Leishmania mexicana recombinant lamentous acid phosphatase as carrier for Toxoplasma gondii surface antigen 1 expression in Leishmania tarentolae

Leishmania tarentolae has been used to produce recombinant intracellular and secreted proteins for their easy handling and posttranslational modications. Filamentous acid phosphatase is a multimeric protein complex composed of many subunits assembled in a linear highly glycosylated lament, which is secreted in vast amounts into the culture supernatant via the agellar pocket of Leishmania mexicana promastigotes. This suggested that the protein could be used as a carrier for other proteins for easy purication to generate a protein complex decorated with multiple SAG1 subunits suitable for immunisation. Surface Antigen1 protein of a Toxoplasma gondii has an immunodominant structure that is involved in binding to host cells. Previous studies used this surface protein for vaccination for its immunological importance for triggering a type 1 immune response in the host. This study aims to determine the production of recombinant lamentous protein carried subunits of the surface protein of Toxoplasma gondii for vaccination purposes. Leishmania codon-optimised SAG1 was cloned as a fusion construct into pLEXSY-ble2.1 and introduced into Leishmani. tarentolae to generate recombinant cell lines expressing of a lamentous fusion protein called SAP2SAG1. PCR conrmed the correct integration into the small ribosomal subunit RNA gene locus of Leishmania tarentolae. Immunouorescences and Immunoblot analyses were used to detect the fusion protein in the sediment of culture supernatants of recombinant L. tarentolae promastigotes after purication by ultracentrifugation. The yield of puried protein was low that suggested further investigations of other methods for scaling large production of secreted protein.


SAP2 Protein of Leishmania mexicana
Leishmania mexicana is a protozoan parasite of the genus Leishmania, which are the causative agents of a range of diseases called leishmaniasis mostly found in tropical and subtropical regions (Dey et al., 2014). L. mexicana insect stage (promastigotes) secrete a lamentous protein of long acid phosphatases (SAP2). SAP2 protein is composed of 383 serine/threonine amino acids rich regions that are enzymatically active and assemble a laments in the agellar pocket of the promastigotes. Besides, SAP2 protein has a long linear epitope in the COOH-terminal tail that can detect by using monoclonal antibody (mAb) LT8.2.

. Leishmania tarentolae
Leishmania tarentolae, is a non-pathogenic trypanosomatid protozoan parasite living as promastigotes in the lumen of the intestine of lizards such as the gecko (Tarentola annularis) (Wilson & and Southern, 1979). L. tarentolae is distinguished by lacking 250 genes that are expressed in the intracellular amastigote stage. Hence, L. tarentolae has been used for heterologous gene expression and recombinant protein production (Raymond et al., 2012). L. tarentolae has been used as protein expression system for both cytoplasmic and secreted proteins. Therefore, the aim of this work is to determine the effectiveness of a novel eukaryotic lamentous protein expression system based on the L. mexicana SAP2 in L. tarentolae for vaccine development.

Toxoplasma gondii Surface Antigen 1 (SAG1) Protein
Surface antigen 1 (SAG1) is considered the major surface antigen in T. gondii and comprises more than 5% of the total amount of protein of the tachyzoite stage of T. gondii (Lekutis et al., 2001). SAG1 is a tachyzoite that facilitate the adhesion and invasion of the host cells (Tang et al., 2016). SAG1 exists as a monomer of 250 amino acids with an approximate molecular weight of 30 kDa. It can form homodimer structures, which are attached to the parasite surface by glycosylphosphatidylinositol (GPI) anchors (He et al., 2002). Previous studies have focused on SAG1 for vaccination because it has a critical importance for the immune response in the initial stage of infection involving both the humoral and cellular immune response (Abdizadeh et al., 2015;Lekutis et al., 2001). Vaccination of mice with SAG1 has provided encouraging results with signi cant protection as measured by a reduction of the number of cysts in vaccinated animals compared with control animals (Lekutis et al., 2001). SAG1 was puri ed from tachyzoites and produced as a recombinant protein in E. coli or in a yeast (Wang & Yin, 2014).
The aim of this was preparation and expression of recombinant protein of lamentous secreted acid phosphatase 2 fused with SAG1 (SAP2SAG1) in L. tarentolae for immunogenic importance in vaccination studies.

