Characterization of aspartyl aminopeptidase from Toxoplasma gondii

Aminopeptidases have emerged as new promising drug targets for the development of novel anti-parasitic drugs. An aspartyl aminopeptidase-like gene has been identified in the Toxoplasma gondii genome (TgAAP), although its function remains unknown. In this study, we characterized TgAAP and performed functional analysis of the gene product. Firstly, we expressed a functional recombinant TgAAP (rTgAAP) protein in Escherichia coli, and found that it required metal ions for activity and showed a substrate preference for N-terminal acidic amino acids Glu and Asp. Then, we evaluated the function and drug target potential of TgAAP using the CRISPR/Cas9 knockout system. Western blotting demonstrated the deletion of TgAAP in the knockout strain. Indirect immunofluorescence analysis showed that TgAAP was localized in the cytoplasm of the wild-type parasite, but was not expressed in the knockout strain. Phenotype analysis revealed that TgAAP knockout inhibited the attachment/invasion, replication, and substrate-specific activity in T. gondii. Finally, the activity of drug CID 23724194, previously described as targeting Plasmodium and malarial parasite AAP, was tested against rTgAAP and the parasite. Overall, TgAAP knockout affected the growth of T. gondii but did not completely abolish parasite replication and growth. Therefore, TgAAP may comprise a useful adjunct drug target of T. gondii.


Generation of TgAAP-knockout and complemented parasites.
To investigate the function of AAPs in T. gondii, we proceeded to generate a TgAAP-knockout mutant (Δ TgAAP). A TgAAP gene-targeting plasmid, designated pCD-TgAAP (Fig. 2a), was constructed using specific gRNA with a dihydrofolate reductase (DHFR) selectable marker cassette. Plasmid pBluescript II containing codons synonymous to TgAAP fused with mCherry and a hypoxanthine xanthine guanine phosphoribosyl transferase (HXGPRT) expression cassette derived from pHXNTPHA plasmid, designated pBH, were used to construct a TgAAP-complementing plasmid pBH-synoTgAAP (Fig. 2b). After electroporation and selection, Δ TgAAP, complemented, and Cas9 control strains were verified by western blot analysis (Fig. 2c). Protein band ~56 kDa was observed in the lysate of the Cas9 control cell line, but not in the Δ TgAAP strain. The size corresponded to the predicted size (55897 Da) in ToxoDB. An ~95 kDa (TgAAP fused with the mcherry tag) band was observed after probing the complemented strain lysate with anti-TgAAP serum. This was confirmed using anti-mCherry monoclonal antibody. We also performed a PCR assay (Fig. 2S) to verify TgAAP knockout. All these experiments confirmed the absence of TgAAP in the Δ TgAAP strain. Cellular localization of TgAAP. Confocal laser scanning microscopy detection of TgAAP with fluorescently labeled anti-TgAAP mouse polyclonal antibodies indicated that TgAAP localized in the cytoplasm of the T. gondii wild-type RH strain, but was not expressed in the Δ TgAAP strain (Fig. 3).

Loss of TgAAP affects attachment/invasion and growth in vitro.
Aminopeptidases are exopeptidases that catalyze sequential removal of amino acids from the N-termini of peptides. These enzymes play major roles in regulating the balance between catabolism and anabolism in all living cells 20 . Since aminopeptidases can affect cellular metabolism, we anticipated that TgAAP plays a role in parasite growth. To assess this, we tested the ability of TgAAP-knockout strains to form plaques on human foreskin fibroblast (HFF) monolayers. The Δ TgAAP strain was able to produce plaques (Fig. 4a), suggesting that the TgAAP gene is not essential for growth.  During repeat experiments, plaques produced by the Δ TgAAP strain were always smaller and less numerous ( Fig. 4a,b) than those produced by the Cas9 control strain (Student's t-test, P < 0.05, n = 3). These results suggested that the parasite attachment/invasion and replication were reduced after TgAAP deletion. To further investigate whether the complemented strain would recover the ability to invade, intracellular parasite numbers were scored by microscopic observation (Fig. 4c). The mean number of parasites per field of view was significantly lower in the Δ TgAAP strain when compared with the Cas9 control strain 2 h post infection (Student's t-test, P < 0.01, n = 3). These phenomena were reversed in the complemented strain. We also performed growth assays and scored the number of parasites per vacuole (Fig. 4d). Growth assays showed that a significant percentage of vacuoles in the Δ TgAAP-infected group contained tachyzoites that exhibited a reduced ability to divide 24 h post infection. Unlike the control and complemented strains, significantly more vacuoles containing four tachyzoites were seen in Δ TgAAP compared with the Cas9 control strain (Student's t-test, P < 0.01, n = 3), with a significantly reduced number of eight-tachyzoite vacuoles (Student's t-test, P < 0.01, n = 3). More parasitophorous vacuoles (PVs) containing more than eight tachyzoites were detected in the Cas9 control and complemented parasites. By contrast, fewer PVs containing more than eight tachyzoites were detected in the cells inoculated with Δ TgAAP parasites (Student's t-test, P < 0.01, n = 3). Together, these results indicate that TgAAP plays an important role in parasite attachment/invasion and growth.

