This work was funded by the Secretar&#237;a Nacional de Ciencia, Tecnolog&#237;a e Innovaci&#243;n (SENACYT), Panam&#225;, grant number NI-177-2016, and Sistema Nacional de Investigaci&#243;n (SNI), Panam&#225;, grant numbers SNI-169-2018, SNI-008-2022, and SNI-060-2022.

To keep the samples sterile, all steps involving parasite culture should be performed inside a biosafety level 2 (BSL-2) hood or according to local health and safety regulations. A graphical summary of this protocol can be found in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64939/64939fig01.jpg" /> 
Figure 1: Summary scheme of the protocol for generating eGFP-expressing L. panamensis and L. donovani parasites using the pLEXSY-2.1 vector family. All six major sections described in the article are depicted here. 1) Amplification and insertion of the eGFP gene into the pLEXSY-2.1 vector: the target gene is amplified, adding the recognition sites for the enzymes KpnI and BglII, and both the eGFP amplicon and pLEXSY plasmid are sequentially digested with both enzymes for subsequent ligation with T4 ligase. 2) Linearization of the pLEXSY expression plasmid: the pLEXSY + eGFP construct is digested with SwaI for linearization and purification of the 7-8 kbp expression cassette. 3) Transfection and 4) polyclonal selection: Leishmania promastigotes are transfected with the expression cassette by electroporation and put back in culture for antibiotic selection of recombinant parasites. Parasite fluorescence is confirmed through flow cytometry. 5) Confirmation of genomic integration and 6) cloning by limiting dilution: integration of the pLEXSY-eGFP expression cassette is confirmed by diagnostic PCR, using a forward primer hybridizing to the expression cassette and a reverse primer hybridizing to a chromosomal sequence absent in the expression cassette. Transfected cultures could be enriched for fluorescent parasites by cloning by limiting dilution, and clones with the highest mean fluorescent intensities can be selected for further applications. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64939/64939fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Amplification and insertion of the eGFP gene into the pLEXSY-2.1 vector
<ol>
	<li>Amplification of the eGFP gene 
	NOTE: As this protocol uses the pLEXSY-2.1 vector family (see Table of Materials) for the constitutive expression of target proteins following the integration of the expression cassette into the chromosomal 18S rRNA locus (ssu) of Leishmania species, the first step is to introduce in the eGFP gene the sequences containing the restriction sites that allow its insertion into pLEXSY-2.1 vectors. In this case, as eGFP is only required to be expressed cytosolically, the restriction sites for the enzymes BglII and KpnI were added in the 5&#39; and 3&#39; ends of the eGFP gene, respectively. Cloning with KpnI results in the fusion of the target protein to a C-terminal polyhistidine tag of six residues, followed by a stop codon encoded in the pLEXSY-2.1 plasmid for further purification of the protein of interest. For this reason, the reverse primer sequence does not contain a stop codon.
	<ol>
		<li>Depending on the plasmid source for eGFP, analyze the target sequence using a restriction analysis tool such as NEBcutter (http://tools.neb.com/NEBcutter/index.php3)31 to make sure that it does not contain internal sites for the restriction enzymes used for cloning&#160;(BglII and KpnI) or for SwaI, which is the enzyme used for vector linearization prior to transfection.</li>
		<li>Design forward and reverse primers for eGFP amplification containing the BglII and KpnI restriction sequences. As an example, the eGFP source for this protocol was the pEGFP-N1-1X plasmid32, and the primer sequences were those reported by Bolhassani et al.27: 
		Forward primer (EGFP1): 5&#39;-ATGATATCAAGATCTATGGTGAGCAAGGGC-3&#39;&#160; (BglII restriction site in bold). 
		Reverse primer (EGFP2): 5&#39;-GCTCTAGATTAGGTACCCTTGTACAGCTCGTC-3&#39;&#160; (KpnI restriction site in bold).</li>
		<li>Amplify the target gene using a high-fidelity polymerase to ensure the preservation of the coding sequence. As an example, for the primers EGFP1 and EGFP2, run the PCR cycling protocol as reported by Bolhassani et al.27. Run an initial denaturation of 2 min at 94 &#176;C, followed by 30 cycles of 30 s at 94 &#176;C, 30 s at 57 &#176;C, and 1 min at 72 &#176;C. Run a final extension step of 10 min at 72 &#176;C. Add the primers at a final concentration of 0.2 &#181;M.</li>
		<li>Purify the fragment using a conventional PCR product purification kit, or standard sodium acetate and ethanol precipitation, as described elsewhere33.</li>
	</ol>
	</li>
	<li>Insertion of the eGFP into the pLEXSY-2.1 expression vector
	<ol>
		<li>Trim the ends of the eGFP-PCR product using BglII and KpnI, following the manufacturer&#39;s instructions.
		<ol>
			<li>First, set up the reaction for the enzyme requiring the lowest salt concentration (in this case, KpnI), according to the manufacturer&#39;s instructions, and incubate at 37 &#176;C for 1 h.</li>
			<li>Then, add 100 mM NaCl and 10 units of BglII and incubate for 15 min. Purify the reaction, as described in step 1.1.4.</li>
		</ol>
		</li>
		<li>Digest the pLEXSY expression vector with BglII and KpnI, following the sequential digestion protocol described in step 1.2.1.</li>
		<li>Ligate the pLEXSY vector and eGFP gene with T4 ligase using a molar ratio of 1:3 (vector:insert). Briefly add 100 ng of digested pLEXSY and 28 ng of digested eGFP product to a 20 &#181;L reaction containing ligase buffer at a final concentration of 1x and 3 units of T4 DNA ligase. Incubate overnight at 4 &#176;C.</li>
		<li>Transform competent E. coli cells, such as XL-10, DH5&#945;, or DH10B, with the ligation product obtained in step 1.2.3 using standard transformation protocols33. Plate the transformed cells in Luira-Bertani (LB)-ampicillin agar and incubate for 24 h at 30 &#176;C to select recombinant clones (pLEXSY plasmids are more stable in E. coli at 30 &#176;C than at 37 &#176;C).</li>
		<li>Screen for the presence of the insert in the plasmids by colony PCR.
		<ol>
			<li>Pick individual colonies, resuspend in 20 &#181;L of nuclease-free water, heat at 95 &#176;C for 15 min, and take 1 &#181;L of the lysate as a DNA template for colony PCR. Use the forward primer for eGFP amplification, described in step 1.1.2 (in this example, EGFP1), and the primer A264 (5&#39;-CATCTATAGAGAAGTACACGTAAAAG-3&#39;) as a reverse primer29.</li>
			<li>The primer A264 is designed to anneal 80 bp after the stop codon of the insert in pLEXSY-2.1 expression vectors. As an example, for the primer pair EGFP1 + A264, use a PCR cycling protocol consisting of an initial denaturation of 2 min at 94 &#176;C, followed by 30 cycles of 30 s at 94 &#176;C, 30 s at 50 &#176;C, and 1 min at 72 &#176;C. Run a final extension step of 10 min at 72 &#176;C. The primers are added at a final concentration of 0.2 &#181;M. 
			NOTE: The expected product using this primer set is 859 bp long. The annealing sites for the primers EGFP1 and A264 are depicted in Figure 2.</li>
		</ol>
		</li>
		<li>Prepare the purified plasmid DNA from a positive clone for subsequent transfection using a commercial plasmid isolation kit or standard alkaline precipitation33. Use 50 mL of an overnight culture at 30 &#176;C for isolation of a minimum of 10 &#181;g of plasmid/positive clone.</li>
	</ol>
	</li>
</ol>
2. Linearization of the pLEXSY expression plasmid
<ol>
	<li>Use at least 10 &#181;g of the purified pLEXSY-eGFP plasmid for digestion with SwaI (recognition site: 5&#39;-ATTTAAAT-3&#39;). Digest for 3-4 h at 25 &#176;C and heat-inactivate at 65 &#176;C for 20 min.</li>
	<li>Run the digestion product in agarose gel electrophoresis to separate the resulting fragments. This digestion will generate two fragments, a 2.9 kbp fragment representing the elements necessary for replication and selection in E. coli, and a 7-8 kbp fragment representing the linearized expression cassette.</li>
