Functional Characterization Of X-prolyl Aminopeptidase From Toxoplasma Gondii RH

Toxoplasma gondii is an obligate intracellular, parasitic protozoan that causes the disease Toxoplasmosis. Found worldwide, Toxoplasma gondii is capable of infecting virtually all warm-blooded animals, which can cause severe consequences in animals and humans. hi humans, T. gondii is one of the most common parasites in developed countries; serological studies estimate that 25% of the global population has been exposed to and may be chronically infected with T. gondii. Athough infection rates differ significantly from country to country. Toxoplasma gondii infections often have relatively asymptomatic because their immune system usually allows the parasite coexisting in organism to maintain homeostasis. However, this pathogen can cause cytopathogenic induces host responses and indirectly damages the immunity system, when existence at a disadvantage healthy systems. As everyone knows Toxoplasma infection may result in severe immunosuppressed and neuropsychiatric diseases. The parasite is also often gennerate associated with other disease, HIV-infected can caused symptoms that include fever, headache, Proteases encoded by T. gondii could be possible targets to develop chemotherapeutic agents to control this disease. Aminopeptidases are exopeptidases which hydrolyze substrates derived from the protein degradation pathway. In addition, they also contribute to a wide range biological processes, including cell skeleton assembly, protein maturation and modulation of gene expression in vivo. Currently, there is no an effective candidate vaccine prevent to Toxoplasma infection. Most healthy people recover from toxoplasmosis without treatment. However, aminopeptidases P in T. gondii have no received much attention to date. We have evaluated the potential of TgAPP phenotypic in T. gondii by CRISP/Cas9 method. Here, we success expressed X-prolyl aminopeptidase in T.gondii and analyzed the chararacteristic of recombinant protein in enzymatic activity. We preliminary explored the function of T. gondii X-prolyl aminopeptidase by CRISP/Cas9 knockout method. Therefore, we expect that APP would be expand the knowledge of aminopeptidases family of biological processes in T. gondii.1. Bioinformatics analysis of APP gene and protein in Toxoplasma gondii RH strain.The RH strain of T. gondii was passaged as tachyzoites in monolayers of vero cells. The cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and penicillin-streptomycin at 37℃ in 5% CO2 environment. To purify T. gondii tachyzoites, the host cells were passed through a 27-gauge needle and strained through filters with 5.0μm pores. TgAPP specific primers were designed based on the TgAPP sequence in ToxoDB (accession number, TGGT1261600). respectively. The full length TgAPP cDNA was amplified by using the RT-PCR kit and cloned into thepMD18-T vector. The GENETYX version 7.0 software and BLAST search at the NCBI database were used for nucleotide acid sequence analyses. Then the protein sequence was sent for analysis with the NCBI/BLAST program. Comparison of the translated TGGT1261600 cDNA sequence with the primary sequences of APP from other organisms was performed using CLUSTALX. A phylogenetic tree was generated from homologs of the full-length APP amino acid sequences using MEGA6 software. The SIGN ALP 3.0 server was used to search for signal peptide sequences.2. Indirect immunofluorescence assay for APP protein in Toxoplasma gondiiX-prolyl aminopeptidases are metalloproteases of the M24 family, also called aminopeptidases P (APP) is a peptidase that specifically removes proline in the penultimate N-terminal position. The metalloproteinase display common pita-bread fold structural features, the X site is any aminoacyl residue. The X-Pro containing peptides are not easily accommodated in the active sites of broad specificity aminopeptidases, APP homologs exhibit high specificity for proline in the second position of the substrate and catalyze the hydrolysis of the X-Pro amide bond. APP is a multifunctional protease that plays an important role in biosynthesis and degradation of collagen. Aminopeptidases P has been characterized from diverse sources including bacteria, parasite and tissues from several mammalian species. A knockout vector targeting TgAPP, designated pGCD-TgAPP was first constructed. The backbone