Specific enzyme activity in ΔTgAAP.
Previous studies demonstrated that AAPs have a strict preference for the N-terminal acidic amino acids Glu and Asp 12 . We performed substrate-cleaving assays to investigate this specific enzymatic activity in the TgAAP deletion strain. Specific fluorescent substrates H-Asp-MCA and H-Glu-MCA were used. H-Leu-MCA and eight other substrates were used to detect the activities of other enzymes in Δ TgAAP, Cas9 control, and complemented strains. The results indicated that TgAAP deletion led to a significant decrease in AAP-specific enzymatic activity compared with the Cas9 control (V max ; Student's t-test, P < 0.01, n = 3) but the cleavage of H-Glu-MCA and H-Asp-MCA in the complemented strain was restored to Cas9 control levels (Fig. 5a). Exopeptidase activities and reaction rates with respect to Ala-, Arg-, Cys-, Lys-, Met-, Phe-, Tyr-, Trp-, and Leu-MCA were higher in Δ TgAAP, Cas9 control, and complemented strains. To further examine whether TgAAP knockout impacted other enzyme activities in T. gondii RH, enzyme activity in Δ TgAAP was assessed using 11 substrates (Fig. 5a) and compared with the Cas9 control (Fig. 5b). Except for a decreased activity toward H-Glu-and H-Asp-MCA (8.33% and 10.37% of the Cas9 control, respectively), no significant difference was observed between the two strains with respect to enzymatic activities toward other substrates, thus indicating the substrate specificity of TgAAP.
Inhibitor assay. In vitro inhibition assay of rTgAAP using CID 23724194, identified as a PfM18AAP inhibitor 21 , is shown in Fig. 6a. rTgAAP activity was partly inhibited by 100 μ M CID 23724194. The activity of rTgAAP was inhibited by more than 50% in the presence of 1 mM drug. The effect of drug concentration on parasite growth is shown in Fig. 6b. As the concentration of the drug was increased, no change in the percentage of Toxoplasma-infected host cells was observed (Student's t-test, P > 0.05, n = 3). This indicated that CID 23724194 inhibited rTgAAP, but this did not affect the growth of the parasite.

Discussion
Similarly to PfM18AAP, TgAAP is the only AAP encoded in the genome of T. gondii. A recent study proved that T. gondii ingests and digests host cytosolic proteins. Disruption of this process attenuates the virulence of the parasites 22 . In addition, deletion of TgLAP severely affects the growth of the parasites. Taken together, these phenomena suggest the importance of protein digestion by these enzymes in parasite development. This study reports the characterization of TgAAP. The amino acid sequence of TgAAP was predicted to contain conserved Zn-binding sites and substrate-binding/catalytic sites. The structure of PfM18AAP revealed a dodecameric assembly that forms a tetrahedron shape 23 . The dodecameric enzyme is formed by interactions of four PfM18AAP trimers with an internal M18 family active site cavity comprising four trimer cones, each with three active sites 19 .