Generation and Puri cation of Expression Plasmid
A single positive colony was inoculated to 100 ml of LB broth with Carbenicillin (100 μg/ml) and incubated 16-18h at 37°C, 220 rpm. The plasmid DNA was puri ed from the overnight culture by using the Nucleobond® Xtra Midi High copy plasmid protocol (Macherey-Nagel, Germany) until the elution step following the manufacturer's instructions. DNA pellets were resuspended in a total volume of 120 μl ddH 2 O and stored at -20°C for transfection into L. tarentolae cells.

Leishmania Cell Culture
L. mexicana and L. tarentolae wild type promastigotes were grown at 27°C without CO 2 in complete SDM79 media and re-inoculated every 3-4 days. Leishmania promastigotes were counted by using a haemocytometer under a light microscope (40× objective lens), and the averaged of cells were calculated per milliliter (number of cells/ml = number of counted cells × dilution factor (100) × 10 4 ).

Transfection of DNA into Leishmania tarentolae by using Electroporation
Transfection of linearised DNA into L. tarentolae cells was performed using a Nucleofector II (Amaxa Biosystems, Lonza, Germany). A maximum volume of 10 μl of puri ed linearised plasmid DNA was mixed with the electroporation buffer before the cells were resuspended in it. L. tarentolae promastigotes were grown until a density of approximately 3×10 7 cells/ml. The cells were harvested by centrifugation for 2 min, at 5,600 × g and 4°C. The supernatant was gently removed from sedimented L. tarentolae cells and immediately replaced with 100 μl electroporation buffer by gentle pipetting. The re-suspended cells were transferred to a pre cooled electroporation cuvette with no air bubbles present. Cells were electroporated using Nucleofector II program V-033 or X-001 and left on ice for 10 minutes.
Electroporated cells were transferred from the cuvette to 10 ml SDM79 medium supplemented with 8% v/v heat-inactivated fetal calf serum, 0.3% v/v hemin and incubated at 27°C for 24 hours. On the second day, the electroporated cells were diluted 1:40 with supplemented SDM79 medium and the appropriate selective antibiotic was added to the diluted cell cultures. 1:40 dilutions were pipetted into 96 well plates using a multi-channel pipette (200 μl per well) and incubated at 27°C for 10-14 days until turbid wells indicated growth of cloned parasites. Turbid wells were checked using an inverted microscope to con rm the presence of Leishmania cells in the well. 200 μl of positive clones identi ed on the 96 well plate were transferred to 12 well plates containing 2 ml supplemented SDM79 medium with the appropriate selective antibiotic and incubated at 27°C for 72 h until turbidity was visibly observed in the well. Subsequently 40 μl from positive clones identi ed in 12 well plates were used to inoculate 10 ml of supplemented SDM79 medium with the selective antibiotic and cultured at 27°C for approximately 96 h until the culture reached the logarithmic growth phase.

Isolation of Genomic DNA from cloned L. tarentolae Cells
The "isolate II genomic DNA kit from Bioline, UK" was used to isolate genomic DNA from L. tarentolae cells following the manufacturer's instructions. 3 ml of a stationary phase culture of Leishmania promastigotes (approximately 10 7 parasites) was used. Genomic DNA pellets were resuspended in 100 µl TE buffer, the concentration was determined using a NanoDrop2000 ® (Thermo Scienti c, Wilmington USA), and stored at 4°C until required.

