Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum

Abstract

Parasite of the genus Leishmania is reliant on the salvage pathway for recycling of ribonucleotides. A class I nuclease enzyme also known as P4 nuclease is involved in salvage of purines in cutaneous Leishmania species but the relevant enzymes have not been characterized in Leishmania infantum (L. infantum). The aim of this study was to clone and characterize the gene encoding class I nuclease in L. infantum. DNA extracted from L. infantum was used for amplification of P4 nuclease gene (Li-P4) by PCR. The product was cloned, sequenced, and expressed in E. coli for further characterization. Analysis of the sequence of Li-P4 revealed that the gene consists of an ORF of 951 bp. Sequence similarity analysis indicated that Li-P4 has a high homology to relevant enzymes of other kintoplastids with the highest homology (88%) to p1/s1 class I nuclease from L. donovani. Western blotting of antirecombinant Li-P4 with promastigote and amastigote stages of L. infantum showed that this nuclease is present in both stages of parasite with higher expression in amastigote stage. The highly conserved nature of this essential enzyme in Leishmania parasites suggests it as a promising drug target for leishmaniasis.

Stage-Specific Class I Nucleases of Leishmania Play Important Roles in Parasite Infection and Survival

“…Western blot analyses have shown that the P4 nuclease of L. infantum is expressed in both promastigotes and amastigotes. However, in accordance with previous studies involving this enzyme, its expression is significantly higher in amastigote parasites (Farajnia et al, 2011).…”

“…Comparative sequence analyses have shown high homology to the P4 nucleases of L. donovani, L. major and L. pifanoi. The alignment results confirmed the existence of the five conserved domains of class I nucleases (Farajnia et al, 2011). Western blot analyses have shown that the P4 nuclease of L. infantum is expressed in both promastigotes and amastigotes.…”

“…Posteriorly, a homolog of P4 nuclease was identified in L. infantum, the causative agent of visceral leishmaniasis. The P4 nuclease gene was cloned, sequenced, and heterologously expressed for further characterization (Farajnia et al, 2011). Comparative sequence analyses have shown high homology to the P4 nucleases of L. donovani, L. major and L. pifanoi.…”

Molecular Cloning of Leishmania major gp63 Gene in BALB/c Mouse CT26 Cell Line

“…So far, different studies have been conducted to clone, express or manipulated Leishmania genes. Cloning and characterization of a gene encoding class I nuclease in L. infantum has already been carried out by Farajnia et al [20]. Also, there are other reports on cloning and expression of different Leishmania genes such as HSP70 and glucose-regulated protein 94 (GRP94) gene from L. infantum [21,22], surface protein encoding genes from L. major [23,24] and L. donovani avastin gene [25].…”

Current trends in targeted therapy for drug-resistant infections

“…At present, chemotherapy is the most common therapeutic strategy against leishmania. However, emergence of drug-resistant parasites has limited conventional therapeutic approaches (Farajnia et al 2011) (Rahbarnia et al 2012). In this respect, researchers have examined the application of RNAi technology to harness leishmaniasis disease.…”