	<li>Purify the expression cassette using a commercial agarose gel extraction kit.</li>
</ol>
3. Transfection of L. panamensis and L. donovani by electroporation
NOTE: Leishmania culture is performed as described elsewhere34. In this example, L. panamensis and L. donovani promastigotes were cultured in Schneider&#39;s insect medium, supplemented with 20% (v/v) fetal bovine serum (FBS) and 50 &#181;g/mL gentamicin, and incubated at 26 &#176;C.
<ol>
	<li>Grow the parasite cultures until there are enough log-phase or early stationary-phase promastigotes to use approximately 4 x 107 parasites per transfection. The maximum cell density per culture flask before reaching the stationary phase may vary depending on the Leishmania species and how well the strains are adapted to laboratory conditions. Pool the contents of multiple culture flasks if required.</li>
	<li>Centrifuge the parasite culture at 2,000 x g for 3 min at room temperature (RT).</li>
	<li>Resuspend the pellet in electroporation buffer at 4 &#176;C (21 mM HEPES, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, and 6 mM glucose; pH 7.5)27 to get a concentration of 1 x 108 parasites/mL. Put on ice for 10 min. 
	NOTE: The electroporation buffer must be sterilized by filtering before use.</li>
	<li>In parallel, pre-chill tubes with 2-10 &#181;g of linearized pLEXSY-eGFP construct in a maximum volume of 50 &#181;L of water or 10 mM tris buffer (pH 8.0) and electroporation cuvettes of d = 2 mm.</li>
	<li>Add 350 &#181;L of pre-chilled parasites to the tube with the linearized plasmid and transfer the entire 400 &#181;L to the electroporation cuvette on ice. In parallel, electroporate the parasite cells without plasmid DNA as a negative control.</li>
	<li>Electroporate using one of these two protocols: 
	Exponential decay: 450 V, 450 &#181;F, one pulse. 
	Time constant: 450 V, T = 3.5 ms, one pulse. 
	NOTE: These protocols were run using a commercial gene pulser (Table of Materials) with PC and CE modules.</li>
	<li>Put the cuvette back on ice for 10 min and transfer the electroporated parasites to 5 mL of the appropriate culture medium. This example used Schneider&#39;s insect medium supplemented with 20% (v/v) FBS. Incubate at 26 &#176;C for 20 h.</li>
</ol>
4. Polyclonal selection in culture
<ol>
	<li>Approximately 20 h post-electroporation, observe the cultures under a microscope. At least half of the parasite population should show visually good morphology and motility (drop-like promastigotes with oscillating flagellum moving through the media and/or forming active parasite aggregates). Add the appropriate selective antibiotic depending on the pLEXSY plasmid used. In this example, pLEXSY-sat2.1 is used, which contains streptothricine acetyltransferase as a selection marker. Therefore, use nourseothricin as a selective antibiotic at a 0.1 mg/mL concentration.</li>
	<li>Follow the cultures microscopically until a clear difference between the parasites electroporated with and without plasmid DNA is seen.</li>
	<li>Verify the parasite fluorescence through flow cytometry.
	<ol>
		<li>Centrifuge 1 mL of the stationary-phase culture at 2,000 x g for 3 min at RT. Wash twice in PBS and resuspend the final pellet in 1 mL of PBS. 
		NOTE: The fluorescence of eGFP can be detected in the FL1 channel of most commercial cytometers. Gain settings for forward and side scatter, and the FL1 channel may vary between cytometers. For this example, a CyFlow space (Sysmex) cytometer was used.</li>
		<li>Run the samples, collecting 20,000 events at a speed of 0.5 &#181;L/s, and set the gain values for the forward scatter (FSC), side scatter (SSC), and fluorescence 1 (FL-1) channels as 225.0, 200.0, and 520.0, respectively. Run a control sample with non-fluorescent parasites to determine the parasite population in a FSC versus SSC dot-plot.</li>
		<li>Create a gate (G1) containing the parasite population and filter the FL-1 channel through that gate for determining the autofluorescence of promastigotes and setting a range gate (G2) for the FL-1 channel.</li>
		<li>Run the transfected parasites to verify if there is fluorescence. Record the percentage of parasites in G1 that are fluorescent and the mean of fluorescence intensity in G2.</li>
	</ol>
	</li>
</ol>
5. Confirmation of genomic integration
NOTE: Integration of the pLEXSY-eGFP expression cassette can be confirmed by diagnostic PCR, using a forward primer hybridizing to the expression cassette (varies depending on the pLEXSY-2.1 vector used) and the reverse primer F3002 (5&#39;-CTGCAGGTTCACCTACAGCTAC-3&#39;) hybridizing to a chromosomal ssu-flanking sequence absent in the expression cassette. In this example, the F2999 forward primer (5&#39;-CCTAGTATGAAGATTTCGGTGATC-3&#39;) is used as it hybridizes in the pLEXSY-sat2.1 expression cassette. A schematic representation of the integrated expression cassette and primer annealing sites for diagnostic PCR is depicted in Figure 2.
<ol>
	<li>Purify the genomic DNA from 2-5 mL of a stationary-phase parasite culture by conventional phenol/chloroform extraction35 or with a commercial kit.</li>
	<li>Perform the diagnostic PCR for pLEXSY-sat2.1 using a cycling protocol consisting of an initial denaturation of 2 min at 94 &#176;C, followed by 30 cycles of 30 s at 94 &#176;C, 30 s at 53 &#176;C, and 1 min at 72 &#176;C. Run a final extension step of 10 min at 72 &#176;C. The primers are added at a final concentration of 0.2 &#181;M.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64939/64939fig02.jpg" /> 
Figure 2: Schematic representation of the integrated expression cassette. Boxes labeled as Chr-S (gray) represent the adjacent chromosomal sequences of the 18S rRNA locus (ssu) into which the expression cassette is integrated. The integrated expression cassette consists of 5&#39; and 3&#39; sequences (blue) for homologous recombination into the ssu locus, an additional Leishmania ribosomal promoter (red) for enhanced protein production, the eGFP gene (green), a selection marker gene (yellow), and three untranslated regions (light gray), namely utr1, 2, and 3, which provide the splicing signals for post-transcriptional mRNA processing for optimized expression of eGFP and the selection marker in Leishmania parasites. Annealing sites for forward and reverse primers used for verification of the insert presence by colony PCR (step 1.2.5) are marked by the black arrows labeled as cpcr-fwd (EGFP1 primer) and cpcr-rev (A264 primer) respectively, which delimit an 859 bp product. The annealing sites for forward and reverse primers used for the verification of genomic integration by diagnostic PCR (step 5) are marked by the black arrows labeled as sat-fwd (F2999 primer) and ssu-rev (F3002 primer), respectively, which delimit a 2,271 bp product. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64939/64939fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Cloning by limiting dilution (optional)
<ol>
	<li>After confirmation of fluorescence through flow cytometry, prepare a dilution of recombinant parasites at a concentration of five promastigotes/mL36.</li>
	<li>Add 100 &#181;L of this dilution into each well of a 96-well plate. In this manner, the plates are seeded at an average density of 0.5 promastigotes/well, which ensures that some wells receive a single parasite while minimizing the probability of having more than one parasite per well36.</li>
	<li>Leave the plate undisturbed for at least 12 h. Follow the plate microscopically at a minimum magnification of 100x until wells with parasite growth are detected.</li>
	<li>When a plate well is full of parasites, use the content to inoculate a 5 mL culture. This takes 1-2 weeks.</li>
	<li>When clone-seeded cultures reach the stationary phase, verify fluorescence using flow cytometry, as described in step 4.3. Select the clones to be used for further in vitro and in vivo assays based on the percentage of fluorescent parasites and the mean fluorescence intensity measured in the FL-1 channel. Aim for clones with 98%-99% of fluorescent parasites and select those with the highest mean fluorescence intensity.</li>
</ol>