Secreted Acid Phosphatase Assay
An acid phosphatase assay was used for detection of the protein expression in Leishmania cell-free culture supernatants, which were prepared from 25 μl culture at late logarithmic stage by sedimentation at 5600 × g, 4°C, 2 min. The total volume per well of a 96-well plate was 100 μl composed of 20 μl supernatant/media sample, 70 μl (50 mM p-nitrophenylphosphate in 100 mM sodium acetate pH 5.0). The plate was incubated at 37°C for 30 min. The reaction was stopped by addition 10 μl of 2M sodium hydroxide solution leading to a visible colour change (yellow, positive reaction; pink, no activity). Absorbance readings for the assay were measured at 405 nm using a spectrophotometer (Spectramax M5, Molecular Devices, USA).

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Proteins were separated by SDS-PAGE consisting of a 10% (v/v) resolving gel and 4% (v/v) stacking gel.
Protein samples were diluted in sample buffer with DDT (40 mM), boiled at 95°C for 10 min. Samples were electrophoresed at 30 mA/gel until the front dye had left the gel (~1hr). A protein marker was also loaded into one of the lanes of the gel. Gel running buffer was used in electrophoresis. Gels were placed in Coomassie stain for 20 min. Then destaining solution was applied on the gel 4-5 times for 20 minutes until the protein bands were clearly visible.

Immunoblot Analysis
The production of SAP2SAG1 protein in the culture supernatant was determined using immunoblot analysis. Proteins were transferred from a 10% SDS-gel to an Immobilon-P Polyvinylidene Di uoride (PVDF) membrane by electroblotting system (Biometra) at a current of 4 mA/cm 2 of gel for 90 min. After transfer the membrane was incubated for one hour at 37°C in blocking solution (5% (w/v) milk powder in PBST, 20 mM Tris-HCl pH 7.5). This was then replaced with blocking solution containing the primary monoclonal antibody (mAb) LT8.2 (1:2,000) in blocking solution and incubated one hour with gentle agitation at 37°C. The membrane was then washed four times for 5 min with 1 × PBST at room temperature before goat anti-mouse IgG HRP-linked1:2,000-1:5,000 (DAKO, Hamburg, Germany) was added in blocking solution and incubated for one hour at 37°C. Three times washes for 5 min were carried out with 1 × PBST and two with 1 × PBS. The blot was incubated and exposed to X-ray lms with solutions from the SuperSignal West Pico Chemiluminescent Substrate Kit (Fischer Scienti c, Loughborough, UK).

Immuno uorescence Microscopy
Leishmania cell culture samples were prepared and xed on a 10 well-masked slide. Each sample well on the slide was prepared by washing with 50 μl of 70% ethanol followed by the application of 20 μl of 0.1 mg/ml polylysine in 1× PBS and incubation at room temperature for 15 min. After the incubation was completed each individual well on the slide was washed with 50 μl per well 1× PBS solution twice. 20 μl of total cell culture or cell culture supernatant in a cell density range of 1-2 × 10 7 cells/ml was xed to each well with 20 μl of 4% p-formaldehyde solution by incubation for 15 min at room temperature. After the incubation was completed each well was washed once for 15 min at room temperature with 50 μl per well 1× PBS solution, 50 mM NH 4 Cl, 0.1% Saponin. 50 μl per well of blocking buffer (1× PBS solution, 2% Bovine Serum Albumin (BSA), 1× PBS, 0.1% Saponin) was applied to the wells and incubated for further 15 min at room temperature. 20 μl of the primary antibody mAb LT8.2 in a 1:40 dilution in blocking buffer was applied for 1h at room temperature followed by two wash steps with 50 μl per well 1× PBS solution. Then the wells were incubated with 20 μl per well of a 1:500 dilution of the secondary antibody (goat anti mouse IgG -conjugated uorescent tag) in blocking buffer at room temperature for 1 h. The slide was washed three times with 50 μl 1× PBS solution. 10 μl Mowiol/DABCO solution was applied to each well before the slide was sealed with a cover slip and left to dry for 15 min. Fluorescence was detected using an Epi uorescence microscope at 60× magni cation.