To assess the multimeric nature of TgAAP, rTgAAP was analyzed by native PAGE, which revealed that our recombinant enzyme exists in solution mainly as a dodecamer, partially in a trimeric and monomeric form. The multimeric nature of TgAAP is similar to that of PfM18AAP. We also predicted a 3D model of TgAAP that exhibited the highest homology with the crystal structure of human AAP (DNPEP, template: 4dyo.1.A, sequence identity: 44.9%) 24 using the auto-model method (Fig. 3S). It is risky to develop new drugs targeting this enzyme. However, it is possible to identify drugs that target the non-conserved part of this enzyme. TgAAP trimers and monomer structures were predicted separately from the tetrahedron by comparison with the X-ray crystal structure of a PfM18AAP monomer. Structural prediction of the TgAAP model suggested that the protein is a canonical member of the M18 aminopeptidase family. PfM18AAP is expressed in the cytosol and is also trafficked out of the parasite into the surrounding PVs. It has therefore been speculated that PfM18AAP plays a role in processing proteins or peptides in transit from the PV to the parasite cytosol 12 . We found that TgAAP is also expressed in the cytosol. We previously characterized another toxoplasma aminopeptidase, M17 leucine aminopeptidase TgLAP 25 , an exopeptidase with specificity toward N-terminal hydrophobic residues Leu and Phe that also localizes to the PVs. These two enzymes function in concert in protein catabolism in mammalian cells 15 . The free amino acids released during the process are likely used in anabolism and facilitate protein synthesis in the rapidly growing intracellular parasite. Previous biochemical assays of the malarial AAP, PfM18AAP, demonstrated that the enzyme requires metal ions for its activity and has a strict preference for N-terminal acidic amino acids Glu and Asp 12 . To investigate substrate specificity of TgAAP, we performed H-Glu-/Asp-MCA substrate activity assays using recombinant TgAAP expressed in E. coli. The results of an assay testing the effect of metal ions on enzyme activity indicated that Co 2+ was the preferred metal cofactor for rTgAAP, followed by Mn 2+ . This is different from the reported activity of PfM18AAP 12 , enhanced only by Co 2+ . The substrate specificity assay indicated that rTgAAP had a strict preference for H-Asp-MCA and H-Glu-MCA, although the catalytic efficiency of the enzyme was higher with H-Glu-MCA than with H-Asp-MCA. This was consistent with the finding that the initial reaction rate with H-Glu-MCA was higher than with H-Asp-MCA.
In P. falciparum, antisense knockdown experiments had previously identified a lethal phenotype associated with PfM18AAP, suggesting that the enzyme is a valid target for new antimalarial therapies 26 . Another study reported that this enzyme is not dispensable for parasite growth through the disruption of this gene in the genome of Plasmodium 18 . To assess whether TgAAP may be a candidate drug target for parasite growth control, we constructed a TgAAP-knockout strain and analyzed its phenotype. The absence of TgAAP hindered parasite invasion and growth but was not lethal to T. gondii. Therefore, we conclude that either the low activity of other aminopeptidases is able to sustain slower growth of the parasites or the AAP activity is not essential for parasite growth. High throughput screening was used to identify potent and selective inhibitors of AAPs. Compound CID 23724194 showed anti-parasite potency in malaria growth assays 11 . CID 23724194 efficiently inhibited PfM18AAP activity in parasite lysates (96% at 10 μ M) and efficiently inhibited parasite growth (IC50 = 1.3 μ M). However, this inhibitor is almost completely ineffective against TgAAP (more than 50% Glu-MCA cleavage inhibition at 1 mM). This may be due to differences in the protein sequence or spatial structure between PfM18AAP and TgAAP. The parasite growth inhibition assay revealed no significant difference between the percentages of Toxoplasma-infected host cells with increasing drug concentrations. In general, CID 23724194 is not a lead compound. Therefore, our results suggest that TgAAP may only be used as an adjuvant drug target. Previously, we reported the function of LAP by deleting TgLAP using the CRISPR/Cas9 genome editing technique. Our results indicated that TgLAP was not essential, but played important roles in growth, invasion, and virulence. Accordingly, from the perspective of new anti-parasite drug development, M18AAP, along with M1 and M17, remains a protein of high interest 27,28 . Until now, we have only tested two metal aminopeptidases, TgLAP and TgAAP, and neither was essential to the parasite. Considering the importance of aminopeptidases for parasite growth and invasion, we believe that TgLAP and TgAAP, together or with other aminopeptidases, may serve as ideal targets. Recently, an "open collaboration" has been pursued in search of new drugs to manage neglected tropical diseases. Such initiatives advance the screening of additional molecular targets in Plasmodium, including PfM18AAP, PfM17LAP, and PfM1 28 . These studies confirmed the feasibility of simultaneous targeting of two or more drug targets. It is yet to be determined whether aminopeptidases are suitable candidates for such drug development strategies for toxoplasmosis treatment.
It has been reported that AAPs of the M18 family are required for the removal of N-terminal acidic amino acid residues, Glu and Asp, that cannot be removed by M17 LAP or other aminopeptidases 29,30 . Similar conclusions may be drawn from our study: the substrate activity (H-Glu-and H-Asp-MCA) was almost completely lost in the Δ TgAAP strain. This also indicated that no other enzyme complemented the TgAAP activity. These studies revealed the presence of additional aminopeptidases that could remove amino acids such as Ala, Arg, and Lys, not cleaved by TgAAP or TgLAP. Other aminopeptidases encoded in the malaria genome, for example, M1 membrane aminopeptidase N 30 , can cleave off these amino acids (Ala, Arg, and Lys), and thus complete the line-up of exopeptidases required for the total degradation of proteins to free amino acids in the parasite cytosol.