sequence except the single guide RNA (gRNA) cassette was modified from pSAGl::CAS9-U6::sgUPRT and self-ligated with Pme I. Then a DHFR cassette was inserted into the Kpn I site. Finally, a gRNA cassette targeting TgAPP under the regulation of the TgU6 promoter was cloned into the Pme I site. Transfection of T. gondii tachyzoites was performed by electroporation as previously described. The pGCD-APP vector was used to prepare ATgAPP parasites and the vector without gRNA cassette was used to prepare control parasites expressing GFP. Selection based on pyrimethamine was performed as described earlier. The intracellular parasites were stained with anti-TgAPP polyclonal mouse antibodies and observed through a confocal laser scanning microscope. The results revealed a diffuse staining characteristic of TgAPP localization in the parasite cytosol. It has been suggested that APP is important for the maturation and degradation of peptide hormones, neuropeptides, and tachykinins。3. Prokaryotic and eukaryotic expression of TgAPP proteinThe TgAPP cDNA was cloned into the prokaryotic expression vector, pColdTM III with a GST tag and transformed into an E. coli BL21 strain. One liter culture of transformed E. coli cells was grown at 37℃ to an OD600 of 0.5, then switched to 18℃ and induced isopropyl-β-D-galactoside. Purification of the rTgAPP was performed with Glutathione Resin according to the manufacturer’s instructions. The rTgAPP fused with GST was eluted with 20mM reduced glutathione. The protein concentration was quantified with the BCA protein assay kit. The protein purity was evaluated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and stained with Coomassie Brilliant Blue R-250. Female ICR mice were immunized intraperitoneally with purified rTgAPP。emulsified with an equal volume of Freund’s adjuvants to prepare a polyclonal serum. Sera were collected 14 days after the last immunization. The serum antibody titer up to 1:5000 by ELSIA method. To identify native TgAPP, polyclonal antibodies against recombinant enzyme were produced in mice. the anti-TgAPP sera recognized with a size of molecular mass of 78 kDa in Toxoplasma gondii lysate.4. Activity and enzyme kinetic analysis of recombinant TgAPP proteinThe peptide, lysyl (N·-2-aminobenzoyl)-prolyl-proline-4-nitroanilide,2-aminobenzoic acid as fluorophore and 4-nitroaniline as quencher are very useful in the development of internally quenched proteases substrates. APP cleaves the Lys-Pro peptide bond separating the fluorogenic aminobenzoyl residue and the internal quenching residue 4-nitroanilide. Lys (Abz)-Pro-Pro-NA was dissolved in water at 25℃ to prepare 10mM stocks and stored at-20℃. Assays were carried out in 96-well black plates using an EnSpire Multimode Plate Reader at a wavelength of 340-430 nm for both emission and excitation. The total reaction volume was 200μL in Tris-HCl containing 1mM MnC12 in the presence of Lys(Abz)-Pro-Pro-NA fluorogenic substrates at 37℃. One unit of activity was defined as the amount of enzyme hydrolyzing 1μmol substrate/min. To assay the native TgAPP activity in knockout and control lines, total protein was extracted from the parasites using lysis buffer and quantitated using a BCA protein assay kit. Parasite protein was added to 200μL of Tris-HCl buffer before the specific substrate was added to measure enzyme activity. The relative fluorescence levels were assessed for 36 min. For pH optimization,50mM Tris buffer over a pH range from 4.0 to 11.0 containing 1 mM MnC12, rTgAPP and 0. 1mM Lys(Abz)-Pro-Pro-NA was used at 37℃. Cation sensitivity was investigated by assaying the rTgAPP activity after pre-incubating the enzyme at 37℃ for 30min in 50mM Tris-HCl (pH 8.0) containing a metal chloride. The Km (Michaelis constant) and Vmax (maximum velocity) values of rTgAPP were determined by incubating the enzyme in the reaction mixture in the presence of increasing different fluorogenic substrate concentrations. The kcat was determined from kcat-Vmax/[E]. The initial velocity was calculated from the slope of the linear range of fluorescence versus the time curve. Activity was measured under the same conditions as described above and expressed as the mean of three different experiments. Bestatin is a potent aminopeptidase inhibitor for cytosol aminopeptidase, aminopeptidase N, zinc aminopeptidase. To check the effect of inhibitors, rTgAPP was pre-incubated