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K., Sundar, S., Dube, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+Leishmania+donovani+clinical+isolates+expressing+green+fluorescent+protein+constitutively+for+rapid+and+reliable+ex+vivo+drug+screening.">Transgenic Leishmania donovani clinical isolates expressing green fluorescent protein constitutively for rapid and reliable ex vivo drug screening.</a> Journal of Antimicrobial Chemotherapy. 64 (2), 370-374 (2009).</li><li>De Rycker, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+a+high-throughput+high-content+intracellular+Leishmania+donovani+assay+with+an+axenic+amastigote+assay.">Comparison of a high-throughput high-content intracellular Leishmania donovani assay with an axenic amastigote assay.</a> Antimicrobial Agents and Chemotherapy. 57 (7), 2913-2922 (2013).</li><li>Pe&#241;a, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+compound+sets+identified+from+high+throughput+phenotypic+screening+against+three+kinetoplastid+parasites:+an+open+resource.">New compound sets identified from high throughput phenotypic screening against three kinetoplastid parasites: an open resource.</a> Scientific Reports. 5, 8771 (2015).</li><li>Bolhassani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Leishmania+species:+development+of+stable+GFP+expression+and+its+application+for+in+vitro+and+in+vivo+studies.">Fluorescent Leishmania species: development of stable GFP expression and its application for in vitro and in vivo studies.</a> Experimental Parasitology. 127 (3), 637-645 (2011).</li><li>Breitling, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-pathogenic+trypanosomatid+protozoa+as+a+platform+for+protein+research+and+production.">Non-pathogenic trypanosomatid protozoa as a platform for protein research and production.</a> Protein Expression and Purification. 25 (2), 209-218 (2002).</li><li>Pulido, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+the+green+fluorescent+protein+reporter+system+in+Leishmania+spp.+for+the+in+vitro+and+in+vivo+screening+of+antileishmanial+drugs.">Improvement of the green fluorescent protein reporter system in Leishmania spp. for the in vitro and in vivo screening of antileishmanial drugs.</a> Acta Tropica. 122 (1), 36-45 (2012).</li><li>Bastos, M. S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achievement+of+constitutive+fluorescent+pLEXSY-egfp+Leishmania+braziliensis+and+its+application+as+an+alternative+method+for+drug+screening+in+vitro.">Achievement of constitutive fluorescent pLEXSY-egfp Leishmania braziliensis and its application as an alternative method for drug screening in vitro.</a> Mem&#243;rias do Instituto Oswaldo Cruz. 112 (2), 155-159 (2017).</li><li>Vincze, T., Posfai, J., Roberts, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NEBcutter:+A+program+to+cleave+DNA+with+restriction+enzymes.">NEBcutter: A program to cleave DNA with restriction enzymes.</a> Nucleic Acids Research. 31 (13), 3688-3691 (2003).</li><li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+beacon-based+approach+for+live-cell+imaging+of+RNA+transcripts+with+minimal+target+engineering+at+the+single-molecule+level.">A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level.</a> Scientific Reports. 7 (1), 1550 (2017).</li><li>Green, M., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. Fourth Edition. , (2012).</li><li>Santar&#233;m, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+distinct+culture+media+in+Leishmania+infantum+biology+and+infectivity.">The impact of distinct culture media in Leishmania infantum biology and infectivity.</a> Parasitology. 141 (2), 192-205 (2014).</li><li>Medina-Acosta, E., Cross, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+DNA+from+trypanosomatid+protozoa+using+a+simple+&amp;#34;mini-prep&amp;#34;+procedure.">Rapid isolation of DNA from trypanosomatid protozoa using a simple &amp;#34;mini-prep&amp;#34; procedure.</a> Molecular and Biochemical Parasitology. 59 (2), 327-329 (1993).</li><li>Ye, M., Wilhelm, M., Gentschev, I., Szalay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+limiting+dilution+method+for+monoclonal+stable+cell+line+selection+using+a+real-time+fluorescence+imaging+system:+a+practical+workflow+and+advanced+applications.">A modified limiting dilution method for monoclonal stable cell line selection using a real-time fluorescence imaging system: a practical workflow and advanced applications.</a> Methods and Protocols. 4 (1), 16 (2021).</li><li>Varela, M. R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leishmania+(Viannia)+panamensis:+an+in+vitro+assay+using+the+expression+of+GFP+for+screening+of+antileishmanial+drug.">Leishmania (Viannia) panamensis: an in vitro assay using the expression of GFP for screening of antileishmanial drug.</a> Experimental Parasitology. 122 (2), 134-139 (2009).</li><li>Komura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+stress+induced+impaired+TLR+signaling+and+macrophage+differentiation+of+human+monocytes.">ER stress induced impaired TLR signaling and macrophage differentiation of human monocytes.</a> Cellular Immunology. 282 (1), 44-52 (2013).</li></ol>