To investigate the essentiality of TgAAP in T. gondii, we created a knockout/transgenic parasite line using the new DNA editing technology, the CRISPR/Cas9 system [31][32][33] . TgAAP deletion was successful, as evidenced by the absence of a corresponding specific protein band in the western blotting experiment and significantly decreased Enzyme activity curves of whole protein lysates from various parasite strains are shown and were generated using GraphPad Prism v. 5.0c (GraphPad Software, San Diego, USA). Enzyme activities were recorded at 3 min intervals until maximum activity levels were reached. Samples devoid of protein lysates were used as negative controls. All experiments were independently repeated three times (each in triplicate), and representative curves are shown. (b) Activities in TgAAP-knockout parasites toward different substrates (x-axis) calculated as percentages of the activity in Cas9 control lysates (y-axis). Student's t-test was used for calculation of significance between groups. *P < 0.05, **P < 0.01 (n = 3).
Scientific RepoRts | 6:34448 | DOI: 10.1038/srep34448 AAP-specific enzyme activity when compared with Cas9 control parasites. The method was also highly specific because the relative activity of TgLAP was unaffected in the TgAAP deletion strain. Phenotype assay revealed that TgAAP deletion inhibited cell growth, leading to smaller parasite plaques, and resulting in a reduced substrate cleavage rate. However, the phenotype of the TgAAP deletion mutant was not as pronounced as the phenotype of the corresponding PfM18AAP knockdown mutant 12 . Whether knocking out TgAAP or TgLAP would impact the anabolism of critical invasive proteins involved in parasite invasion requires further investigation.
Aminopeptidases have been treated as tractable Plasmodium targets for antimalarial drug development 34,30,28 . The function of other T. gondii aminopeptidases should also be investigated in the future, to establish a foundation for designing multiple-target anti-toxoplasma drugs to explore malaria treatments.

Materials
Parasite strains and growth conditions. The T. gondii RH strain (with HXGPRT deleted by homologous recombination; the strain is a generous gift from Dr. X. Xuan) and parasite mutants used in this study were maintained by growth in Vero cells or HFFs cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Invitrogen) supplemented with fetal bovine serum (FBS; 2%), penicillin/streptomycin (1%), gentamicin (10 μ g/mL), and glutamine (10 mM; Thermo, Fisher Scientific, Waltham, MA), at 37 °C in an air/CO 2 (5%) environment. To purify T. gondii tachyzoites, parasites and host cells were washed in cold phosphate-buffered saline (PBS), and the final pellets were suspended in cold PBS and passed three times through a 27-gauge needle. The parasites were then passed through 5.0 μ m-pore filters (Millipore, USA), washed twice with PBS, and stored at − 80 °C until use.
Recombinant TgAAP and anti-TgAAP polyclonal antibody production. TgAAP cDNA was amplified using the following primers: ColdTgAAPFwd, 5′ -CTCGAGGGATCCGAATTCATGCAAA TGCAGACTGGCAC-3′ (EcoRI site underlined); and pColdTgAAPRev, 5′ -GTCGACAAGCTTGAATTCTTAC ATGCCCTTGTAGCTAT-3′ (EcoRI site underlined). The PCR fragment fused with GST at the N-terminus was cloned into the pCold III vector (Takara Bio Inc., Dalian, China). The verified plasmids were transformed into E. coli BL21. The culture were induced by treatment with 1 mM IPTG at an OD 600 of ~0.5 and cultivated at 25 °C for 20 h. After centrifugation, cells were resuspended in PBS and lysed by ultrasonic treatment. Purification of rTgAAP was performed using glutathione resin (GenScript, Piscataway, USA), according to the manufacturer's instructions. rTgAAP fused with GST was eluted with a buffer containing reduced glutathione (20 mM; GE Healthcare, Piscataway, USA) and dialyzed against Tris-HCl (50 mM, pH 8.0). The purified rTgAAP was resolved on SDS-PAGE and native PAGE (Novex ® System, Invitrogen), as previously described 35 . The concentration of purified rTgAAP was measured with the BCA Protein Assay Kit (Thermo Scientific Pierce, USA). Mice were immunized three times at 2 week intervals with GST-tagged rTgAAP peptide (100 μ g per injection) formulated in Freund's Complete and Incomplete Adjuvant 6 . Sera were collected 14 days after the last immunization.
