The gene encoding type II DNA topoisomerase from the kinetoplastid hemoflagellated protozoan parasite Leishmania donovani (LdTOP2) was isolated from a genomic DNA library of this parasite. DNA sequence analysis revealed an ORF of 3711 bp encoding a putative protein of 1236 amino acids with no introns. The deduced amino acid sequence of LdTOP2 showed strong homologies to TOP2 sequences from other kinetoplastids, namely Crithidia and Trypanosoma spp. with estimated identities of 86 and 68%, respectively. LdTOP2 shares a much lower identity of 32% with its human homologue. LdTOP2 is located as a single copy on a chromosome in the 0.7 Mb region in the L.donovani genome and is expressed as a 5 kb transcript. 5'-Mapping studies indicate that the LdTOP2 gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon occurring at -639 from the predicted initiation site. Antiserum raised in rabbit against glutathione S-transferase fusion protein containing the major catalytic portion of the recombinant L.donovani topoisomerase II protein could detect a band on western blots at approximately 132 kDa, the expected size of the entire protein. Use of the same antiserum for immunolocalisation analysis led to the identification of nuclear, as well as kinetoplast, antigens for L.donovani topoisomerase II. The in vitro biochemical properties of the full-length recombinant LdTOP2 when overexpressed in E.coli were similar to the Mg(II) and ATP-dependent activity found in cell extracts of L.donovani.

Morphological and functional heterogeneity of the splenic macrophage (Mf) population was studied in Leishmania donovani (LD) infected BALB/c mice. On a discontinuous percoll gradient two distinct Mf populations were separated. They differed significantly in size as evident from Scanning Electron Microscopy (SEM). Morphologically, the bigger Mf (LM) showed surface projections, whereas the smaller Mf (SM) was round. As regards the antigen-presenting abilities, the LM of infected animals showed defective antigen-presenting abilities at a later stage of the disease, i.e. 6 months post infection ( 6 I-LM) but not earlier, whereas the SM population remained functionally intact throughout the course of the infection. Further, the 6 I-LM showed a much enhanced A d status as compared to their controls. Interestingly, both the 6 I-LM and the control set showed a comparable level of binding of a known A d restricted peptide. Despite the presence of sufficient A d molecules and the ability to bind the appropriate peptide, 6 I-LM were unable to stimulate peptide specific T-cell hybridoma. Further, the 6 I-LM showed an increase in membrane fluidity and distorted morphology with membrane fissure and blebs as evident from SEM. It is possible that an increase in the membrane fluidity may lead to the defective antigen-presenting ability of 6 I-LM. Thus, the LD infection functionally keep the 6 I-LM out of antigen presentation and this may contribute to the defective cell mediated immune response in leishmaniasis.