The authors have nothing to disclose.

The advantages and disadvantages of various reporter genes have been studied in several protozoan parasites. Among them, GFP and eGFP are intrinsically fluorescent and allow easy quantification and imaging. The fluorescent activity of these proteins can be detected with minimal manipulation using fluorescence microscopy, fluorimetry, or flow cytometry. Few studies have been carried out for generating GFP-expressing L. (Viannia) strains, despite the demonstrated robustness of GFP and their derivatives as reporter genes in Old World Leishmania species15,16,18,24. Varela et al.37 developed L. panamensis strains that express GFP as an episomal transgene. Flow cytometry analysis of both axenic promastigotes and intracellular amastigotes showed the usefulness of these parasites for drug screening assays. However, fluorescence was very heterogeneous, and the number of fluorescent parasites in the transfected population was dependent on constant selective pressure with the antibiotic tunicamycin. These facts restrict using these parasites as intracellular amastigotes for drug-screening assays, given that tunicamycin cannot be added to infected macrophages38, leaving parasites with low fluorescence present in the population of intracellular amastigotes that could bias the quantification of infected macrophages.
The use of vectors such as pLEXSY constitutes a powerful and reliable tool for the stable modification of Leishmania species parasites through the integration of transgenes by means of homologous recombination28. The pLEXSY cassette is integrated into the 18S rRNA locus (ssu), whose transcription is under the control of RNA polymerase I strong promoter, leading to higher transcription rates20. The use of this system has proven to allow stable and homogeneous expression of the target protein27. These characteristics favor the expression of eGFP and its use in intracellular assays with homogenous levels of fluorescence in infected cells, as well as its use for experimental infections in murine models. The pLEXSY-2.1 vector family used in this protocol has been designed with an additional Leishmania ribosomal promoter in front of the expression cassette, enhancing protein production29,30.
In this protocol, there are several critical steps for obtaining optimal results. Firstly, cloning of the target gene should be performed using a high-fidelity polymerase33, and digestion of the target DNA and expression vector should be done using a sequential digestion protocol when using the BglII/KpnI combination for cloning. The latter is because there is no buffer in which both BglII and KpnI exhibit optimal activity, although both enzymes are optimally active at the same temperature (37 &#176;C). Secondly, the selection of recombinant clones after transformation in E. coli should be carried out at 30 &#176;C, as pLEXSY plasmids are more stable at this temperature in E. coli28,29. Additionally, the growth rates of L. panamensis and L. donavani could be quite different. During the performance of the experiments described in this article, L. panamensis usually took longer to reach the stationary phase. Growth rates can also vary from strain to strain in different laboratories; therefore, it is necessary to characterize the growth rates of the laboratory strains for estimation of the time required to reach the parasite concentration required for electroporation. Using the buffer reported by Bolhassani et al.27 for electroporation is an important modification that significantly increases the survival of the electroporated parasites in comparison with direct electroporation in a culture medium, as suggested by the original Jena Bioscience protocol. During polyclonal selection, it is extremely important not to let the electroporated cultures overgrow before adding the selective antibiotic; otherwise, non-recombinant parasites will outgrow the recombinant ones27. It will take one or two consecutive passages (1:10 inoculum) into fresh medium with the appropriate antibiotic to get a turbid culture of antibiotic-resistant recombinant parasites and a clear negative control. Passages should be done within 5 days of the first antibiotic addition. Finally, there are notably differences in the efficiency of transfection and integration of the pLEXSY expression cassette between L. panamensis and L. donovani24,37. While in L. donovani almost 98% of the total population analyzed through flow cytometry after polyclonal selection shows fluorescence, in L. panamesis, the percentage of fluorescent parasites is around 80%. For this reason, the optional step of cloning by limiting dilution is a modification mostly required to obtain an L. panamensis population where more than 95% of the parasites analyzed through flow cytometry are fluorescent. The use of pLEXSY plasmids is limited to genomic modification of Leishmania species, as they were originally designed for using L. tarentolae as protein production platforms28. Although pLEXSY has proven to work in several Leishmania species different from L. tarentolae21,24,27, it is important to verify the conservation of the ssu sequence when working with a new Leishmania species to make sure that recombination sites in the pLEXSY plasmid will work.
The method described in this article allows the production of recombinant Leishmania parasites with a constitutive and stable expression of eGFP or any other reporter of interest. In these parasites, fluorescence is homogeneous, and it is maintained in intracellular forms. Therefore, strains generated through this process could be used for standardizing high-throughput drug-screening assays to evaluate the potential anti-leishmania activity of molecules of natural and synthetic origin.