with bestatin and metal ion at 37℃ for 15 min before substrate addition. Relative inhibition of rTgAPP was assessed using bestatin at various concentrations. The synthetic substrate Lys(Abz)-Pro-Pro-NA was efficiently hydrolyzed by rTgAPP with a Km value of 0.255μM, kcat values of 35.6 s-land kcat/Km of 139.6x105 M-1s-1. Activity of the recombinant enzyme was tested in the pH range from 4 to 11, and optimum activity was observed at pH 8.0. The activity of rTgAPP was influenced by the addition of metal ions to the reaction mix. Activity was markedly enhanced in the presence of Mn2+. Other divalent ions (Zn2+, Cu2+, Fe2+) also showed a slight increase in activity. However, the activity of rTgAPP was significantly low in the absent of metal ions. Similar results were also reported in Lactococcus lactis. The inhibitor sensitivity of purified rTgAPP with a variety concentrations of bestain inhibitors was examined. Optimal inhibition was observed at 100mM. The rTgAPP activity slightly affected by addition of EDTA.5. Construction knockout vector and parasite of APP geneA knockout vector targeting TgAPP, designated pGCD-TgAPP was first constructed modified from pSAGl::CAS9-U6::sgUPRT. Then a DHFR cassette was inserted into the pGCD-TgAPP vector. Transfection of T. gondii tachyzoites was performed by electroporation as previously described. The pGCD-APP vector was used to prepare ATgAPP parasites and the vector without gRNA cassette was used to prepare control parasites expressing GFP. Selection based on pyrimethamine was performed as described earlier. Stable clones were isolated using serial dilutions in 96-well plates and confirmed by western blot analysis. We used the CRISPR/Cas9 system to generate a knockout mutant of TgAPP. The TgAPP-deficient parasites were selected with pyrimethamine, and screened by western blotting using mouse anti-TgAPP serum. The Lys(Abz)-Pro-Pro-NA was also used to test the hydrolyzing ability of whole protein extracts from the ATgAPP parasites. The results indicated that the enzyme activity of TgAPP was completely lost against the substrate in ATgAPP parasites.6. Phenotypic analysis of APP gene deficient in Toxoplasma gondiiTo determine whether the ATgAPP parasites had defects invasion, the parasite fluorescence was measured on a flow cytometer. The results revealed a significant difference in invasion between Cas9 control and ATgAPP parasites. This difference was significant after inoculation for 2 hours (P< 0.05) and 4 days (P< 0.01). To further confirm this result, we directly count the population of invasion cells by fluorescence microscope. Similar results show that the invasion rate of Cas9 control parasites is much more than ATgAPP parasites. To confirm an intracellular replication, we performed replication assays by scoring the number of parasites formating plaques foci. Knockout the APP gene effect the plaques foci formative of T.gondii compared to the Cas9 control. After incubation for 24 hours, It was found that the number of parasitophorous vacuoles containing eight tachyzoites peaked in Cas9-control parasites. We could occasionally find vacuoles containing more than eight tachyzoites in ATgAPP parasites. The results indicated that parasite are effected by deleting TgAPP gene.In previous reports, aminopeptidase could release free and small amino acids from the final step of protein catabolism. The substrates of this aminopeptidase might be peptides from proteasomal protein degradation pathways or provide nutritional support parasite to invasion and replication. Possibly recycle the circulating amino acid metabolism and degraded in PV by endoproteases and sent to cytoplasma. There have mang questions need to explore. To investigate the essential nature of the TgAPP we created a knockout transgenic parasite line by transfecting parasites with an Cas-9 knockout plasmid. Our study provides direct evidence that TgAPP plays an important role in parasite invasion and replication. Here, we success expressed X-prolyl Aminope-ptidase in T.gondii and analyzed the chararacteristic of recombinant protein in enzymatic activity. We preliminary probe the function of T. gondii X-prolyl aminopeptidase by CRISP/Cas9 knockout method. Therefore, we expect that APP would be expand the knowledge of aminopeptidases family of biological processes in T. gondii. We will continue to about other aminopeptidase on its biology.