LEXSY in Parasitology

Leishmania tarentolae is a close relative of all pathogenic Leishmania species as well as of other pathogens such as Trypanosomes, Plasmodium and Toxoplasma. Due to this evolutionary proximity, the LEXSY technology is efficiently expressing parasite proteins with


• High yields
• Correct protein folding
• Native post-translational modifications
 
Figure 18 stammbaum parasites

LEXSY was used for overexpression and purification of functional parasite proteins. Insert A: Western blot of 93 kDa J-binding protein (JBP1) of Leishmania sp. Lane 1 = host control, lane 2 = induced culture, lane 3 = non-induced culture (Courtesy of S. Vainio, NCI Amsterdam). Insert B: Coomassie stain of immunoreactive surface proteins SAG1 (28 kDa) and SAG2 (15 kDa) of Toxoplasma gondii. Lanes 1 and 5 = host controls, lanes 2 and 3 = SAG1, lane 4 = SAG 2 secreted to the culture medium (Courtesy of M. Ebert, FZMB, Erfurt)


 

In addition, the expression vectors developed for LEXSY can be used for creation of transgenic strains of other Leishmania species including L. amazonensis, L. donovani, L. infantum, L. major, L. mexicana and also Crithidia sp. as well as the plant parasite Phytomonas serpens. These features of LEXSY enable functional characterization of parasite proteins, investigation of parasite-host interactions, in vivo and in vitro screening of anti-leishmanial drugs and vaccine development.


 
Figure 19 applic parasitology
 

A: Expression and functional analysis of the catalytic domain of α-N-acetylglucosaminyltransferase from Trypanosoma cruzi (TcOGNT2cat) in LEXSY by Western blotting (left) and enzymatic activities (right). P10 = non-transfected host strain; wt = P10 expressing wild-type TcOGNT2cat; D234A and D234N = single point mutants (from Heise et al. 2009). B: Subcellular localization of ferrous iron transporter LIT1 expressed in L. amazonensis Δlit promastigotes using pLEXSY constructs. Immunofluorescence demonstrated different targeting of wild type and mutant proteins to the plasma membrane. LIT1 immunofluorescence = green, parasite DNA = blue, FITC = anti-LIT1 IF on fixed/non-permeabilized promastigotes, FITC-LIVE = anti-LIT1 IF on live promastigotes (from Jacques et al. 2010). C: EGFP imaging in L. major reporter strain stably transfected with pLEXSY-egfp construct by Epi-fluorescence microscopy of recombinant L. major promastigotes (left) and intracellular amastigotes in bone marrow-derived macrophages (right) (from Bolhassani et al. 2011). D: Expression of protozoon RabGTPases originating from L. tarentolae or P. falciparum in PCR-based In Vitro LEXSY. Coomassie stained SDS-PAGE gel loaded with EGFP-Rab GTPases eluted from a GFP binding matrix. For details see In Vitro LEXSY manual (adapted from Kovtun et al. 2010).


 

Reference listing LEXSY in parasitology

Kumar et al. (2012) Overexpression of S4D Mutant of Leishmania donovani ADF/Cofilin Impairs Flagellum Assembly by Affecting Actin Dynamics. Eukaryotic cell 11: 752
 

Balaña-Fouce et al. (2012) Indotecan (LMP400) and AM13-55: Two novel indenoisoquinolines show potential for treating visceral leishmaniasis. Antimicrobial Agents and Chemotherapy doi: 10.1128/AAC.00499-12
 
de La Llave et al. (2011) A combined luciferase imaging and reverse transcription polymerase chain reaction assay for the study of Leishmania amastigote burden and correlated mouse tissue transcript fluctuations. Cellular Microbiology 13: 81
 
Ghosh et al. (2011) Valeriana wallichii root extracts and fractions with activity against Leishmania spp. Parasitology Research 108: 861
 
Bolhassani et al. (2011) Fluorescent Leishmania species: Development of stable GFP expression and its application for in vitro and in vivo studies. Experimental Parasitology 127: 637
 
Sadlova et al. (2011) Visualisation of Leishmania donovani Fluorescent Hybrids during Early Stage Development in the Sand Fly Vector. PLoS ONE 6: 5 e19851
 
Jacques et al. (2010) Functional characterization of LIT1, the Leishmania amazonensis ferrous iron transporter. Molecular & Biochemical Parasitology 170: 28
 
Lecoeur et al. (2010) Early Curative Applications of the Aminoglycoside WR279396 on an Experimental Leishmania major-Loaded Cutaneous Site Do Not Impair the Acquisition of Immunity. Antimicrobial Agents and Chemotherapy 54: 984
 
Smirlis et al. (2009) Leishmania donovani Ran-GTPase interacts at the nuclear rim with linker histone H1. Biochemical Journal 424: 367
 
Xingi et al. (2009) 6-Br-5methylindirubin-3'oxime (5-Me-6-BIO) targeting the leishmanial glycogen synthase kinase-3 (GSK-3) short form affects cell-cycle progression and induces apoptosis-like death: Exploitation of GSK-3 for treating leishmaniasis. International Journal for Parasitology 39: 1298
 
Heise et al. (2009) Molecular analysis of a UDP-GlcNAc:polypeptide α-N-acetylglucosaminyltransferase implicated in the initiation of mucin-type O-glycosylation in Trypanosoma cruzi. Glycobiology 19: 918
 
Bhattacharya et al. (2009) Role of a differentially expressed cAMP phosphodiesterase in regulating the induction of resistance against oxidative damage in Leishmania donovani. Free Radical Biology & Medicine 47: 1494
 
Lecoeur et al. (2007) Optimization of Topical Therapy for Leishmania major Localized Cutaneous Leishmaniasis Using a Reliable C57BL/6 Model PLoS Neglected Tropical Diseases 1: e34
 
Lukeš et al. (2006) Translational initiation in Leishmania tarentolae and Phytomonas serpens (Kinetoplastida) is strongly influenced by pre-ATG triplet and its 5' sequence context. Molecular & Biochemical Parasitology 148: 125
 
Lang et al. (2005) Bioluminescent Leishmania expressing luciferase for rapid and high throughput screening of drugs acting on amastigote-harbouring macrophages and for quantitative real-time monitoring of parasitism features in living mice. Cellular Microbiology 7: 383
 
Barak et al. (2005) Differentiation of Leishmania donovani in host-free system: analysis of signal perception and response.
Molecular & Biochemical Parasitology 141: 99