Analysis of sequences of minicircles of kDNA gotten of clinical samples (active and healed injuries) of patients with American cutaneous leishmaniasis in Pernambuco, Brazil
Some species of the genus Leishmania sp. are causative agents of the leishmaniasis, that are important public health problems. American cutaneous leishmaniasis (ACL), caused by Leishmania (Viannia) braziliensis presents cutaneous lesions that heal spontaneously or after specific treatment. In the present study, in vitro and in silico approaches were performed to analyse the mitochondrial genome. Two hundreds and ninety complete Leishmania sp. kDNA minicircles obtained from clinical samples (lesions and scars) of patients with ACL were studied. This work demonstrate multiple alignment of minicircle sequences that size polymorphisms (ranging from 518-797 bp) were present, as well as sequence polymorphisms, indicating significant heterogeneity of classes, particularly in minicircles amplified from active cutaneous lesions. The minicircle sequences obtained from scars were grouped in one cluster, indicating some degree of homogeneity, possibly due to clonal selection. The compositional analysis showed A + T % 70%. Palindromes were mapped and identified in two clones, showing that the variable region of minicircles was richer in A + T than the conserved region. Direct repeated alternated motifs composed of TA, AT, TT e AA represented 55% of these sequences. Fifty five polimorphic microsatellites were mapped in both the conserved and variable regions of the minicircles. The mapping of palindromes, direct alternated repeats and microsatellites were not diferentially present, suggesting that represent common structural features of this molecule. Specific motifs were identified and mapped in the conserved and variable regions of kDNA minicircles, and classified according to frequency as pertaining to scar minicircles or lesion minicircles. For the same motif, the frequency was higher in the scar minicircles. In conclusion, some molecular features seems to be common to all minicircles. On the other hand, the classes of minicircles obtained from scars are more similar, as judged by the multiple alignment and cladogram analyses, possibly due to the predominance of specific motifs. In future work, it would be interesting to precisely identify the regions responsible for the genetic differences between minicircles obtained from scars and cutaneous lesions. The regions encoding gRNA deserve a particular interes, as different types of gRNA may be essential to the survival of Leishmania in the biological context of healed tissue or cutaneous lesions. However, the possible biological consequences of our findings for Leishmania persistence need further investigations aiming at a better understanding of this complex issue.
Advisor:Octavio Fernandes da Silva Filho; Frederico Guilherme Coutinho Abath
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Cutaneous Leishmaniasis DNA Sequence Analysis, Kinetoplast Genetic Polymorphism
Date of Publication:04/20/2006