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with a wide range of clinical manifestations. This disease is prevalent in 98 countries, and its annual incidence is estimated at 0.9 to 1.6 million cases1. Leishmania species that are pathogenic to humans are divided into two subgenera, namely L. (Leishmania) and L. (Viannia). Infection with some species belonging to the L. (Leishmania) subgenus, such as L. donovani and L. infantum, may result in visceral leishmaniasis (VL), which is fatal if left untreated2. Species belonging to the L. (Viannia) subgenus are associated with most cases of cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) in Central and South America, particularly in Panama, Colombia, and Costa Rica, with L. panamensis being the main etiological agent of these clinical presentations3,4.
Existing anti-leishmania chemotherapy that includes drugs such as pentavalent antimonials, miltefosine, and amphotericin B is highly toxic and expensive. Furthermore, increased drug resistance during recent decades has been added to the factors that interfere with the effective treatment of patients worldwide5. Substantial differences have been demonstrated across species of the genus Leishmania in relation to drug susceptibility, especially between New and Old World species6,7. For these reasons, it is necessary to direct efforts to the identification and development of new anti-leishmania drugs, paying special attention to species-specific approaches. Studying large libraries of drug candidates with traditional methodologies is quite difficult, given that these methodologies are very laborious for evaluating the activity of compounds against intracellular amastigotes or for performing in vivo experiments8; therefore, it has been necessary to develop new techniques that reduce these disadvantages, including the implementation of reporter genes and development of high-content phenotypic screening assays9.
The use of reporter genes has shown the potential to increase the efficiency of the drug screening process as it facilitates the development of high-throughput and in vivo assays. Recombinant Leishmania parasites expressing several reporter genes have been generated by various research groups. Reporter genes, such as &#946;-galactosidase, &#946;-lactamase, and luciferase, have been introduced in several Leishmania species using episomal vectors, showing limited utility for drug screening in extra- and intra-cellular forms of the parasite10,11,12,13,14,15. These approaches have the limitation of requiring a strong selective pressure in culture to avoid the elimination of the episomal construct, as well as the use of additional reagents to reveal the activity of the reporter gene. Conversely, the green fluorescent protein (GFP) and its variant, the enhanced green fluorescent protein (eGFP), have been used in the generation of a large number of transgenic Leishmania strains for in vitro drug screening assays due to their flexibility and sensitivity, as well as the possibility of automating the screening process using flow cytometry or fluorometry15,16,17,18,19. Despite promising results, cultures of these transgenic strains were highly heterogeneous in their fluorescence levels, since the number of copies of the GFP gene was not the same in all the parasites. Furthermore, maintaining fluorescence required constant selective pressure on the parasites in culture, since the GFP gene was introduced in an episomal construct.
For the reasons stated before, many efforts have focused on developing new methodologies for producing stable recombinant strains. These efforts have mostly relied on the integration of reporter genes into ribosomal loci, taking advantage of the higher transcription rates of ribosomal genes20. Strains of L. infantum and L. amazonensis have been generated having integrated the genes coding for &#946;-galactocidase21, IFP 1.4, iRFP22, and tdTomato23, and they have been evaluated for their usefulness in drug screening assays. Various groups have developed L. donovani strains that express GFP constitutively by integrating its coding gene into the 18S ribosomal RNA locus (ssu locus) through homologous recombination24,25; they showed stable and homogeneous GFP expression in the transfected population, including intracellular amastigotes24,25, and they were successfully implemented in drug screening assays24,25,26. Bolhassani et al.27 developed strains of L. major and L. infantum expressing GFP as an integrated transgene. They used the integration vector pLEXSY, originally designed for the transgenic expression of proteins in a system using L. tarentolae as the host28. The pLEXSY-GFP vector has shown to be very efficient for the generation of different Leishmania strains constitutively expressing GFP24,25,27,29,30. In these parasites, fluorescence is homogeneous and maintained in the intracellular forms, being able to be detected in footpad lesions of infected mice27.
In this work, we describe the methodology used for generating L. panamensis and L. donovani strains expressing the gene encoding for eGFP as an integrated transgene using the pLEXSY system. The strains generated through this process are used in our laboratory for performing drug-screening assays that evaluate the potential anti-leishmania activity of molecules of natural and synthetic origin.