Generation of pLPhSAP2SAG1 and Transfection of L. tarentolae Promastigotes
A 7984 bp fragment was isolated from pLEXSY-ble2.1 cleaved with NcoI + NheI. pSSUPacSAP2SAG1 was cleaved by NcoI + XbaI and the 3386 bp and 3164 bp fragments isolated, respectively (Figure 3.1 ). pLPhSAP2SAG1 was produced from ligation of 3386 bp with the 7984 bp fragments. pLPhSAP2SAG1 was transformed into E. coli DH5α, ampli ed, isolated and cleaved with BamHI, NotI and HindIII to con rm the identities of the plasmids ( gure 3.2 A). To generate a Linear DNA Fragments for Electroporation, pLPhSAP2SAG1 was cleaved with SwaI to isolate the 7207 bp under a sterile condition.

Con rmation of Integration of Constructs into the 18S rRNA Gene Locus by Polymerase Chain Reaction (PCR)
To test the correct integration of the construct into the rRNA gene locus speci c oligonucleotide primers were chosen for PCR in order to amplify fragments of distinctive size. Ampli cation of an 862 bp DNA fragment proved the correct integration of SAP2SAG1 construct into the rRNA gene locus (Figure 3. 3 A).
In a second PCR, a 696 bp amplicon con rmed the fusion of the secreted acid phosphatase gene (SAP2) to SAG1 coding region (Figure 3.3 B). L. tarentolae wild type, which served as negative control in the PCR reactions.
3.3. Detection of Secreted Acid Phosphatase Activity in the Culture Supernatants of Recombinant L. tarentolae Promastigotes Secreted acid phosphatase (SAP2) activity in culture supernatants was used to determine the presence of the recombinant fusion proteins. A quantitative spectrophotometric assay was performed to measure the SAP2 activity from different culture supernatants of recombinant L. tarentolae promastigotes grown in SDM79 medium supplemented with 10% iFCS and 7.5 μg/ml hemin. For comparison purposes, the supernatants of two controls, L. mexicana wild type (positive control) and L. tarentolae wild type (negative control), were also measured. Four cell lines ( A11, E11, F7, F11) of L. tarentolae promastigotes transfected with PhleoSAP2SAG1 presented enzymatic activity of acid phosphatase activity (Figure 3.4).
To determine the levels of recombinant protein in supplemented culture supernatants, the cells were grown over three days and the cell density and enzymatic activity were measured. At day one a low cell density and enzymatic activity was found. The second and third day presented similar enzymatic activity, but the cell density on day three suggested that the cells were already in stationary growth phase ( Figure  3.5 A and B). Therefore, for the following experiments the second day of culture was selected to enrich the fusion protein from the culture supernatants of recombinant cells. In order to make puri cation of the fusion protein from culture supernatants as simple as possible, it was attempted to grow the promastigotes in non-supplemented SDM79 medium. Four L. tarentolae cell lines transfected with PhleoSAP2SAG1, L. tarentolae and L. mexicana wild type were grown in supplemented and nonsupplemented SDM79 medium to determine the enzyme activity in the media. When compared, no activity was observed from promastigotes cells growing in non-supplemented media for two days; in contrast, growing cells in supplemented media showed high enzymatic activity (Figure 3.6).

Detection of Recombinant Protein in Culture Supernatants by Immuno uorescence Microscopy
Immuno uorescence analysis of cells and cell culture supernatants was used to detect the SAP2SAG1 fusion protein secreted by L. tarentolae promastigotes into their culture supernatants by using the mAb LT8.2. L. mexicana wild type promastigotes secrete large amounts of secreted acid phosphatase, served as positive control. No material detectable with mAb LT8.2 from L. tarentolae promastigotes (negative control) in their culture supernatants. Supernatants from L. tarentolae promastigotes transfected with PhleoSAP2SAG1 showed green uorescence less than those from L. mexicana in their culture supernatant (Figure 3.7).

