| Plague is a severe epidemic caused by Y. pestis and has caused three pandimes in human history. The pathogenic mechanism of Y. pestis depends on its interactions with the hosts. Revealing the protein-protein interactions (PPI) between Y. pestis and its host will not only lay a significant foundation for thoroughly understanding of its pathogenic mechanism, but also provide clues to develope efficient means for plague preventing and controlling. There are three parts in this research. First, by applying a high throughput Yeast Two-Hybrid (Y2H) System, the predicted virulence-related proteins of Y. pestis were used as baits to screen human spleen cDNA library. This study aims to obtain a primary PPI network between Y. pestis and huamn and a set of important interactions, so as to provide targets for deeply revealing the pathogenic mechanism of Y. pestis. Second, we systemically analyze the protein interactions within Y. pestis T3SS using yeast two hybrid system. Our results will help us to elucidate the structure and function of T3SS. Third, to better understand the role of negative regulator LcrG on T3SS, we compared the transcriptional profiles from wild-type strain and ?lcrG mutant by using whole-genome microarrays.PART 1 Investigation of interactions between Y.pestis virulence related proteins and human proteomeWe identified 152 Y. pestis proteins (about 3.75% of the Y. pestis full genome) that might interact with human proteins according to the results of previous studies carried in our lab, including the comparative genomics of Y. pestis and Y. pseudotuberculosis, the gene expression profile under different stimuli, serum antibody profile of infected hosts, and the current progress of Y. pestis research from references. Our research adopted the ProQuest Two-Hybrid System (Invitrogen) based on the Gateway Recombination Technology, which facilitate the high throughput parallel operation. Through the gateway recombination technology, 154 bait vectors corresponding to 152 ORFs of Y. pestis proteins (two of them were fragmented) were constructed. Three of them (YPCD1.26c, YPCD1.31c and YPMT1.61c) were self-activating baits.High-throughput Y2H screening were carried out using sequential transformation method. 151 non-self-activating baits were screened against the human spleen cDNA library for at least once. The candidate clones grown on the selective plates were selected further by analyzing the auxotrophic phenotypes of reporter genes his and ade and the X-Gal assay to identify the phenotype of reporter gene lacZ, respectively. The prey-vectors form positive clones were isolated and sequenced. Total of 1087 preys were successfully sequenced. The BLAST program of the NCBI website was used to search the database, 833 clones containing the inframe inserts of full or partial ORFs were identified, which were corresponding to 185 non-redundant prey molecules. These 185 prey formed 359 interactions with 92 proteins of Y. pestis, which constituted our core data set. No known interactions reported previously were screened out in our experiment. Among these 359 interaction pairs, 60 occurred twice and 91 occurred three times or more, which represented 16.7% and 25.5% of the total PPIs number, respectively, suggesting that our screening results should be credible.Subsequently, experimental validation and bioinformatics analysis were adopted to determine the reliability of those interactions. In the experimental part, we evaluated the technical false positive by plasmid retransformation in yeast and GST pull-down assay. Plasmid retransformation assay were carried out in 375 pairs PPIs and 208 pairs shown to be positive through phenotypic identification, with a positive rate of 57.9% that is comparable with the published YTH work . We obtained 208 PPIs between 67 baits and 109 preys. Among the 208 PPIs that shown to be positive in the plasmid rescue from yeast experiment, 21 were selected for GST pull-down assay, representing 10% of the total retransformation-positive protein pairs (208 pairs). Of these 21 interaction pairs, six prey proteins were involved, and 7 interaction pairs were unable to be validated because of the inability of a prey protein to release into the supernatant of cell lysate. Of the rest 14 interaction pairs, 13 were validated positive and one negative by GST pull-down assay, resulting in a positive rate of 92.8%, indicating that the data set of 208 pairs of protein-protein interactions between Y. pestis and human protein had comparatively high reliability. With the thoroughly mining the current public database (BIND, BioGRID, DIP, GeneRIF, HPRD, IntAct, MINT and Reactome etc.), we construct a network using the data set consisting of 208 PPIs, and analyzed the characteristics of its structure and function. Network between HYT-HYT (HYT, human protein targeted by Y.pestis proteins) were also obtained by extracting HYT and their interactions from human interaction networks form existing references. These two networks were fused with each other to form a network with 176 nodes and 252 lines, representing 252 pairs of interactions involving 67 Y. pestis proteins and 109 human proteins, which including 44 pairs of interactions between 109 HYT, which was extracted form the known human protein interaction