The Study Of Interaction Of Anaplasma Phagocytophilum Msp2、Ats-1Proteins And Host Cell Potential Protein(s)

Human granulocytic anaplasmosis(HGA) is a emerging infectious disease caused by Anaplasmaphagocytophilum(A. phagocytophilum),whcih is an obligate intracellular parasitic bacterium and a smallGram-negative pleomorphic coccus. The bacterium can exist in several species of mammals and Ixodesticks. Human being granulocytess is one of the most important target cell for survival, reproduction for A.phagocytophilum. HGA is characterized by fever, headache, myalgia, leucopenia, thrombocytopenia, andincreased serum aminotransferase liver enzyme activity. Clinical signs and/or symptoms of HGA appearmost similarity to those of certain viral infection. HGA is commonly misdiagnosed and may potentiallyresult in fatal complications. Clinical signs and/or symptoms of granulocytic anaplasmosis in domesticanimals, such as horse, dog and ruminants was basically similar to that of HGA. It was reported that A.phagocytophilum replicates in inclusions/morulae of human granulocytes, and interferes withinclusions/morulae trafficking to avoid fusion of lysosomes. A. phagocytophilum infects and subvertsgranulocytes innate defenses in order to survive and replicate inside its hostile host cells. It is important forA. phagocytophilum to utilize itself some proteins, such as surface proteins, secreted proteins, membraneproteins of inclusions, to establish a right niche for its survival and replication during infection. Currently,the molecular pathogenesis of the pathogen is not fully understood. In recent years, the increasing cases ofHGA have caused widespread concern in many countries and areas of the world. According to the studieson epidemiology, HGA cases, which were caused by A. phagocytophilum isolate from China, appearserious clinical outcomes. Bacteria isolate were carried out and identified from HGA patients. Therefore, itis interesting to detect anaplasmosis in China and investigate surface proteins, secreted proteins from A.phagocytophilum. In this study, the main research works follow as:1. In order to know prevalent characterization of Anaplasma in Qujing of Yunnan and Lvliang ofShanxi, we collected and extract genome DNA from spleens of rodents, and then carried out nested PCR totest Anaplasm in those spleens samples, suspected positive PCR products were send to sequencing, andsequencing results were analyzed by NCBI website. The results showed that dominant rodent species wereRattus norvegicus and Rattus tanezumi in Qujing Yunnan and was Apodemus agrarius in Lvliang Shanxi.Tested lots of bacteria species in Yunnan included Rickettsia, Ehrlichia, Anaplasma, Wolbachia etc, totalpositive rate of tested bacteria was1.7%. Tested bacteria species in Shanxi only included Ehrlichiachaffeensis, total positive rate was5.9%. It was a conclusion that rodents may be natural reservoirs ofmembers of Rickettsiales in two areas above.2. The cDNA library construction from THP-1cells takes place directly in S. cerevisiae strain Y187.Total RNA extracted from THP-1cells was used to generate cDNA by reverse transcription, usingSMART technology. cDNA and linearized pGADT7-Rec vector were cotransformed into S. cerevisiaeStrain Y187and were reassembled by potent homologous recombination machinery in vivo. cDNA inserts size was initially analyzed by PCR with specific primers. The results showed that cDNA library wassuccessfully generated in the work, the library technical indicators included1.02×106for its capacity, the96.1%for recombinant rate,100-3000bp for size of its inserts, most of inserts is between500-2000bp.3. For purpose of screening target proteins of THP-1cell interaction with Msp2、Ats-1proteins from A.phagocytophilum, we chose surface proteins Msp2and secreted proteins Ats-1of A. phagocytophilumisolate as the proteins investigated. Bait inserts msp2, ats-1from genome DNA of A. phagocytophilumisolate were amplified, sequenced and subcloned into linear pGBKT7DNA-BD vector obtained bydigestion with NdeI and BamHI to generate bait plasmids pGBKT7-msp2, pGBKT7-ats-1, which wereintroduced into S. cerevisiae strain Y2HGold. The results showed that all bait constructs does notautonomously activate the reporter genes in Y2HGold and is not toxic to the yeast cells. Through yeasttwo-hybrid systems, we screened THP-1potential proteins interation with Msp2protein have7proteinsincluding Homo sapiens NADH dehydrogenase (ubiquinone)1alpha subcomplex13(NDUFA13), Homosapiens ZFP36ring finger protein-like2(ZFP36L2), Homo sapiens ribosomal protein L11(RPL11), Homosapiens prothymosin, alpha (PTMA), Homo sapiens chromosome19open reading frame10(C19orf10),Homo sapiens cathepsin G (CTSG), Homo sapiens ribosomal protein S25(RPS25); THP-1potentialproteins interation with Ats-1protein have also7proteins including Homo sapiens eukaryotic translationelongation factor1alpha1(EEF1A1), Homo sapiens TIMP metallopeptidase inhibitor1(TIMP1), Homosapiens ZFP36ring finger protein-like2(ZFP36L2), Homo sapiens lectin, galactoside-binding, soluble,1(LGALS1), Homo sapiens prothymosin, alpha (PTMA), Homo sapiens Williams Beuren syndromechromosome region22(WBSCR22), Homo sapiens attractin (ATRN).4. Genuine interactions between bait proteins Msp2, Ats-1and THP-1potential proteins wereconfirmed via co-immunoprecipitation, western blot assays to eliminate false positive interaction. Surfaceprotein Msp2from A. phagocytophilum isolate interact with CTSG, ZFP36L2proteins of THP-1cells,secreted proteinAts-1from the bacterium interaction with LGALS1, TIMP1proteins of THP-1cells.In summary, The results of pathogen Anaplasma tested in Quqing Yunnan and Lvliang Shanxiconfirmed existence of many members of Rickettsiales in rodents, it is indispensable to monitor, preventionand control HGA in two areas above. a few of potential proteins from THP-1cells were screened andverified interaction with bait proteins Msp2, Ats-1from A. phagocytophilum isolate, respectively. Theseprotein-protein interactions may extremely effect on invasion, survival and replication of A.phagocytophilum inside its hostile host cells. The datum on the study are useful to understand the molecularpathogenesis of A. phagocytophilum isolate, to screen new therapy and prevention candidates to HGA.