Precise and sensitive identification methods of Leishmania are required for efficient treatment of leishmaniasis. Detection of Leishmania species approves the efficiency of the applied therapy. Current diagnosis methods, such as culture and microscopy methods, are time-consuming and have limited sensitivity, and are laborious when encountering lots of samples. Although molecular methods can identify Leishmania species, their diagnosis depends on individual responsiveness. Therefore, rapid, sensitive and low-cost methods are required for identification of Leishmania species. In this study, a new method was developed based on hybridization of gold nanoparticles linked a specific single stranded DNA probe from non-protein coding region ( AB678349.1 ) of Leishmania major minicircle kinetoplast DNA (kDNA). Dispersion or aggregation of the gold nanoparticles-probe conjugates in the presence or absence of a complementary DNA sequence leads to an obvious and sensitive change in the UV–vis spectra and the solution color. The results indicated that this method is useful for diagnosis of Leishmania major from other non- Leishmania species with a detection limit of 7.0 pg μL −1 . The efficacy of the present PCR-free assay was evaluated to identify genomic DNA extracted from Leishmania major and clinical samples.

Gold nanoparticles-based biosensing of Leishmania major kDNA genome: Visual and spectrophotometric detections

We have compared the sequences of a major class of kinetoplast DNA (kDNA) minicircle (pLURkE3) of Leishmania strain UR6 with other minicircle sequences from different Leishmaniaspecies. Alignment of these sequences allowed the selection of a pair of oligonucleotides suitable as primers in polymerase chain reaction (PCR) which is specific for Leishmaniaparasites. PCR with this genus-specific primer set is capable of detecting 1 femtogram of kDNA. These primers have been tested with kDNAs from both old world and new world Leishmaniaspecies. The results indicate that the primers may be suitable for detection of any kind of leishmaniasis.

Development of a genus specific primer set for detection of Leishmania parasites by polymerase chain reaction

DNA topoisomerase II is a multidomain homodimeric enzyme that changes DNA topology by coupling ATP hydrolysis to the transport of one DNA helix through a transient double-stranded break in another. To investigate the biochemical properties of the individual domains of Leishmania donovani topoisomerase II, four truncation mutants were generated. Deletion of 178 aminoacids from the C-terminus (core and LdΔC1058) had no apparent effect on the DNA-binding or cleavage activities of the enzymes. However, when 429 aminoacids from the N-terminus and 451 aminoacids from the C-terminus were removed (LdΔNΔC), the enzyme was no longer active. Moreover, the removal of 429 aminoacids from the N-terminus (LdΔNΔC, core and LdΔN429) render the mutant proteins incapable of performing ATP hydrolysis. The mutant proteins show cleavage activities at wide range of KCl concentrations (25–350 mM). In addition, the mutant proteins, excepting LdΔNΔC, can also act on kDNA and linearize the minicircles. Surprisingly, the mutant proteins fail to show the formation of the enhanced cleavable complex in the presence of etoposide. Our findings suggest that the conformation required for interaction with the drug is absent in the mutant proteins. Here, we have also identified Tyr775 through direct sequencing of the DNA linked peptide as the catalytic residue implicated in DNA-breakage and rejoining. Taken together, our results demonstrate that topoisomerase II are functionally and mechanistically conserved enzymes and the variations in activity seem to reflect functional optimization for its physiological role during parasite genome replication.

Characterization of the DNA-binding domain and identification of the active site residue in the ‘Gyr A’ half of Leishmania donovani topoisomerase II

Characterization of kinetoplast DNA minicircles of an Indian isolate of Leishmania donovani.

Kinetoplastid topoisomerase IB is an unusual bisubunit enzyme where reconstitution of the large (LdTOPIL or L) and small (LdTOPIS or S) subunits shows functional activity. It is yet to be deciphered whether one subunit or both navigate the heterodimer to its cellular DNA targets. Tethering a specific DNA-binding protein to topoisomerase I alters its site specificity. The chimeric constructs UMSBP-LdTOPIL/S or U-L/S (fusion of UMSBP to the N-terminus of L and reconstituted with S) and LdTOPIL/UMSBP-LdTOPIS or L/U-S (fusion of UMSBP to the N-terminus of S and reconstituted with L) exhibit relaxation activity. Only U-L/S shows altered site specificity and enhanced DNA-binding affinity for the universal minicircle sequence (UMS) containing substrate. This proves that L alone serves as the 'molecular steer' for this heterodimer. Reconstituted U-L/S also induces cleavage close to UMS and causes minicircle linearization. The differential properties of the reconstituted chimeras U-L/S and L/U-S reveal the structural and functional asymmetry between the heterodimer. Therefore this study helps in a better understanding of the mechanistic details underlying topoisomerization by this bi-subunit enzyme.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.2007.06002.x

The large subunit of Leishmania topoisomerase I functions as the ‘molecular steer’ in type IB topoisomerase

We have determined the nucleotide sequences of the minicircles representing a major (pLURkE3) and a minor (pLURkH13) class populations from the kinetoplast DNA ofLeishmania strain UR6. These minicircles have sequence organization similar to other kinetoplastid parasites, however, they have some unique structural features. These features include the following: (i) imperfect inverted repeat in the variable regions, similar to the conserved sequence elements of guide RNA genes in African trypanosomes, (ii) tandem and non-tandem direct repeats of 8 bp or longer scattered throughout the minicircles, (iii) non uniform strand distribution of bases throughout the minicircles and (iv) high TG content, more than half of the molecules being extremely (T + G) versus (A + C) strand biased.

Analysis of sequences of two different classes of Kinetoplast DNA minicircles of a Leishmania Spp.

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Analysis of sequences of two different classes of Kinetoplast DNA minicircles of a Leishmania Spp.