Enzyme activity and kinetics. Enzymatic activity of rTgAAP was determined by measuring the rate of Glu or Asp release from fluorogenic substrates H-Glu-MCA or H-Asp-MCA (Bachem, Bubendorf, Switzerland), respectively. MCA release was measured using the EnSpire Multimode Plate Reader (PerkinElmer, Turku, Finland), at 355 and 460 nm for both emission and excitation. The experimental data were plotted using GraphPad Prism v. 5.0c (GraphPad Software, San Diego, USA). To determine enzyme sensitivity to metal ions, TgAAP activity was investigated after pre-incubating the enzyme (0.3 μ M) at 37 °C for 30 min in Tris-HCl (50 mM, pH 7.5) containing 1 mM specified Co 2+ (Sigma-Aldrich, St Louis, USA).
K m (Michaelis-Menten constant) and V max (maximum velocity) values of rTgAAP were determined by incubating the enzyme (0.3 μ M) in the presence of increasing concentrations of various fluorogenic substrates (Bachem, Bubendorf, Switzerland) at 37 °C. The initial velocity was calculated from the slope of the linear range of fluorescence vs. time curve. The average K m and V max values were calculated with standard errors from three independent experiments.
Construction of T. gondii transgenic knockout and complementing plasmids. The structure of the pSAG1::CAS9-U6::sgUPRT plasmid (Addgene, #54467) has been described previously 31 . In this vector, Cas9 protein fused with GFP is expressed under the control of a SAG1 promotor. We constructed a knockout vector targeting TgAAP by modifying this plasmid. The backbone sequence, except the single guide RNA (gRNA) cassette, was amplified from pSAG1::CAS9-U6::sgUPRT and self-ligated after digestion with PmeI. A DHFR cassette was then inserted into the KpnI site, and the resultant plasmid was designated pCD. Finally, a gRNA cassette targeting TgAAP (gRNA sequence: 5′ -AGAACGAGGATATCGTTGAG-3′ ) under the control of the TgU6 promoter was cloned into the PmeI site. The final plasmid was designated pCD-TgAAP.
Plasmid pBluescript II containing the HXGPRT expression cassette (derived from pHXNTPHA plasmid, a generous gift from Dr. X. Xuan) and a GFP cassette under the control of the GRA1 promotor (derived from PDMG 33 ) was used to construct the complementing plasmid. To validate and identify the monoclonal complemented strain, the GFP expression cassette was inserted into a the plasmid. The plasmid was designated pBH. To construct the complementing plasmid, the synonymous codon TgAAP gene (synoTgAAP, containing synonymous codons GTG and GAA in the target gRNA sequence adjacent to the PAM site) was amplified using primers synoTgAAPFwd (5′ -GAACGAGGATATCGTGGAATGGGACTTGTG-3′ ) and synoTgAAPRev (5′ -CACAAGTCCCATTCCACGATATCCTCGTTC-3′ ), and inserted into the PmeI site of the pBH vector using the ClonExpress ™ II One Step Cloning Kit. In the constructed plasmid, synoTgAAP expression was regulated by the GRA1 promoter. The C-terminus of synoTgAAP was fused to mCherry tag (synthesized by GeneScript Co., Ltd.) by hierarchical fusion PCR. The final plasmid was designated pBH-synoTgAAP.

Transient transformation and Cas9-mediated gene disruption and complementation. Transient
transformation with the plasmid (pCD-TgAAP) was achieved by transfecting freshly lysed tachyzoites with circular DNA (10 μ g) 36 , and immediate infection of Vero cells. Selection of the transfected parasites that acquired resistance to pyrimethamine (1 μ M, in ethanol) was performed as described earlier 37 . For cloning purposes, the transformed parasites were serially diluted into a culture of Vero cells grown on 96-well plates without selection. Single-plaque wells were replica-passaged for propagation or for western blotting, and indirect immunofluorescence detection with anti-rTgAAP antibodies was used to determine whether the TgAAP gene was disrupted. To generate Δ TgAAP/synoTgAAP complemented parasites, 10 7 Δ TgAAP parasites were transfected with pBH-synoTgAAP plasmid (10 μ g) and selected with mycophenolic acid (25 μ g/mL) and xanthine (50 μ g/mL). After three generations, parasites expressing synoTgAAP were used in in vitro invasion and replication assays. We also constructed and screened a T. gondii RH strain that was transformed with plasmid pCD-HXGPRT (HXGPRT gRNA sequence: 5′ -GTGGAGACCATTGGGTCACT-3′ ), named Cas9 control. The resultant strain was used as a control.