Detection of Recombinant Proteins in Culture Supernatants by Immunoblot Analysis
Immunoblot analysis was used to detect the recombinant SAP2SAG1 fusion protein in the sediment of culture supernatants of recombinant L. tarentolae promastigotes after puri ed by ultracentrifugation using mAb LT8.2. Strong bands were detected by mAb LT8.2 around 175 kDa in culture supernatants and promastigote cells for L. tarentolae transfected with the PhleoSAP2SAG1 construct. L. mexicana samples showed a band of 89 kDa corresponding to SAP1 (Martin Wiese et al., 1999), while no band was detectable when using L. tarentolae as a negative control (Figure 3.8).

DNA Constructs Generation
Because of its unique features, L. tarentolae has previously been used to express secreted proteins. These features entail, easy growth of parasites, protein puri cation from culture supernatants, and the presence of eukaryotic post-translational modi cations (Basile & Peticca, 2009;Kovtun et al., 2011). We have constructed a novel lamentous protein expression system using a DNA cassette for integration into the 18S ribosomal RNA gene locus using pLEXSY-ble2.1 expression vector. Previously, different proteins have been successfully expressed in high levels using pSSU constructs in Leishmania, e.g. b-galactosidase and green uorescent protein (Mißlitz et al., 2000). Human laminin-332 and c-reactive proteins were produced into the culture supernatant of L. tarentolae (Dortay et al., 2011;Phan et al., 2009). SAP2 from L. mexicana including its N-terminal phosphatase domain, the serine/threonine-rich repeats and the mAb LT8.2 epitope was used as a carrier for T. gondii proteins, SAG1, which were genetically fused to its Cterminus.

E ciency of Electroporation
Trypanosomatids can be e ciently transfected by electroporation of in vitro cultivated promastigotes (Beverley & Clayton, 1993). Moreover, selection of recombinant parasites can easily be performed using antibiotic resistance genes, such as the genes for phleomycin binding protein, which was also used in this investigation. To generate permanent and stable transfectants, linear DNA has to be used for chromosomal integration (Beverley & Clayton, 1993). However, when using higher DNA amounts homologous integration of multiple copies of the transfected DNA could be observed (Kianmehr et al., 2016). The recombinant DNA cassette is integrated into the L. tarentolae genome through homologous recombination (Kianmehr et al., 2016). To con rm integration into the small subunit rRNA gene locus of the L. tarentolae genome by homologous recombination diagnostic PCRs were performed. The PCR results proved both the fusion of SAG1 gene to the SAP2 gene and the integration into the proposed site. It is important to note that transcription in this chromosomal location is under the control of RNA polymerase I, which allows high level transcription (Teixeira, 1998). 4.3. Gene Expression in L. tarentolae SAP of L. mexicana promastigotes was previously expressed in L. donovani (Ilg et al., 1991) and L. major (Martin Wiese et al., 1999). It is easier to isolate and purify recombinant proteins if they are exported outside the cell. In this study, laments composed of SAP2SAG1 fusion protein was generated and expressed in L. tarentolae. To improve protein yield transfection of L. tarentolae with multiple copies of the gene of interest integration into different gene loci could have been attempted (Breitling et al., 2002). The recombinant constructs for gene expression were integrated into the 18S small subunit ribosomal RNA gene locus to achieve strong transcription. Interestingly gene regulation, in trypanosomatids, occurs on a post-transcriptional level through intergenic untranslated regions (UTRs) (Clayton, 1999). Therefore, the choice of suitable UTRs is crucial for the construction of an e cient expression cassette suitable for large-scale recombinant protein production (Breitling et al., 2002). The commercial plasmid, pLEXSY-ble2.1, contains three different untranslated regions (UTR1, UTR2 and UTR3), which were supposed to increase the expression level of recombinant proteins in L. tarentolae. UTR1 belongs to the adenine phosphoribosyl transferase gene of L. tarentolae; UTR2 is derived from the intergenic region of the calmodulin cluster and the UTR3 intergenic region is from the dihydrofolate reductase-thymidylate synthase gene locus of L. major. The LEXSY system has been successfully used to express small secreted proteins, like IFN- (Davoudi et al., 2011) andIL-29 (Taromchi et al., 2013).

