Screening And Functional Study Of The Protein-protein Interaction Network Between Mycobacterium Tuberculosis And Host

Tuberculosis(TB)which caused by mycobacterium tuberculosis(M.tb)is one of the most leading infectious chronic zoonosis that severely disturbs human health and development of the stock farming.However,there is still vast amount of challenge on the prevention and curing of the tuberculosis,many concern areas remain unclear like the molecular mechanism of M.tb's intracellular parasitism,resistance to host phagocytosis and escape from the host cell immunity system,which strictly limit the accuracy and the target selection of screening the anti-TB drug.Our study utilize a Recombination-based“library vs library”yeast two-hybrid system(RLL-Y2H),by which multi-library screening can be accomplished in a single pool without any individual treatment.This system is based on the phi C31/att integrase-mediated integration between bait and prey plasmids.After improving the system,we constructed the M.tb bait library and host cell prey library respectively.We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells.The sub-set networks between M.tb and different pathway proteins of host cells were extracted from the entire PPI network.Results are double-checked by Y2H,indirect immunofluorescence,bimolecular fluorescence complementation and co-immunoprecipitation assay.The preliminary study on the function of the interaction proteins enabled us acquire deeper understanding of the unknown molecular mechanism of M.tb resistance to host phagocytosis and escape from the host cell immunity system.Furthermore,the construction and improvement in RLL-Y2H system not only lay the foundation of M.tb and host cell interactome,but can also promote the interactome study of other organisms.It makes the study of the protein interaction network more systematically,leads to a better understanding of metabolic pathway.The detailed experimental results are shown as follows:(1)Development of a novel of RLL-Y2H screening system.To test the efficiency of this RLL-Y2H system,positive controls consisting of a well-known interacting protein pair,murine p53 and SV40 T-antigen,were inserted into the pm GBKT7 and pm GADT7 of RLL-Y2H system,respectively.Then Y187 and Y2H gold are transferred by these two plasmids for mating,we found that the inserted unit in RLL-Y2H system causes no impact on protein's expression and interaction.At last we randomly picked up the colony and done the amplification of integrated fragments,the amplicons are sent for sequencing and the result shows that the integration efficiency is100%and the sequence is completely specific,which is evident for purpose of our novel high-throughput RLL-Y2H system.(2)Construction and screening of M.tb bait library and host prey libraryThe brain,liver,kidney and lymph nodes were extracted from specific pathogen-free6–8-week-old BALB/c mice for the total RNA isolation.RNA was revers-transcripted to c DNA.We designed 374 pairs of primer that contains the homologues arm and Mme I restriction site and used the c DNA that mentioned previously as the template for the homologues recombination and result in the construction of 316 host protein ORFs were amplified,which contains autophagy-related genes,SNARE genes,TB susceptible genes,Toll like receptor signaling genes,NF-k B signaling genes,Jak/Stat Signaling:IL-6Receptor Family genes,B cell receptor genes,T Cell Receptor Signaling genes.These ORFs were cloned into the pm GADT7 vector individually with a Mme I restriction site at the end of it.Each gene in prey library was mated with Y2H gold harboring pm GBKT7 to rule out those showing sticky interaction or self-activation.The rest were pooled and used as the prey library.M.tb bait library was successfully constructed and preserved in our group,which containing a total of 542 M.tb(H37Rv)lipoprotein,membrane and secretory protein ORFs were amplified and cloned into pm GBKT7 vector individually with a Mme I restriction site at the end of it by homologous recombination.The eukaryotic expression plasmids that H37Rv protein ORF was ligated to the vector pc DNA3.1-V5/His B were used as template.Self-activation genes were excluded in the following experiments.(3)Delineation and confirmation of PPI network between M.tb and the hostThe bait library was mated with the prey library.All the colonies on SD/-AHLT selective plates were pooled for plasmid extraction and low cycle PCR amplification.A smear band representing integrated Prey-Mme I-ATTL-Mme I-Bait fragments were observed and then harvested for Mme I digestion,which generated a?110bp band comprising of the bait Mme I end,the att L linker and the prey Mme I end.These digested products were ligated with Illumina P5 and P7 adapters to generate a high-throughput sequencing library.Among 46 million raw reads,14 million reads could be mapped as M.tb-host interacting protein pairs.We identified a total of 1,438 PPIs between 82 M.tb proteins and128 host proteins.The interaction frequencies of M.tb-host protein pairs were calculated based on the number of corresponding aligned reads.The interactions with 5 or less reads in the sequencing result are excluded for further study.Meanwhile,we also excluded the interactions with 45 or more reads in the sequencing result for further study.Finally,we delineated a PPI network between M.tb proteins and host proteins containing 441 PPIs.To systematically evaluate the false positive rate,269 randomly selected protein interaction pairs were transformed into Y187 and Y2HGold yeast strains,respectively,and were mated to confirm their interaction individually.As shown in Supplementary Fig.2 and Table 5,189 out of 269(70.3%)protein interaction pairs can be confirmed.(4)Delineation of PPI sub-set networks between M.tb and different pathway proteins of host cells.The sub-set networks between M.tb and different pathway proteins of host cells were extracted from the entire PPI network,which contain the PPI sub-set network between M.tb and toll-like receptor signalling pathway proteins,T cell receptor signalling pathway proteins,NK-?B signaling pathway proteins,B cell signaling pathway proteins,Jak/Stat signaling pathway proteins,SNAARE proteins and TB susceptibility proteins of host cells.(5)Identification of the M.tb proteins interfering autophagy in host cells.Our lab mate Qili Yao used CRISPR/Cas9 system to construct the RAW 264.7 cell line,which stably express GFPph-m Cherry-LC3(G_phML in short)for autophagy assay.All of the 38 M.tb genes in this M.tb-autophagy PPI sub-network or a control(empty vector)was individually transfected into this stable cell lin.Autophagy was induced by rapamycin and then subjected to florescence assay.Of note,transfection of Rv2427c could significantly rescue the rapamycin-induced decrease of GFP/m Cherry fluorescence ratio of aggregation dots,suggesting Rv2427c can interfere with autolysosome formation.(6)The preliminary study on the function of M.tb Rv3722c.We selected uncharacterized Rv3772c from the interaction pairs for further study.We confirmed that Rv3722c interact with TRAF3 according to the results of Y2H,IFA,Bi Fc and Co-immunoprecipitation.According to the confocal microscopy,we found that Rv3722c promotes the intracellular survival of M.tb H37Ra in mice macrophage cells RAW 264.7 cells;the result of luciferase reporter gene assay indicated that Rv3722c promotes NF-?B as well as TNF-?induced NF-?B promoter activity in HEK 293T cells.Inhibiting the expression of TRAF3 in HEK 293T cells can inhibit the activation of NF-?B as well as TNF-?induced NF-?B promoter activity by Rv3722c.The further study showed that Rv3722c do not affect the ubiquitination of TRAF3,neither the degradation of IKB?nor the phosphorylation and nuclear translocation of p65.