Global Identification Of Mycobacterium Tuberculosis Serine/Threonine Protein Kinase Interaction Proteins And Function Analysis

Mycobacterium tuberculosis(Mtb)is the causative agent of tuberculosis,the leading cause of death among all infectious diseases.One of the most significant reasons for the slow progress in discovering and characterizing appropriate vaccine candidates and drug targets is the general lack of knowledge about basic biological pathways in Mtb.It is well known that phosphorylation is widely recognized to play a significant role in signal transduction in many pathways in both eukaryotes and prokaryotes,no exception for Mtb.There are 11 eukaryotic-like serine/threonine protein kinases(STPKs)in Mtb,which are thought to play pivotal roles in cell growth,division and pathogenesis.However,their underlying mechanisms remain largely uncharacterized.For now,only a few of STPK regulated proteins have been found,which are far from enough for understanding the complex regulation role of STPK.Thus,there is an urgent need to discover STPK interacted proteins with a systematic way.Proteome microarray is a high-throughput,direct,simple and fast technology for studying protein-protein interactions.Based on proteome microarray,three related studies have been conducted.1,global identification of STPK interacting proteins in Mtb.2,functional analysis of one candidated interaction,i.e.,PknB and RmlA.3,global identification of PknG interacting proteins in human.Firstly,using Mtb proteome microarray,we have globally identified the binding proteins in Mtb for all of the STPKs,and constructed the STPK-Protein Interaction(KPI)map which includes 492 binding proteins and 1,027 interactions.Bioinformatics analysis showed that the interacting proteins reflect diverse functions,including roles in two-component system,transcription,protein degradation,and cell wall integrity.Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan(PG)biosynthesis,e.g.,MurC.The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb,thus facilitating drug development and effective control of Mtb.Secondly,based on our first part project,we discovered a new protein RmlA,a crucial L-rhamnose biosynthesis enzyme,which is a substrate of PknB in Mtb.MS analysis revealed that the phosphorylation sites of RmlA occurred at Thr-12,Thr-54 and Thr-197,and we also found that Thr-12 is located close to the catalytic triad of RmlA.We hypothesized that RmlA phosphorylation could affect cell wall formation and virulence.To this end,biochemical and phenotypic analysis of two RmlA mutants,T12A/T12 D,indicated diminished RmlA activity and cell wall formation.Moreover,virulence of RmlA phosphomimetic mutant was attenuated in a macrophage model.Overall,these results provide evidence for the role of PknB-dependent RmlA phosphorylation in regulating cell wall formation in Mtb,with significant implications for pathogenicity of this important human pathogen.Lastly,a human proteome microarray was applied to globally identify the host proteins that PknG bind,a list of PknG interactors were successfully discovered,which belong to a variety of functional categories.Among the newly identified interactors,human protein CypA strongly binds to PknG.This interaction was validated both in vitro and in vivo.Functional study showed that PknG could reduce protein level of CypA through phosphorylation,and consequently,inhibits inflammatory response through down-regulation of NF-κB and ERK1/2 pathways.Phenotypically,overexpression of PknG reduced the level of cytokines and promoted the survival of Mycobacterium smegmatis(Msm)in macrophages.Besides the specific interaction of PknG-CypA,the PknG interactors identified in this study could serve as a solid base for systematical understanding of the critical role that PknG plays in Mtb-host interaction.Taken together,in this study,applying the Mtb and human proteome microarray,we have systematically identified the STPK interacting proteins in Mtb and host and also performed functional studies for two newly identified protein-protein interactions,i.e.,PknB-RmlA and PknG-CypA.Meanwhile,this study also expands the knowledge for understanding the role of STPK in Mtb and pathogen-host interaction.We believe that this study will shed light on further research for STPK phosphorylation pathway and drug discovery.