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Leishmania: A Brand New Chassis
One of the interesting aspects of our project is the use of a new chassis: Leishmania!
Leishmania is a genus of protozoans with about 40 species described, exclusively parasitic, that belongs to the Tripanossomatidae family. This group also comprises the genus Trypanosoma, responsible for diseases such as African trypanosomiasis (Sleeping Sickness, caused by Trypanosoma brucei) and South American trypanosomiasis (Chagas Disease, caused by Trypanosoma cruzi).
Leishmania is an intracellular parasite and presents two different life forms, in a heteroxenic life cycle (cycle is complete only when it has two hosts). Sandflies (belonging either to the genus Phlebotomus, in the Old World, or Lutzomyia, in the New World) are the invertebrate host of Leishmania. Inside the insect, the parasite is in an extracellular flagellated form, called promastigote. In mammalian hosts, Leishmania is in the amastigote form and infect macrophages.
Figure 1 - Harhay et al., 2011. Complete life cycle of Leishmania.
The complete life cycle of Leishmania can be seen in these WHO animations:
http://www.who.int/leishmaniasis/leishmaniasis-life-cycle.swf
http://www.who.int/leishmaniasis/leish-sandfly.swf
The first complete genome sequence of a Leishmania was accomplished in 2005 (Leishmania major) and, since then, five other species were also sequenced (Leishmania infantum, Leishmania braziliensis, Leishmania mexicana, Leishmania donovani and Leishmania amazonensis).
Leishmaniasis: The Dark Side…
Leishmaniasis is the disease caused by the infection of vertebrates with certain species of Leishmania. It is widespread in 98 countries, but the great majority of the cases occurs in developing countries, specially India, Bangladesh, Sudan, Ethiopia and Brazil.
Leishmaniasis can present itself as different pathologies, according to the infectant species and the host health status (Beverley, 2003). These pathologies can be classified in three different forms (WHO, 2015):
Visceral: also known as kala-azar. It is the most serious form of the disease, and can be fatal if left untreated. It is characterized by irregular bouts of fever, weight loss, enlargement of the spleen and liver, and anaemia. Visceral leishmaniasis is highly endemic in the Indian subcontinent and in East Africa. About 200,000 to 400,000 new cases occur worldwide each year, of these, over 90% of new cases occur in 6 countries: Bangladesh, Brazil, Ethiopia, India, South Sudan and Sudan.
Cutaneous: is the most common form of leishmaniasis and causes skin lesions, mainly ulcers, on exposed parts of the body, leaving life-long scars and serious disability. About 95% of the cases occur in the Americas, the Mediterranean basin, the Middle East and Central Asia. Over 2/3 of new cases occur in 6 countries: Afghanistan, Algeria, Brazil, Colombia, Iran (Islamic Republic of) and the Syrian Arab Republic. An estimated 0.7 million to 1.3 million new cases occur worldwide annually.
Mucocutaneous: leads to partial or total destruction of mucous membranes of the nose, mouth and throat. Almost 90% of mucocutaneous leishmaniasis cases occurs in Bolivia, Brazil and Peru.
Each year, around 0.3 million new cases for visceral leishmaniasis and more than 1 million new cases for cutaneous leishmaniasis are reported worldwide. Between 20,000 to 30,000 deaths occur annually. Due to the fact that the disease affects mostly poor communities, not too much effort has been applied by the scientific community in finding effective treatments and vaccines for this disease.
Figure 2 - WHO, 2015. World prevalence of leishmaniasis.
But can we bring it to light?
Of course that, in view of all harm that this parasite can cause, we do not intend to use it in its natural form. A non virulent strain of Leishmania donovani has already been created and thoroughly characterized.
To be able to work safely with this organism, we decided to use Hira Nakhasi’s FDA-approved centrin-1 double knockout Leishmania donovani. Centrin-1 is a cytoskeletal protein involved in the duplication of centrosomes in some eukaryotes.
This specific strain was made by homologous recombination with two different antibiotic-resistant genes as selectable markers ― hygromycin (hyg) and geneticin (neo), as seen below ―, as Leishmania is diploid throughout its life cycle.
Figure 3 - obtained from Selvapandiyan et al., 2004.
