Los genes HSP70 de Leishmania

Abstract

Protozoan parasites of the genus Leishmania are aetiological agents of leishmaniasis, one of the major health problems worldwide. During its digenetic life cycle, Leishmania is exposed to environmental changes, including a temperature shift, which is believed to be a triggering factor affecting gene expression and promoting stage differentiation. This temperature upshift encountered by Leishmania promastigotes in the mammalian host induces a typical heat-shock response, but due to the absence of transcriptional control in these organisms, Leishmania does not respond to heat shock by increasing the transcription of genes coding for heat shock proteins (HSPs). Instead, the regulation of the heat shock response occurs exclusively at the post-transcriptional level and often involves sequences within the 3’-untranslated regions (3’UTR) of mRNAs that modulate RNA stability and/or translational efficiency. The HSP70 locus in L. infantum is composed by six copies arranged in a head-to-tail tandem differing in their 3’UTRs. Although all genes are transcribed at similar rates, the abundance of the mRNAs derived from HSP70 genes 1 to 5 (HSP70-I) increases following heat shock, while the mRNAs derived from HSP70 gene 6 (HSP70-II) remain unaffected. We have demonstrated that exposing L. infantum promastigotes to heat shock temperatures leads to an increase in the de novo synthesis of HSP70. Thus, a 5-fold increase was detected after 1 hour of incubation at 37ºC when compared with HSP70 synthesis rate at 26ºC. Furthermore, a 3-fold increase in the synthesis of HSP70 was detected in promastigotes incubated at 39ºC, which represents a severe heat shock temperature. To investigate the contribution of both types of HSP70 genes to the enhanced synthesis of HSP70 during heat shock, we have investigated the translational efficiencies of HSP70-I and HSP70-II transcripts at the temperatures that the parasite encounters in the insect (26ºC) and in the mammalian host (37ºC). Interestingly, the HSP70-I transcripts were found associated with ribosomes at normal and heat shock temperatures, whereas the HSP70-II transcripts are bound to ribosomes only at heat shock temperatures. To analyze the function of UTRs in the HSP70 gene regulation, we created a set of plasmid constructs. Our data indicate that the elements responsible of the translational control are localized in the 3’UTRs. Thus, the 3’UTR of HSP70-II gene is involved in the translational silencing of HSP70-II transcripts at 26ºC. Furthermore, a null mutant for both HSP70-II alleles was created by targeted disruption (Δhsp70-II). This mutant synthesizes HSP70 at a lower rate than the wild type parasites. Overall, our data suggest that the role of the HSP70-II gene is to top up HSP70 levels under conditions of stress. Important implications of HSP70-II gene have been uncovered after characterization of the Δhsp70-II mutant line. These mutant parasites have morfological alterations, cell cycle distortion and a retarded growth when arriving the stationary phase. Leishmania promastigotes lacking the HSP70-II gene are able to invade monocytes but, inside the cells, they proliferate at a lower rate than the wild type parasites. Moreover, when the mutant parasites were inoculated into six BALB/c mice, only in two of them the infection was established, and the parasite load in the infected animals was lower than that one detected in mice infected with the wild type parasites.

Finally, we have analyzed the organization and expression of the HSP70 locus in representative Leishmania species. The organization of the HSP70 loci was found to be well conserved between L. infantum and the other species analyzed, with the exception of L. braziliensis that showed the most divergent organization. The two types of HSP70 genes were present in all Leishmania species except for L. braziliensis that lacks the HSP70-II gene. Based on the polymorphisms in the HSP70 loci is possible to differenciate the Old and New World species within the subgenus Leishmania. The expression analysis indicated that the temperature-dependent accumulation of the HSP70-I mRNAs is also conserved among Leishmania species except for L. braziliensis. However, in this species the analysis of the HSP70 synthesis revealed that the HSP70 mRNAs are also preferentially translated at heat shock temperatures.