Study On Protein-Protein Interactions Between Yersinia Pestis And Murine Macrophage

Yersinia pestis is the aetiological agent of plague, a disease of human that has resulted in three worldwide pandemics in history. Interactions between Yersinia pestis and host macrophages, a key component of innate immunity system, are very importanct and essential parts in pathogenesis of this pathogen. Here, we attempted to get more understandings of pathogenesis of Yersinia pestis through the investigation of protein-protein interactions between the bacterium and the murine macrophage. A protein chip method was established and optimized, and based on this technique a protein chip containing 149 virulence related proteins of Yersinia pestis was used to screen the protein-protein interactions occurred between proteins from the pathogen and the J774A.1 cell. A total of 17 proteins were selected out, among which LcrG, YscW and YscB are known to be components of low calcium stimulon that is very important for full virulence of Yersinia pestis, and katY is a catalase/peroxidase. All the other proteins are putative proteins encoded by bacteriophage genes or putative unknown function proteins. Indirect immunofluorescence and confocal analysis confirmed that the 8 proteins, namely LcrG, YscB, YscW, YpPCP1.06, YpMT1.25ac, YpMT1.24c, YPO2090 and YPO2108 that have the most intensive signals in protein chip screening, show the reaction activity with J774A.1 cells, attesting that the protein chip screening results are deserved to be trusted. Mutant strains with the corresponding genes knocked-out were constructed by λ Red homologous recombination method, comparisons of the cytotoxicity to J774A. 1 and LD₅₀ in BALB/c mouse between the mutants and the wild type Yersinia pestis demonstrate that LcrG and YscB mutants are much less virulent. To identify the target proteins of the virulent proteins, a cDNA library of J774A. 1 was constructed in pTRG plasmid and then screened using LcrG and YscW as bait proteins. It was shown that Rpl39 and Trx-2 can interact with LcrG, and Rnfl49 with YscW. Trx-2 is a thioredoxin, and Rnfl49 is a putative protein related to ubiquitin ligase containing a RING domain, both of them are very important in fundamental cellular functions and related with cell signaling and immune modulation. We will go on studying the protein-protein...