<table><tbody><tr><td>96 Well Microplates</td><td>Corning</td><td>CLS3340</td><td>Flat bottom clear, black polystyrene, sterile, lid</td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A4718</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma-Aldrich</td><td>A8351</td><td>BioXtra, suitable for cell culture</td></tr><tr><td>&lt;em&gt;Bgl&lt;/em&gt;II restriction enzyme</td><td>New England BioLabs</td><td>R0144S</td><td>2,000 units. 10,000 units/mL</td></tr><tr><td>Cell Culture Flasks</td><td>Corning</td><td>CLS430168</td><td>Surface area 25 cm&lt;sup&gt;2&lt;/sup&gt;, canted neck, cap (plug seal)</td></tr><tr><td>ChemiDoc Imaging System</td><td>Bio-Rad</td><td>17001401</td><td></td></tr><tr><td>CyFlow Space</td><td>Sysmex</td><td>Not available</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td>powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, &amp;ge;99.5%</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>Gel Loading Buffer</td><td>Sigma-Aldrich</td><td>G2526</td><td>&amp;nbsp;The rate of migration varies with gel composition. Dilute 1:3 to 1:6 with sample before loading.</td></tr><tr><td>Gene Pulser Xcell Electroporation System</td><td>Bio-Rad</td><td>1652660</td><td>The system is composed of a main unit, two accessory modules, the capacitance extender (CE module) and the pulse controller (PC module), and a ShockPod cuvette chamber.</td></tr><tr><td>Gene Pulser/MicroPulser Electroporation Cuvettes</td><td>Bio-Rad</td><td>1652086</td><td>Pkg of 50, 0.2 cm&amp;ndash;gap sterile electroporation cuvette, for use with the Gene Pulser and MicroPulser Systems, for mammalian and other eukaryotic cells</td></tr><tr><td>Gentamicin solution</td><td>Sigma-Aldrich</td><td>G1397</td><td>50 mg/mL in deionized water, liquid, 0.1 &amp;mu;m filtered, BioReagent, suitable for cell culture</td></tr><tr><td>GoTaq Long PCR Master Mix</td><td>Promega</td><td>M4021</td><td></td></tr><tr><td>HEPES solution</td><td>Sigma-Aldrich</td><td>H0887</td><td>1 M, pH 7.0-7.6, sterile-filtered, BioReagent, suitable for cell culture</td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IXplore Standard</td><td></td></tr><tr><td>&lt;em&gt;Kpn&lt;/em&gt;I-HF restriction enzyme</td><td>New England BioLabs</td><td>R3142S</td><td>4,000 units. 20,000 units/mL</td></tr><tr><td>LB Broth with agar</td><td>Sigma-Aldrich</td><td>L3147</td><td>Highly-referenced nutrient-rich microbial growth powder medium with Agar, suitable for regular&amp;nbsp;E.coli&amp;nbsp;culture.</td></tr><tr><td>LB Broth&amp;nbsp;</td><td>Sigma-Aldrich</td><td>L2542</td><td>Liquid microbial growth medium</td></tr><tr><td>Mini-Sub Cell GT Horizontal Electrophoresis System</td><td>Bio-Rad</td><td>1640300</td><td>Mini horizontal electrophoresis system, includes 8- and 15-well combs, 7 cm x 10 cm UV-transparent tray</td></tr><tr><td>pEGFP-N1-1x</td><td>Addgene</td><td>172281</td><td>Expressing eGFP mRNA fused with 1 tandem repeat of a 50-base sequence</td></tr><tr><td>pLEXSYcon2.1 expression kit</td><td>Jena Bioscience</td><td>EGE-1310sat</td><td>Contains integrative constitutive expression vector pLEXSY-sat2.1. Antibiotic selection of transfectants with Nourseothricin (NTC, clonNAT). Contains all primers for diagnostic PCRs and sequencing.</td></tr><tr><td>Potassium Chloride</td><td>Millipore</td><td>529552</td><td>Molecular Biology Grade - CAS 7447-40-7 - Calbiochem</td></tr><tr><td>PureYield Plasmid Miniprep System</td><td>Promega</td><td>A1222</td><td>Up to 15 &amp;mu;g of Transfection-Ready Plasmid from 3 mL cultures.</td></tr><tr><td>Schneider&amp;prime;s Insect Medium</td><td>Sigma-Aldrich</td><td>S0146</td><td>Medium used in our laboratory for culturing Leishmania.</td></tr><tr><td>SOC Medium</td><td>Sigma-Aldrich</td><td>S1797</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S3014</td><td>for molecular biology, DNase, RNase, and protease, none detected, &amp;ge;99% (titration)</td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich</td><td>RDD038</td><td>BioReagent, suitable for cell culture, suitable for insect cell culture, &amp;ge;99.0%, free-flowing, Redi-Dri</td></tr><tr><td>SwaI restriction enzyme</td><td>New England BioLabs</td><td>R0604S</td><td>2,000 units. 10,000 units/mL</td></tr><tr><td>Syringe filters</td><td>Corning</td><td>CLS431212</td><td>regenerated cellulose membrane, diam. 4&amp;nbsp;mm, pore size 0.2&amp;nbsp;&amp;mu;m</td></tr><tr><td>T100 Thermal Cycler</td><td>Bio-Rad</td><td>1861096</td><td>Thermal cycler system, includes 96-well thermal cycler, power cord, tube support ring</td></tr><tr><td>T4 DNA Ligase</td><td>Promega</td><td>M1801</td><td>Joins two DNA strands with cohesive or blunt ends</td></tr><tr><td>Tris-Borate-EDTA buffer</td><td>Sigma-Aldrich</td><td>T4415</td><td>BioReagent, suitable for electrophoresis, 10&amp;times; concentrate</td></tr><tr><td>Wizard Genomic DNA Purification Kit</td><td>Promega</td><td>A1120</td><td></td></tr><tr><td>Wizard SV Gel and PCR Clean-Up System</td><td>Promega</td><td>A9285</td><td></td></tr><tr><td>XL10-Gold Ultracompetent Cells</td><td>Agilent</td><td>200317</td><td>XL10-Gold Kanr Ultracompetent Cells, 10 x 0.1 mL. Features the kanamycin-resistance gene on the F&#039; episome, for extremely demanding cloning in chloramphenicol-resistant vectors. Efficiency: &gt; 5 x 10 9 transformants/&amp;micro;g pUC18 DNA.</td></tr></tbody></table>

development of leishmania species strains with constitutive expression of egfp

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people worldwide. Infection with L. donovani can result in fatal visceral disease. In Panama, Colombia, and Costa Rica, L. panamensis is responsible for most of the reported cases of cutaneous and mucocutaneous leishmaniasis. Studying a large number of drug candidates with the methodologies available to date is quite difficult, given that they are very laborious for evaluating the activity of compounds against intracellular forms of the parasite or for performing in vivo assays. In this work, we describe the generation of L. panamensis and L. donovani strains with constitutive expression of the gene that encodes for an enhanced green fluorescent protein (eGFP) integrated into the locus that encodes for 18S rRNA (ssu). The gene encoding eGFP was obtained from a commercial vector and amplified by polymerase chain reaction (PCR) to enrich it and add restriction sites for the BglII and KpnI enzymes. The eGFP amplicon was isolated by agarose gel purification, digested with the enzymes BglII and KpnI, and ligated into the Leishmania expression vector pLEXSY-sat2.1 previously digested with the same set of enzymes. The expression vector with the cloned gene was propagated in E. coli, purified, and the presence of the insert was verified by colony PCR. The purified plasmid was linearized and used to transfect L. donovani and L. panamensis parasites. The integration of the gene was verified by PCR. The expression of the eGFP gene was evaluated by flow cytometry. Fluorescent parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity were selected using flow cytometry.

