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
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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
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