networks. The relatively high reliability of those interactions was validated by topological analysis and functional GO analysis. Among the 112 human proteins that were found to interact with Epstein-Barr virus by YTH, 26 also interacted with Y. pestis (26/112=23%), which suggested that the virus and the bacteria might use similar strategy to attack the important nodes of human protein interaction network and the host's defending system. This result was consisted with the current understanding of pathogen-host protein interaction. When processing GO biological process ID enrichment analysis, proteins involved in interspecies interactions and multi-species interactions, cell adhesion proteins, some intracellular negative regulatory proteins were significantly enriched. Processing GO molecular function ID enrichment analysis, cytoskeleton proteins, transcription factor binding proteins, integrins and proteasome proteins were significantly enriched. These proteins may play important roles in the pathogenesis of Y. pestis. Finally, we conducted predictive analysis of those interactions based on deeply investigation of related references. Interactions between Y.pestis and its host were coordinated effects produced by a series of Y. pestis and human protein interactions, and further studies are needed to define the roles of those PPIs in the process of Y. pestis infection.This study investigated the PPIs between Y. pestis and its host by high throughput screening method. We obtained a primary interaction network between Y. pestis and human proteins, which had been subjected to topological analysis and functional analysis further. The results revealed the main pattern of protein-protein interactions between Y. pestis and human and provided lots of targets for elucidating the pathogenic mechanism of Y. pestis.PART 2 Protein-Protein interactions within Type III Secretion System of Y. pestispCD1 is a common plasmid owned by three pathogenic Yersinia species. It is the essential virulence determinant of Y. pestis and encodes T3SS that can form injectisome on the surface of the bacteria and inject at least six Yops(Yersinia outer membrane proteins)into the host cell. Yop effectors usually have various protease activities, that render the pathogens'ability to inhibit the normal immune response of the host by alter the cytoskeletal structure of the hots cell, resist phagocytosis and interfere with signal transduction pathways.Identifying the interaction of among the components of T3SS is helpful for predicting and elucidating the structure and function of the Ysc injectisome. This study selected 57 genes from the pCD1 plasmid encoded-genes by excluding those related with plasmid replication and partition, and the putative transposase genes. YTH matrix technique was used to study the interactions within T3SS. Totally 19 pairs of interaction proteins were obtained in the research. Eight pairs of which were reported previously, and the rest 11 pairs hadn't been reported yet. The interactions were classified into three major types: i) interaction between the chaperon and secretory substrate; ii) interaction between secretory regulation complexes; iii) other interactions. Several putative proteins with unknown function were involved in interactions with the known T3SS proteins or another unknown function protein, indicating that they might be new members of T3SS. These results are helpful for elucidating the T3SS structure and function in the future. Further studies are needed to define the precise interaction and significance.PART 3 Study on the regulatory role of LcrG on the T3SS transcriptionYersinia T3SS is tightly regulated by environmental factors that are closely related to the life cycle of the pathogens, such as temperature, calcium and glutamine levels, pH, nutrient availability, and contact with eukaryotic cells. It is known that LcrG is a negative regulator for the secretion of Yops. At mammalian body temperature, i.e. 37℃, ?lcrG mutant consistently secretes Yops in the presence or absence of Ca2+ in vitro, accompanied by growth restriction.In this work, to better understand the roles of LcrG in the regulation of LCRS in Y. pestis, we compared the transcriptional profiles from wild-type strain and ?lcrG mutant by using whole-genome microarrays. It was found that most T3SS genes were up-regulated in the ?lcrG mutant when compared with the wild-type strain, and immunoblotting analysis also revealed that more YopM and LcrV proteins were secreted by the ?lcrG mutant. Furthermore, the virulence of the ?lcrG mutant was dramatically attenuated in both in vitro (HeLa cells and macrophage cells) and in vivo (mice systemic infection) analysis. The capability to inhibit TNF-αsecretion in macrophages was severely impaired and the LD50 in BALB/c mice was increased by approximately 600 folds. Although Yops were overexpressed in the ?lcrG mutant, the translocation of them into the eukaryotic cells was severely hampered, which was consistent with the previous report.We therefore speculate that LcrG could play a negative regulatory role not only in the Yop secretion but also in the transcription of T3SS genes, and this function is possibly exerted through indirect mechanisms. |
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