Secreted Acid Phosphatase Activity
An advantage of the SAP2 fusion protein system is the ease of detecting protein expression by a phosphatase reporter assay directly from cell culture supernatants. The expression level of the SAP2 fusion proteins in L. tarentolae was tested by measuring the secreted acid phosphatase activity in the culture supernatant. Reasonable enzymatic level of the SAP2 fusion proteins from culture supernatant of L. tarentolae growing in 10% iFCS demonstrated the successful expression of SAP2SAG1 in L. tarentolae. The secretion signal sequence of L. mexicana secreted acid phosphatase was used to produce different proteins (Basile & Peticca, 2009). To avoid interference of proteins, present in iFCS (mainly bovine serum albumin) with the puri cation of the recombinant protein, recombinant parasites were grown in nonsupplemented media. No phosphatase activity was detectable in culture supernatants from these cells con rming the importance of iFCS in the culture medium. In fact, serum is required for L. tarentolae growth and for their use as expression system.

Immuno uorescence Analyses
The detection of the recombinant proteins by immuno uorescence con rmed successful secretion of the recombinant proteins into culture supernatants. When comparing the amount of green uorescence obtained from L. mexicana wild type, which served as a positive control, SAP2SAG1 expressed using pLEXSY-ble2.1 revealed lamentous structures along with stronger green uorescence signals. SAG1 is a prototypic member of a superfamily of surface glycoproteins, which is mostly located at the surface of T. gondii (He et al., 2002). This makes it more likely to be secreted normally through the secretory pathway of other protozoan parasites like L. tarentolae.

Immunoblot Analyses
Immunoblot analyses results of SAP2SAG1 using the mAb LT8.2 showed strong bands around175 kDa from supernatants and cell pellets. The signal in the cell pellets is most likely derived from fusion protein retained in the agellar pocket or from protein in transit in the secretory pathway. The 175 kDa bands correspond to the full length recombinant fusion protein. Generally, proteolytic maturation occurs in secretory or transport vesicles carrying proteins from the trans-Golgi network to the cell surface (Lodish et al., 2000). SAP2 forms a lamentous complex with long serine/threonine-rich regions, which are highly glycosylated (M Wiese et al., 1995). These modi cations will protect the protein against proteolytic degradation. Likewise, N-linked glycans are present on the N-terminal phosphatase domain of SAP2 protecting this part of the protein against degradation.

Conclusions
The data gathered by the use of enzyme activity measurement, immunoblot and immuno uorescence analyses in this study provide convincing evidence that the proteins of interest have been expressed successfully. Protein amounts obtained for SAP2SAG1 using pLEXSY-ble2.1 were not determined because of too high serum contaminations in the samples. However, it can be said from the results from the Coomassie-stained gels that the amount of protein is more in the µg than in the mg range. To improve the protein expression levels of the lamentous fusion proteins, further investigation need to be done to increase the expected e ciently of L. tarentolae production of T. gondii protein SAG1. Table   Table 3. 1 Oligonucleotide sequences used in PCR and expected fragment sizes for each PCR reaction. Generation of pLPhSAP2SAG1 from pLEXSY-ble2.1. Comparison of secreted acid phosphatase levels from culture supernatants of Leishmania promastigotes grown in supplemented and non-supplemented cultures. WT L. tarentolae and L. mexicana were used as negative and positive controls, respectively. Measurements were done in triplicate.

Figure 7
Immuno uorescence analysis of Leishmania promastigotes in medium using mAb LT8.2 to detect secreted acid phosphatase. Bright eld microscopy using 40× magni cation on the left and uorescence microscopy using 488 nm excitation with FITC lter at the same magni cation on the right. Size bar, 10 μm.