Nakahasi has observed that the growth arrest was selective for amastigotes, but not for promastigotes. On the other hand, the single allele disrupted has the same behavior as the wild type (WT) Leishmania. Below, we have Leishmania’s life dynamics, where both the WT and the null mutant promastigotes duplicate normally as well as the WT amastigotes. The amastigote double knockout can duplicate its DNA (both nuclear and kinetoplast), but is unable to divide itselves. The result is a multi-nuclear and multi-kinetoplastic cell, which triggers an apoptosis pathway.
Figure 4- Modified figure from Selvapandiyan et al.., 2004.
In the graphic below, we can see a comparison of a WT (+/+) and null mutant (-/-) macrophage infection at 120 h and 240 h of infection. The WT amastigotes duplicate normally, while the null mutant amastigotes are multinucleated at 120 h, and at 240 h of infection we can already see that the mutant amastigotes population inside the macrophages is already dying.
Figure 5- Figure from Selvapandiyan et al, 2004.
This proves that the L. donovani strain used throughout our work is safe to be used in vivo, for it does not replicate itself when in an amastigote stage. The use of this modified strain will enable us to benefit from the advantages of this new chassis, without the deleterious effects it causes.
Can we play with it?
Leishmania is a diploid eukaryote and its genetic manipulation does not follow the principles learnt for bacteria. Therefore, some considerations must be done if we want to genetically manipulate these organisms. Since 1990, techniques for genetic manipulation of parasites have been developing and it is now possible to mutate genes, express recombinant proteins, create knockout strains. By now, transfection can be stably achieved with a 5% efficiency in Leishmania. Homologous recombination for achieving integrative expression is also effective in these parasites and can be used to add drug resistance markers, allowing selection mechanisms. However, since Leishmania is a diploid organism, two rounds of replacement, using different selection drugs, must be performed.
Figure 6 - Adapted from Roberts, 2011. Leishmania can be genetically transformed by homologous recombination, leading to integrative expression of the desired gene. The strategy uses linearized plasmids containing parts of the sequences of known genes of the parasite flanking the sequence of the gene of interest. A resistance sequence (NEO) is also added, in order to monitor effective integration. Since Leishmania is a diploid organism, this procedure has to be performed twice, with different resistance markers (BLE). Double resistant organisms means that the gene of interest has been efficiently integrated to the parasite’s genome.
Notably, Leishmania do not present traditional transcriptional control. Genes are transcribed in a constitutive controlless manner, originating long polycistronic pre-mRNAs that are further processed by trans splicing. So, expression is controlled by other strategies, such as RNA stability, translation and protein turnover. The precise sequence elements responsible for this control have not been completely identified until the moment.
Since transcription is not the main checkpoint for expression control, promoters do not play an important role in genetic manipulation of Leishmania. Instead, elements located 5’ and 3’ of the ORF can play the main part in controlling protein expression. Artificial constructs can be designed having upstream and downstream regions similar to those of a gene expressed in the desired conditions within the organism. For example, if it is desired the constitutive expression of a recombinant protein only in the amastigote form of the parasite, it is necessary to add 5’ and 3’ sequences that flank a native protein from Leishmania expressed in these similar conditions.
For example, see the figure below.
Figure 7 - For constitutive expression of the artificial gene X, its sequence must be inserted between the 5’ and 3’ untranslated regions (UTR) sequences of the native protein A2, amastigote-specific.
Figure 8 - Through eletroporation, plasmids can be added to Leishmania’s cytoplasm. Since expression control does not occur in the transcriptional level, promoters are irrelevant to promote an artificial gene expression. Instead, upstream and downstream sequences (5’ and 3’ UTR) of known Leishmania’s genes can be added flanking the artificial gene, along with resistance genes (NEO) to monitor transformation.
Another strategy has been developed to make it possible to use traditional promoter-controlled transcriptions. It is needed to initially build a genetically modified Leishmania, expressing constitutively RNA polymerases from virus or bacteria that are capable of recognizing promoters. This can be achieved by homologous recombination. After this transformation, an episomal vector containing sequences under the control of promoters recognized by the transfected polymerases can be expressed.
Figure 9 - Traditional promoter-controlled strategies can also be adapted to express artificial genes in Leishmania. To do so, it is necessary to combine integrative transformation (to express a promotor sensitive RNA polymerase) and episomal transfection, with the gene of interest placed downstream a traditional promoter.
