Interaction Mechanism Of Listeria Monocytogenes Phospholipase PlcB With Mitochondrial Inner Membrane Protein Mic60

Listeria monocytogenes(L.monocytogenes)is a zoonotic gram-positive bacterium and a foodborne microorganism.It enters the human body from the digestive tract through contaminated food,can cross the intestinal barrier and enter the blood or act on the corresponding target organs.As an intracellular parasite,L.monocytogenes have a number of key virulence factors involved in the process of infecting cells.Phospholipase PlcB,as one of the important virulence proteins,plays an important role in Listeria infection and phagosome escape,but whether it has the function similar to LLO to manipulate the host-related biological processes is still unclear.Based on this,this study used cell biology and molecular biology methods such as yeast two-hybrid,protein interaction positioning,and laser confocal to screen the host proteins that interact with the virulence factor PlcB.By exploring the molecular mechanism of the interaction between the phospholipase PlcB and the host protein,it lays a foundation for further understanding the mechanism of Listeria escaping the host's natural immunity.1.Construction of bait vector pGBKT7-PlcB and detection of toxicity and self-activation.In the yeast two-hybrid test,Listeria monocytogenes genome was used as a template,and the plc B gene was amplified by high-fidelity PCR and colony PCR was used to verify positive clones.The results showed that the Smart View Pro gel imaging system showed clear and bright bands on the 1%agarose gel,which was consistent with the expected band of 795 bp and the size of the positive control band.The above results indicate the bait plasmid pGBKT7-BD-PlcB was successfully constructed;after the bait plasmid was constructed,the self-activation test of yeast cells was carried out.The results showed that the yeast strain containing the bait plasmid pGBKT7-BD-PlcB and the empty vector pGBKT7 could grow in SDO/X-?-Gal and the colony does not change blue,and there is no growth in SDO/X-?-Gal/Aba.The results show that the bait plasmid pGBKT7-BD-PlcB did not auto-activate in yeast;The results showed that the yeast containing pGBKT7-BD-PlcB and pGBKT7 can grow normally in SDO medium and there is no significant difference in the number and size of colonies,indicating that the bait plasmid pGBKT7-BD-PlcB is in yeast Non-toxic;Finally,the protein expression test of the bait plasmid pGBKT7-BD-PlcB was carried out.The results showed that the protein band expressed by the yeast strain containing the bait plasmid pGBKT7-BD-PlcB was in line with the expected protein size of51.3 k Da and the positive control band was clear.The blank and negative controls have no bands,indicating that the bait plasmid pGBKT7-BD-PlcB can normally express protein in yeast.2.Yeast two-hybrid screening of host proteins that interact with phospholipase PlcBIn this chapter,the quality of the human c DNA library was tested first,and the results showed that the titer of the human c DNA library was 3.73×10~8 CFU/m L,indicating that the library meets the criteria for being a yeast two-hybrid library;Next,the successfully constructed bait plasmid pGBKT7-BD-PlcB was hybridized with the human c DNA library,and through the first round of screening of strictly defective media,a total of 348 blue clones were grown from DDO/X solid media.Through the second and third rounds of screening,341 blue positive clones were grown in QDO/X/A solid medium,which were sequenced and compared to obtain 4 candidate positive clones;During the Anaplerosis assay of bait plasmid and candidate positive plasmid,the results showed that all 4 candidate positive clones were successfully supplemented and verified,indicating that they passed the yeast two-hybrid test.A total of4 host proteins were screened to interact with phospholipase PlcB.3.Study on the interaction mechanism between PlcB and mitochondrial inner membrane protein Mic60Through the method of homologous recombination,the full-length PlcB and Mic60 recombinant plasmids were constructed.The results showed that in the colony PCR test,there are clear and bright bands in the lanes on 1%agarose gel,the size of which is consistent with the expected bands of 795 bp and 1395 bp.That is,the recombinant plasmids p CMV-Myc-PlcB and p CMV-HA-Mic60 were successfully constructed;in the immunoprecipitation test,the expression of exogenous Mic60 and PlcB protein and?-Tubulin internal reference protein in the input sample is normal,the exogenous Mic60has a band in the IP sample and the control IgG has no band.The results indicate that the exogenous Mic60 and PlcB protein can interact directly in vitro;in the indirect immunofluorescence test.Exogenous Mic60 exhibites green fluorescence and PlcB protein shows red fluorescence,and the two fluorescences overlapped each other.The results showed that the exogenous Mic60 and PlcB protein co-localized in the cell.Next,the key domains of phospholipase PlcB and Mic60 are truncated,and the results of co-immunoprecipitation experiments show that phospholipase PlcB has multiple key domains that interact with the inner mitochondrial membrane protein Mic60,and Mic60 has multiple key domains that interact with PlcB,and Mic60(187-296aa)cannot interact with PlcB.In summary,this study used yeast two-hybrid and immunoprecipitation methods to screen for the first time and determined that Listeria virulence protein PlcB can interact with Mic60;phospholipase PlcB has multiple key domains that interact with the inner mitochondrial membrane protein Mic60,and Mic60 has multiple key domains that interact with PlcB,and Mic60(187-296aa)cannot interact with PlcB.This provides a key clue to further elucidate the function and molecular basis of PlcB in the process of Listeria infection regulating mitochondrial structure changes.