 PPIs between 67 Y.pestis proteins and 109 human proteins. Using the data set of 208 PPIs, we construct a PPI network between these two species. Although Y2H has been widely utilized as one of the two powerful tools to screening intraspecies or interspecies PPI and scientists from all over the world establish many databases about PPI on the basis of data from Y2H, due to its intrinsic defect, Y2H may result in false-positive and false-negative PPIs. In order to precluding the possibility of false-positive results, I use GST Pull down to identify the PPIs acquired form Y2H, and found that, of 42 pairs identified, 37 pairs is positive, which demonstrates our data from Y2H is trustful. Additionally, in the PPI network, we find many proteins from Y.pestis interact with human proteins relating to NF-κB and MAPK signaling pathways. Therefore, I apply Reporter Gene Activity Assay to identify the effect of Y.pestis proteins on this two signaling pathways. Our results show that some gene of Y.pestis indeed affect the NF-κB and MAPK signaling pathways, which may suggest interactions between these Y.pestis proteins and their corresponding human proteins. From physical and functional identification with GST Pull down and Reporter Gene Activity Assay, we confirm that our Y2H results is highly reliable and can be used to further analysis of network construction and features. Analysis of PPI network between Y.pestis and human help us to understanding pathogenetic mechanism of Y.pestis and the response of human to bacteiral infection, which would help our searching for better therapeutic methods and vaccines against Y.pestis infection.
|
PDF Full Text Request