Research Projects

Pseudomonas aeruginosa, gram-negative opportunistic pathogen is major cause for nosocomial infection and contributes to increased mortality in hospitalized and immunocompromised individuals. Infection by P. aeruginosa are either acute or chronic and causes several diseases through extension of infection to different sites including burn, respiratory tract, blood stream and surgical site. Several virulence factors like LPS, proteases, lipases and toxins contribute to the pathogenicity of P. aeruginosa. Further P. aeruginosa express pyocyanin which inhibits innate immune cells including neutrophils, T and B lymphocytes and monocytes. Hence understanding the biology of immune cells towards P. aeruginosa infection becomes subject of interest at the global level. Several studies have reported the mechanism of P. aeruginosa induced innate immunity in multiple cell types. During pulmonary infection, lung epithelial cells upon encountering P. aeruginosa recruit several hematopoietic cells to site of infection. These recruited cells are further encountered by P. aeruginosa and execute cell type specific functions. Megakaryocyte was reported to be one of the predominant hematopoietic cells recruited by lungs epithelial cells. However, there was no report on the response of megakaryocytes to P. aeruginosa infection. Hence, in our earlier study we reported that P. aeruginosa infection regulates MAPK signaling pathway and importin for translocation of NFκB and JNK to nucleus and induce cytotoxicity in megakaryocyte. To understand further, the response of megakaryocyte to P. aeruginosa infection, in our pilot studies of global RNA sequencing and quantitative RT-PCR validation, we observed Copine 5 and Vasorin as highly upregulated proteins in P. aeruginosa LPS and ExoA treated megakaryocytes. Interestingly, the involvement of these proteins during bacterial infections are completely novel and their functions are not known. Furthermore, the key transcription factors that are activated for the upregulation of Copine 5 and Vasorin during bacterial infection are not known. Hence, based on our pilot studies and bioinformatics prediction we hypothesis that megakaryocyte execute cell type specific innate signaling mechanism and functions during P. aeruginosa infection in lungs. The hypothesis to be tested in this grant are 1) P. aeruginosa infection induces activation of transcription factors (MIF1 and ADR1, KID3) to upregulate Copine 5 and Vasorin in megakaryocyte. 2) During P. aeruginosa infection increased expression of Copine 5 and Vasorin are highly essential for increased megakaryocyte proliferation, survival, and release of antibacterial factors to execute innate immunity. Hence, this proposal will identify the Key transcription factors, interactive proteins and functions of Copine 5 and Vasorin using DNA pull down assay, Immunoprecipitation, CHIP assay, LC-MS/MS, phosphoflow, immunoblot and cell differentiation assay. Overall, the newly identified transcription factors, Copine 5 and Vasorin and its interactive protein is highly helpful for early diagnosis and to treat the nosocomial infection which in turn contributes to reduced mortality in hospitalized and immunocompromised individuals.