Western blotting. Samples for western blotting were obtained by gentle centrifugation of extracellular parasites and incubating them with RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA) containing a protease inhibitor cocktail (Calbiochem, USA) for 20 min on ice, to lyse them. Next, the samples were centrifuged for 10 min at 15,000 × g at 4 °C, and Laemmli buffer was added to the supernatant. Unless indicated otherwise, an equivalent of 10 7 parasites was loaded per SDS-polyacrylamide gel lane and immunoblotting was performed. Briefly, parasite extracts were resolved on a 10% SDS-polyacrylamide gel, transferred to a PVDF membrane, and probed with mouse anti-rTgAAP serum (1:500) as a primary antibody. The mCherry protein was detected using an anti-mCherry mouse monoclonal antibody (1:5000) (Abbkine, California, USA) as the primary antibody. Then, a horseradish peroxidase-conjugated goat anti-mouse antibody (1:5000) (Jackson Immunoresearch, West Grove, Pennsylvania, USA) was used as the secondary antibody. Chemiluminescent signals were developed with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, USA).
Immunofluorescence. Immunofluorescence assays were carried out as described previously 25 . Briefly, after 16-24 h of incubation, HFF monolayers infected with T. gondii wild-type RH or Δ TgAAP parasites were fixed with paraformaldehyde (4%) prepared in PBS for 15 min and then permeabilized with Triton X-100 (0.3%) prepared in PBS for 10 min. Cells were then incubated with mouse anti-rTgAAP serum (1:200) as a primary antibody. An Alexa 488-conjugated goat anti-mouse fluorescent antibody (1:500) (Jackson ImmunoResearch) was used as the secondary antibody. The parasite nuclei were visualized with DAPI. Samples were examined with a confocal microscope.
Attachment/invasion assay. To measure parasite attachment/invasion rates, freshly lysed Cas9 control, knockout, and complemented parasites were filtered and used to inoculate HFF cell monolayers in 6-well culture plates (Costar, USA; 10 6 parasites/well, three wells per each strain). Parasites were allowed to invade HFF cells for 2 h (37 °C, 5% CO 2 ), and extracellular parasites were washed off with PBS. Then, mean numbers of parasites per field (60 fields/well) were counted under a microscope. Three independent experiments were performed, and each strain was assayed in triplicate within each experiment.

Replication assay.
To directly compare the growth of Cas9 control and knockout parasites, plaque assays were performed. Purified parasites were used to infect fresh HFF monolayers seeded in 6-well plates and grown for 7 days without shaking (100 parasites/well). Cells were then fixed with ethanol (70%) and stained with crystal violet (0.1%). Plaques were scanned using an Epson scanner and analyzed as previously reported 31 . Three independent experiments were performed, and each strain was assayed in triplicate within each experiment. To investigate replication rates of Δ TgAAP, Cas9 control, and complemented parasite lines, freshly lysed tachyzoites were collected, filtered, and used to inoculate Vero cell monolayers in 6-well culture plates (10 6 parasites/well). Parasites were allowed to invade for 2 h under normal growth conditions (37 °C, 5% CO 2 ), following which, extracellular parasites were washed away with PBS, and the parasites were incubated for another 24 h. The number of vacuoles containing the indicated number of parasites (i.e., 1, 2, 4, 8, or > 8 cells) was counted in ≥ 100 vacuoles from three separate wells per experiment. Three independent experiments were conducted, and the results were combined and graphed.
Native enzyme activity assay. To determine the native TgAAP enzyme activity in the knockout and complemented lines, freshly lysed tachyzoites were purified and collected by centrifugation. Proteins were extracted using lysis buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 1% Nonidet P-40, and 2 mM EDTA) and quantified using a BCA Protein Assay Kit (Pierce, Bonn, Germany). Parasite proteins (10 μ g) were added to 200 μ L of Tris-HCl buffer (50 mM, pH 7.5) before a specific substrate (0.1 mM) was added. Relative fluorescence levels were assessed every 3 min for a total of 60 min. To determine the effect of TgAAP deletion on the activity of other cellular aminopeptidases, 11 substrates were used. For each substrate, maximum enzyme activities of Δ TgAAP and Cas9 control strains were compared.