The Interaction And Functional Study Of Mycobacterium Tuberculosis (Mtb) Protein Kinase G And Human Protein PPIA

Tuberculosis is a worldwide spread epidemic.There is almost one-third of the world population carrying the latent tuberculosis.Mycobacterium tuberculosis(Mtb)is the main pathogen causing the disease.Its latency and ability to survive in the host is one of the reasons why it is difficult to eradicate the disease.Mtb has 11 eukaryotic-like serine/threonine protein kinases,only 2 of them are soluble proteins.Among them,protein kinase G(PknG)is a soluble protein with critical function which can modify the inner environment of Mtb and can inhibit the maturation of phagosome and its fusion with the lysosome,providing Mtb's latency and survival in the host.Although PknG is a crucial kinase of Mtb,its signaling pathways and functions in the human body are still unclear and needed to be further studied.To better understand PknG's function and its network in the host,in this research,I used the human proteome microarray constructed of 19191 proteins to globally study the proteins interacting with PknG and have found 38 interactors that are potentially related to PknG.Bioinformatics analysis showed that the interacting proteins are involved in various biological pathways such as protein folding,transportation,carbohydrate biosynthesis and signaling transduction.Based on the microarray results,I observed the human protein PPIA has strong interaction signal with PknG.Protein PPIA is an important regulator in human inflammatory diseases.Its overexpression can activate NF-?B and ERK1/2 pathways in inflammatory responses.In this study,I further validated the interaction between PknG and PPIA by Biolayer Interferometry,Co-IP and Mass Spectrometry.In vitro phosphorylation assay validated that PPIA can be phosphorylated by PknG on Thr107.In addition,PknG can significantly reduce the protein expression level of PPIA in HEK293 T cells as well as its induction of inflammatory response through NF-?B and ERK1/2 pathways.To further study the influence of PPIA on the survival of M.sm in the macrophage,I investigated its role in the M.sm?PknG infection of Phorbol-12-myristate-13-acetate stimulated macrophage differentiation of THP-1 cells overexpressing PPIA by lentivirus transfection.Finally,I found that overexpressing PPIA in THP-1 cells upregulated the mRNA level of cytokines such as TNF-? and IL-4.Meanwhile,the survival of M.sm ? PknG in the differentiated macrophages of THP-1 cells overexpressing PPIA was also decreased.The results indicated that PknG interacts with PPIA through phosphorylation and its downregulation of PPIA suppresses the activation of inflammatory response through NF-?B and ERK1/2 pathways which may contribute to Mtb's survival in the host.