Gene expression analysis of wild Leishmania major isolates: identification of genes preferentially expressed in amastigotes

Abstract

Trying to identify virulence genes of wild Leishmania (L.) major parasites, the species responsible for zoonotic cutaneous leishmaniasis, we compared, using differential display technique, gene expression in two L. major isolates obtained from human lesions and characterized by their contrasting pathogenicity in the BALB/c mouse model. The analysis was performed on amastigotes derived from BALB/c mice lesions. A total of 13 different clones were identified, but the use of reverse transcription and real-time polymerase chain reaction technique did not allow us to confirm any of these clones as differentially expressed. However, the fact that we used the amastigote stage of the parasite led us the identification of amastigote-specific genes, essentially (8 among 13). They are overexpressed, two to seven times, in amastigotes relative to promastigotes. Sequence analysis revealed that two of them namely LPG3 and the ATP dependent RNA helicase correspond to previously described amastigote-specific genes. The others correspond to genes involved in important biological process. Their better characterization could help the development of new drugs targeting the processes in which these molecules are involved.

Oligopeptidase B deficient mutants of Leishmania major

“…1), whereas previous reports indicated that OPB is upregulated in amastigotes of L. braziliensis and L. donovani compared with promastigotes [17, 18]. The higher expression reported for L. braziliensis amastigotes was based on RNA levels detected using differential display methodology [17]; interestingly, OPB was not one of the genes found to have higher expression in L. major amastigotes in another differential display analysis [19]. Microarray analysis of RNA expressed at differential levels between promastigotes and amastigotes of L. mexicana similarly revealed no change for OPB [20].…”

“…Detection of OPB activity in the growth medium demonstrated the release of OPB by L. donovani promastigotes [16]. OPB is up-regulated in the amastigote stage of the life-cycle in L. braziliensis and L. donovani compared with promastigotes [17, 18], whereas there are similar levels of expression of OPB in all stages in L. major and L. mexicana [19, 20].…”

Putative markers of infective life stages in Leishmania (Viannia) braziliensis

“…DD analysis is one of the methods that can be used for comparing large numbers of mRNA between 2 samples. It has been validated in different organisms and, among others, allowed identification of genes preferentially expressed in amastigotes of L. (L.) major (Ouakad et al 2007) as well as a putative virulence factor (Ben Achour et al 2002). This approach is complementary to other gene expression methodological approaches like suppression subtractive hybridization (SSH, Diatchenko et al 1996) or micro-arrays (Saxena et al 2003; Almeida et al 2004).…”

Evidence of RNA editing in Leishmania braziliensis promastigotes

“…However, to verify whether gRNA loss did occur in L. braziliensis strain M2904, a complete characterization of both maxicircle and minicircle sequences needs to be done, and a comparison with those of recent isolates of this species should be carried out. Furthermore, determination of possible differences in RNA editing between promastigotes and amastigotes, using serial analysis of gene expression (Ouakad et al 2007;Li et al 2008), would help to deciphering the actual importance of this process in Leishmania. This is likewise the emphasis in the next stage of our research.…”