After building the pLEXSY-eGFP construct and transforming competent E. coli cells, colonies containing the construct with the eGFP insert will generate an approximately 859 bp product after running the colony PCR described in section 1.2 (Figure 3A). Total digestion of the purified plasmid from positive colonies using SwaI should give two characteristic fragments in gel electrophoresis, a 2.9 kbp fragment that is the portion of the PLEXSY vector containing all the necessary elements for replication and selection in E. coli, and a 7-8 kpb fragment representing the linearized expression cassette that is used for transfection (Figure 3B).
Following transfection, the polyclonal selection is made mostly qualitatively. The status of the parasite cultures during selection with antibiotics is monitored microscopically, bringing special attention to parasite morphology and motility. After successful recovery of a culture of transfected parasites, those effectively expressing eGFP should be detected in the FL-1 channel of a flow cytometer (Figure 4A); transfection efficiencies may vary between different Leishmania species. In this work, after polyclonal selection, 98.44% of L. donovani parasites analyzed by flow cytometry were fluorescent in comparison with 82.00% of L. panamensis (Figure 4B). If the pLEXSY-eGFP expression cassette has been successfully integrated into the ssu locus, a 2.3 kbp product should be observed after running the diagnostic PCR described in section 5 (Figure 4C). These results ensure that recombinant parasites are obtained that constitutively express eGFP as an integrated transgene and will remain stable in subsequent passages of the parasite culture.
Cloning by limiting dilution allows the enrichment of the fluorescent parasite population with lower transfection efficiencies, such as L. panamensis, as well as the selection of clones with the highest fluorescence intensities. Four clones were obtained for each of the Leishmania species used in this work (Table 1), namely Lpan-A7, F11, F12, and H2 for L. panamensis, and Ldon-C1, G4, F2, and H1 for L. donovani. Clones with the highest percentage of fluorescent parasites (Lpan-F11 = 95.74%; Ldon-C1 = 99.16%) and mean fluorescence intensity (Lpan-F11 = 35.63 relative fluorescent units [RFU]; Ldon-C1 = 14.12 RFU) were selected for further use in drug screening assays.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64939/64939fig03.jpg" /> 
Figure 3: Representative results of preparation of the pLEXSY-eGFP construct for transfection. (A) Representative results of the colony PCR. Lane 1: 1 kb ladder (units in base pairs); lanes 2, 10, and 12: negative samples for the presence of the eGFP insert; lanes 3-9 and 13-14: positive samples for the presence of the eGFP insert. (B) Results of the linearization with SwaI. Lane 1: 1 kb ladder (units in base pairs); lanes 2-4: digestion reactions of plasmids purified from three different colonies positive for the presence of the eGFP insert. A 2.9 kbp fragment representing the elements necessary for replication and selection in E. coli, and a 7-8 kpb fragment representing the linearized expression cassette are observed. Both gels were prepared at an agarose concentration of 1%. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64939/64939fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64939/64939fig04.jpg" /> 
Figure 4: Representative results of confirmation of eGFP expression and chromosomal insertion of the expression cassette. (A) Flow cytometry histogram in the FL-1 channel. Red: wild-type cells without eGFP; Blue: transfected L. panamensis parasites with 82.00% of the population expressing eGFP. (B) Comparison of transfection results between L. panamensis (Lpan-1) and L. donovani (Ldon-1). A higher transfection efficiency was observed in L. donovani (98.44%) than L. panamensis (82.00%) after polyclonal selection. (C) Results of the PCR for confirmation of genomic integration. L: 100 bp ladder (units in base pairs); lanes 1-4: samples from four different clones of L. panamensis showing a characteristic 2.3 kbp product for pLEXSY-sat2.1 integration into the ssu locus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64939/64939fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Species</td>
			<td>Clone code</td>
			<td>Percentage of fluorescent parasites</td>
			<td>Mean fluorescence intensity*</td>
		</tr>
		<tr>
			<td rowspan="4">L. panamensis</td>
			<td>Lpan-A7</td>
			<td>90.00</td>
			<td>24.70</td>
		</tr>
		<tr>
			<td>Lpan-F11</td>
			<td>95.74</td>
			<td>35.63</td>
		</tr>
		<tr>
			<td>Lpan-F12</td>
			<td>92.85</td>
			<td>34.44</td>
		</tr>
		<tr>
			<td>Lpan-H2</td>
			<td>85.00</td>
			<td>20.66</td>
		</tr>
		<tr>
			<td rowspan="4">L. donovani</td>
			<td>Ldon-C1</td>
			<td>99.16</td>
			<td>14.12</td>
		</tr>
		<tr>
			<td>Ldon-G4</td>
			<td>99.21</td>
			<td>13.96</td>
		</tr>
		<tr>
			<td>Ldon-F2</td>
			<td>97.96</td>
			<td>11.67</td>
		</tr>
		<tr>
			<td>Ldon-H1</td>
			<td>98.97</td>
			<td>12.90</td>
		</tr>
	</tbody>
</table>
Table 1: Clones obtained by limiting dilution. Four clones were obtained by limiting dilution for each of the Leishmania species used in this work. Percentage of fluorescent parasites and mean fluorescence intensity are reported for each clone. *Fluorescence is expressed in relative fluorescence units (RFU).

Watch this Scientific Journal Video about Development of Leishmania Species Strains with Constitutive Expression of eGFP at JoVE.com

Development of Leishmania Species Strains with Constitutive Expression of eGFP

Here, we describe the methodology used for generating L. panamensis and L. donovani strains expressing the gene for eGFP as a stable integrated transgene using the pLEXSY system. Transfected parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity in both species were selected for further use in drug screening assays.

Development of Leishmania Species Strains with Constitutive Expression of eGFP

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people ...

development-leishmania-species-strains-with-constitutive-expression

Nanyang Technological University

Research

JoVE Journal

Genetics

710 Views.  Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP). Dit protocol is de sleutel voor het genereren van stammen die kunnen worden gebruikt in high-throughput screening assays om het potentieel en de traditionele activiteit van moleculen te evalueren Juana Quintero en Laura Pineda, onderzoeksassistenten van mijn laboratorium, zullen de procedure demonstreren. Om te beginnen, amplify het doelgen met behulp van een high fidelity polymerase om de coderende sequentie te behouden. Voeg de primers toe aan het reactiemengsel in een eindconcentratie van ...

Video: Development of Leishmania Species Strains with Constitutive Expression of eGFP

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Immunology and Infection

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Biochemistry

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to visceral lesions. Currently, 12 million people are estimated to be infected with Leishmania worldwide and over 1 billion people live at the risk of infection. Leishmania amazonensis is endemic in Central and South America and usually leads to the cutaneous form of the disease, which can be directly visualized in an animal model. Therefore, L. amazonensis strains are good models for cutaneous leishmaniasis studies because they are also easily cultivated in vitro. C57BL/6 mice mimic the L. amazonensis-driven disease progression observed in humans and are considered one of the best mice strains model for cutaneous leishmaniasis. In the vertebrate host, these parasites inhabit macrophages despite the defense mechanisms of these cells. Several studies use in vitro macrophage infection assays to evaluate the parasite infectivity under different conditions. However, the in vitro approach is limited to an isolated cell system that disregards the organism&#39;s response. Here, we compile an in vivo murine infection method that provides a systemic physiological overview of the host-parasite interaction. The detailed protocol for the in vivo infection of C57BL/6 mice with L. amazonensis comprises parasite differentiation into infective amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. We propose this well-established method as the most adequate method for physiological studies of the host immune and metabolic responses to cutaneous leishmaniasis.

