DIPEPTIDYL PEPTIDASE III: A NOVEL MARKER TO INVESTIGATE LEISHMANIA SPECIES EVOLUTION AND TAXONOMY.

Introduction and Objectives: Dipeptidyl peptidase III (DPPIII) member of M49 peptidase family is a zinc-dependent metallopeptidase that cleaves dipeptides sequentially from the N-terminus of its substrates. It is absent in trypanosomes, however, in Leishmania, it was reported that the DPPIII with other peptidases, could be key to the parasites' growth and survival. In a previous study, we used a coding sequence annotated as DPPIII to develop a PCR assay that is specific to dermotropic Old World (OW) Leishmania species. Thus, our objective was to further assess use of this gene to identify Leishmania species or to study their evolution. Material and Methods: Orthologous DDPIII genes were searched in all Leishmania genomes and aligned to design primers and to search restriction enzymes. A PCR-RFLP scheme was designed and tested on 51 OW typed Leishmania strains belonging to 10 Leishmania species (L. major (L.m), L. infantum (L.i), L. tropica (L.t), L. donovani (L.d), L. aethiopica (L.aet), L. arabica (L.ar), L. turanica (L.tu), L. chagasi (L.ch), L. archibaldi (L.arch) and L. tarentolae (L.tar)), 7 strains isolated from CL patients, and 50 biological samples taken for diagnosis of suspected Tunisian CL patients. Seventy-one PCR products were further studied by sequence analysis using MEGA and DNA sp softwares to infer evolution and phylogenetic relationship of studied species and strains. Results: DPPIII gene is present in all genomes. Inter- and intra-specific genetic diversity was used to develop universal amplification of a 662bp fragment followed by a double digestion with HaeIII and KpnI. Overall, this PCR-RFLP yielded distinct profiles for each of the species L.m, L.t, L.aet, and L.tar. L.d, L.arch and L.ishared their profile with L.ara and L.tur. Sequence analysis showed 145/589 variable sites (116 being parsimony informative), high haplotype diversity (H=0.81; SD=0.026), and low nucleotide diversity (p=0.04; SD=0.005). Test of natural selection (dN/dS=4.7, Fu and Li's D test (2.79; p<0.02)) inferred a positive selection confirming importance of DPPIII for parasite adaptation to its environment. Phylogenetic relationships confirmed occurrence of 6 clusters congruent to L.m, L.t, L.aet, L.ar, L.tu, L. tar species, and one to the L.i, L.d and L.arch group of species. Conclusion: DPPIII gene is suitable to investigate Leishmania epidemiology and evolution and can contribute to timely disease control. [ABSTRACT FROM AUTHOR]