In spite of these differences, many other standard molecular biology procedures can be used to manipulate Leishmania. Parasites can be transfected by electroporation and typically plated and selected in semi-solid Agar plates. Remarkably, iRNA approaches seem to function poorly and perhaps not at all in Leishmania.
Figure 10 - Adapted from Beverley, 2003
Is it worth it?
Genetic manipulation of Leishmania has been successfully achieved by several studies with L. tarentolae (Bolhassani et al., 2015; Katebi et al., 2015; Pion et al., 2014; Shahbazi et al., 2015) and it shows benefits when compared to traditional chassis. L.tarentolae, although not harmful for humans, can infect reptiles. The centrin-1 knockout L. donovani strain that we are proposing have the additional advantage of being completely apathogenic.
Figure 11 - Pros of using a Leishmania chassis
Leishmania can be cultivated in labs easily;
It can grow on semi-solid medium plates;
It has an optimal culture temperature of 26° C, compatible with room temperature, therefore making it possible to be cultivated with minimum lab equipment;
It can be transformed with relative ease by large episomes;
Its genetic manipulation can be carried out in a reasonable amount of time (doubling times typically of 6-10 h);
Using Leishmania expression system, up to 30 mg/L of recombinant protein can be produced;
It can be cultivated in bioreactors, scaling up to the industrial level;
Protein expression can be life-form specific, according to the regulatory sequences placed upstream and downstream the gene of interest;
Proteins expressed in Leishmania can count on a complete eukaryotic folding and post-translational modification machinery, including a mammalian glycosylation profile.
Figure 12 - Figure Glycosylation pattern in different expression chassis. Adapted from Niimi, 2012.
References
Beverley SM. Protozomics: Trypanosomatid parasite genetics comes of age. Nat Rev Genet. 2003 Jan;4(1):11-9.
Bolhassani A, Shirbaghaee Z, Agi E, Davoudi N. VLP production in Leishmania tarentolae: A novel expression system for purification and assembly of HPV16 L1.Protein Expr Purif. 2015 Aug 21. pii: S1046-5928(15)30044-9.
Cantacessi C, Dantas-Torres F, Nolan MJ, Otranto D.The past, present, and future of Leishmania genomics and transcriptomics.Trends Parasitol. 2015 Mar;31(3):100-8.
Clayton CE.Genetic Manipulation of Kinetoplastida. Parasitology Today, 1999 vol. 15, no. 9.
Harhay MO, Olliaro PL, Costa DL, Costa CH.Urban parasitology: visceral leishmaniasis in Brazil.Trends Parasitol. 2011 Sep;27(9):403-9.
Katebi A, Gholami E, Taheri T, Zahedifard F, Habibzadeh S, Taslimi Y, Shokri F, Papadopoulou B, Kamhawi S, Valenzuela JG, Rafati S. Leishmania tarentolae secreting the sand fly salivary antigen PpSP15 confers protection against Leishmania major infection in a susceptible BALB/c mice model. Mol Immunol. 2015 Oct;67(2 Pt B):501-11.
Niimi T. Recombinant protein production in the eukaryotic protozoan parasite Leishmania tarentolae: a review. Methods Mol Biol. 2012;824:307-15.
Pion C, Courtois V, Husson S, Bernard MC, Nicolai MC, Talaga P, Trannoy E, Moste C, Sodoyer R, Legastelois I.Characterization and immunogenicity in mice of recombinant influenza haemagglutinins produced in Leishmania tarentolae. Vaccine. 2014 Sep 29;32(43):5570-6.
Roberts SC.The genetic toolbox for Leishmania parasites. Bioengineered Bugs. 2011. 2:6, 320-326
Selvapandiyan A, Debrabant A, Duncan R, Muller J, Salotra P, Sreenivas G, Salisbury JL, Nakhasi HL. Centrin gene disruption impairs stage-specific basal body duplication and cell cycle progression in Leishmania. J Biol Chem. 2004 Jun 11;279(24):25703-10.
Shahbazi M, Zahedifard F, Taheri T, Taslimi Y, Jamshidi S, Shirian S, Mahdavi N, Hassankhani M, Daneshbod Y, Zarkesh-Esfahani SH, Papadopoulou B, Rafati S.Evaluation of Live Recombinant Nonpathogenic Leishmania tarentolae Expressing Cysteine Proteinase and A2 Genes as a Candidate Vaccine against Experimental Canine Visceral Leishmaniasis. PLoS One. 2015 Jul 21;10(7).
