Identification Of Anaplasma Phagocytophilum Type IV Secretion Substrates And Novel Diagnostic Antigen For Human Granulocytic Anaplasmosis

Objective:Type IV secretion system(T4SS) plays important roles in the pathogenesis of bacterial infectious diseases by translocation of multiple effector proteins from bacteria into host cells, dysregulating diverse target cell functions, thereby leading to disease development. In order to elucidate the pathogenesis of human granulocytic anaplasmosis(HGA), bioinformatics analysis was employed to identify T4 SS substrates of Anaplasma phagocytophilum(AP), the etiological agent of HGA. In addition, the identification of novel diagnostic antigen for HGA was conducted with the aim to improve the sensitivity of serological test for HGA.Methods:I. Screening, recombinant protein expression and antibody preparation for AP T4 SS effector candidates.1. Screening for AP T4 SS effector candidates. According to the common features shared among T4 SS effectors, the 610 hypothetical proteins encoded by AP genome were screened with bioinformatics analysis as effector candidates.2. Recombinant protein expression. Eight effector candidates were chosen to study in this thesis. DNA fragments encoding seven effector candidates(APH0177, APH0248, APH0261, APH0615, APH0653, APH0812 and APH1127) were amplified by PCR using AP genomic DNA as template, and cloned into prokaryotic expression vector p ET28a(+), along with the gene of known T4 SS effector Ats-1. DNA fragments encoding two segments of APH0215(90th-144 th amino acids, and 77th-144 th amino acids) were amplified by PCR and cloned into prokaryotic expression vector p GEX-4T-1. To facilitate the purification, the nucleotide sequence encoding 6×His tag was incorporated into the reverse PCR primer using for amplification of aph0215. The recombinant plasmids were transformed into Escherichia coli BL21(DE3) competent cells for expression and purification.3. Antibody preparation. The recombinant proteins purified by nickel affinity chromatography were used to immunize ICR mice to prepare polyclonal antibody. The antibody specificity were determined by western blot analysis.4. Affinity purification of anti-APH0215 antibody. A DNA fragment encoding APH0215(77-144) was cloned into the prokaryotic expression vector p MAL-c5 x to express recombinant fusion protein MBP-APH0215. After purification, MBP-APH0215 was conjugated to NHS-resin for antibody affinity purification.II. Amino acid sequence analysis and eukaryotic expression of AP T4 SS effector candidate APH0215.1. Analysis of the properties and prediction of subcellular localization of APH0215 by bioinformatics.2. Molecular cloning. DNA fragment encoding full-length APH0215 was amplified by PCR, and cloned into the eukaryotic expression vector p EGFP-N1.3. Transfection and microscopy. He La cells were transfected with the plasmid expressing APH0215-GFP. The distribution of APH0215-GFP in transfected cells was observed under fluorescence microscope. The impact on cell morphology by APH0215-GFP was also investigated under fluorescence microscope.III. Identification of novel diagnostic antigens for HGA.1. Determination of antigenicity of the purified recombinant AP proteins(APH0177-APH1127, and Ats-1) by western blot analysis.2. Comparison of the immune reaction of 100 serum samples against AP immune dominant outer membrane protein P44 and a selected AP protein(the protein with strong antigenicity was selected according to the previous experiment result).Results:I. Screening, recombinant protein expression and antibody preparation for AP T4 SS effector candidates.1. By bioinformatics analysis, 35 AP hypothetical proteins were identified as T4 SS effector candidates, including APH0177, APH0215, APH0248, APH0261, APH0615, APH0653, APH0812 and APH1127.2. DNA fragments encoding seven T4 SS effector candidates(APH0177, APH0248, APH0261, APH0615, APH0653, APH0812 and APH1127), and Ats-1 were cloned into p ET28a(+). Two DNA fragments encoding one T4 SS effector candidate APH0215 were cloned into p GEX-4T-1. All recombinant plasmids were transformed into E. coli BL21(DE3). Recombinant proteins were expressed, and separated by SDS-PAGE. The induced protein bands with expected molecular sizes were detected after gel staining.3. The recombinant proteins were purified by nickel affinity chromatography. SDS-PAGE showed they all have high purity. The titer of the sera from ICR mice immunized with purified recombinant proteins were determined by western blot analysis, showing that all sera have high titer.4. MBP-APH0215 was expressed, purified, and coupled to agarose resin. Monospecific anti-APH0215 antibody was prepared by affinity purification with immobilized MBP-APH0215.II. Amino acid sequence analysis and eukaryotic expression of AP T4 SS effector candidate APH0215.1. The bioinformatics analysis of APH0215 amino acid sequence was performed, and its subcellular location in host cells after translocation was predicated, suggesting that APH0215 does not have signal peptide and transmembrane, and most likely localizes to lysosomes.2. The eukaryotic plasmid expressing APH0215-GFP was constructed, and transfected into He La cells. Green punctate was found in the cytoplasm of APH0215-GFP-expressing He La cells under fluorescence microscope and the morphology of transfected cells changed apparently.III. Identification of novel diagnostic antigens for HGA.1. Western blot analysis showed that APH1127, APH0812 and APH0653 strongly reacted with AP positive sera.2. Western blot analysis showed that the same serum sample reacted with P44 and APH0812 at different degrees.Conclusions:1. The multiple AP T4 SS effector candidates were identified by bioinformatics analysis, and polyclonal antibodies against these effector candidates were prepared, laying the foundation for characterization of these effector candidates.2. The intracellular punctuate distribution of APH0215 in transfected cells and its effect on the morphology of He La cells indicated that APH0215 may play a role in pathogenesis of HGA by interference of host cell function.3. The strong reaction of APH0812 with AP positive sera and the differential immune reaction of clinical serum samples against P44 and APH812 indicated APH0812 may improve the sensitivity of the serological test for HGA when using together with P44 as antigens.