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While biophysical approaches help understand pore formation, they often rely on reductionist approaches lacking the full complement of membrane lipids and proteins. Cultured human cells provide an alternative system, but their complexity and redundancies in repair mechanisms make identifying specific mechanisms difficult. In contrast, the human protozoan pathogen responsible for cutaneous leishmaniasis, Leishmania major,&#160;offers an optimal balance between complexity and physiologic relevance. L. major is genetically tractable and can be cultured to high density in vitro, and any impact of perturbations on infection can be measured in established murine models. In addition, L. major synthesizes lipids distinct from their mammalian counterparts, which could alter membrane dynamics. These alterations in membrane dynamics can be probed with PFTs from the best-characterized toxin family, cholesterol-dependent cytolysins (CDCs). CDCs bind to ergosterol in the Leishmania membrane and can kill L. major promastigotes, indicating that L. major is a suitable model system for determining the cellular and molecular mechanisms of PFT function. This work describes methods for testing PFT function in L. major promastigotes, including parasite culture, genetic tools for assessing lipid susceptibility, membrane binding assays, and cell death assays. These assays will enable the rapid use of L. major as a powerful model system for understanding PFT function across a range of evolutionarily diverse organisms and commonalities in lipid organization.

Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

Presented here is a protocol using Leishmania major promastigotes to determine the binding, cytotoxicity, and signaling induced by pore-forming toxins. A proof-of-concept with streptolysin O is provided. Other toxins can also be used to leverage the genetic mutants available in L. major to define new mechanisms of toxin resistance.

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While ...

Streamlined Protocol for Leishmania Translational Extract (LTE) Production

Production and Optimization of LTE, a Leishmania tarentolae Derived Cell-Free Protein Expression System for Recombinant Protein Production

This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate Leishmania tarentolae, referred to as Leishmania Translational Extract or LTE. Although this organism originally evolved as a parasite of geckos, it can be cultivated easily and inexpensively in flasks or bioreactors. Unlike Leishmania major, it is non-pathogenic to humans and does not require special laboratory precautions. Another advantage of using Leishmania for CFPS is that the addition of a single antisense oligonucleotide to the CFPS, targeting a conserved splice leader sequence on the 5'-end of all protein-coding RNAs, can suppress endogenous protein expression. We provide procedures for cell disruption and lysate processing, which have been simplified and improved compared to previous versions. These procedures start with simple flask cultures. Additionally, we explain how to introduce genetic information using vectors containing species-independent translation initiation sites (SITS) and how to perform straightforward batch optimization and quality control to ensure consistent protein expression quality.

Leishmania Translational Extract (LTE) is a eukaryotic cell-free protein expression system derived from the single-celled parasite, Leishmania tarentolae. This optimized protocol makes LTE simple and cost-effective to manufacture. It is suitable for various applications focused on the multiparallel expression and study of complex eukaryotic proteins and their interactions.

This protocol outlines the production and optimization of a eukaryotic Cell-Free Protein Expression System (CFPS) derived from the unicellular flagellate ...

Bioengineering

In Vivo Imaging of Leishmania infantum-infected Hamsters by Gingival Inoculation of Axenic Amastigotes Expressing Luciferase

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if not treated; the drugs used in treatment are toxic, and there are cases of resistance. Preclinical tests can represent a bottleneck in discovering new medicines for treatment, depending on the animal model, the strain used, and the inoculum route. The golden hamster stands out for its high susceptibility to subgenera Viannia and Leishmania species, displaying many of the clinical and immunopathological processes observed in human disease.
By hamster anatomy, which has a short tail and limbs, the intracardiac route is usually the choice for intravenous injection of Leishmania. However, it is an inoculum that can lead to bleeding and eventually to animal death. Thus, we standardized an alternative intravenous inoculation route for infection at the gingival vein, which is minimally invasive, allows easy venous access, and causes few local and systemic injuries to the animal. Therefore, hamsters infected by the intraperitoneal (IP) or intragingival (IG) route with Leishmania Infantum expressing luciferase (Luc) were followed up for 22 days by the bioluminescence imaging system and 50 days and 8 months post infection by PCR.
After gingival inoculation of both axenic amastigotes and promastigotes of L. infantum-Luc, bioluminescence was restricted for at least 2 weeks at the site of injection, which is an indicator of infection in the tissues around the gingival plexus. Hamsters infected intraperitoneally with L. infantum-Luc displayed bioluminescence dispersed throughout the abdomen, as expected. However, by the bioluminescence imaging system infection declined until the 50th dpi and was only detectable by PCR.&#160;Axenic amastigotes showed better infection than promastigotes, as evaluated by PCR. Indeed, 8 months after infection, parasites were detected by PCR in the liver of animals inoculated with axenic amastigotes by the intravenous route, which can be a characteristic of the reference strain of L. infantum MHOM/BR/1974/PP75, whose infection progresses slowly and display low parasite burden, below the bioluminescent imaging resolution. Thus, axenic amastigotes can be a better choice for infection and follow-up than promastigotes, and the gingival inoculum is a feasible route for intravenous injection of Leishmania and other pathogens.

Here we present a longitudinal evaluation of golden hamsters infected intraperitoneally (IP) or via intragingival (IG) with L. infantum-Luc by bioluminescent imaging and by PCR. Hamsters were evaluated 1 day post infection (1 dpi), 1 week after infection (8 dpi), and 3 weeks after infection (22 dpi) and euthanized at the 50th dpi and 8 months post infection.

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if ...

Biology

Assessment of Leishmania Virulence within Cultured Macrophages

Source: Sarkar, A. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57486">Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of&#160;Leishmania&#160;Parasites.</a>&#160;J. Vis. Exp.&#160;(2018)
This video investigates Leishmania&#39;s virulence by treating macrophages with metacyclic promastigotes, resulting in the formation of intracellular amastigotes. The confirmation of Leishmania&#39;s virulence is achieved through nucleic acid fluorescent dye staining. This insight can contribute to the development of new therapies for leishmaniasis.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Development of Leishmania Species Strains with Constitutive Expression of eGFP

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