Disclosing the essentiality of ribose-5-phosphate isomerase B in Trypanosomatids

Abstract

Ribose-5-phosphate isomerase (RPI) belongs to the non-oxidative branch of the pentose phosphate pathway, catalysing the inter-conversion of D-ribose-5-phosphate and D-ribulose-5-phosphate. Trypanosomatids encode a type B RPI, whereas humans have a structurally unrelated type A, making RPIB worthy of exploration as a potential drug target. Null mutant generation in Leishmania infantum was only possible when an episomal copy of RPIB gene was provided, and the latter was retained both in vitro and in vivo in the absence of drug pressure. This suggests the gene is essential for parasite survival. Importantly, the inability to remove the second allele of RPIB gene in sKO mutants complemented with an episomal copy of RPIB carrying a mutation that abolishes isomerase activity suggests the essentiality is due to its metabolic function. In vitro, sKO promastigotes exhibited no defect in growth, metacyclogenesis or macrophage infection, however, an impairment in intracellular amastigotes’ replication was observed. Additionally, mice infected with sKO mutants rescued by RPIB complementation had a reduced parasite burden in the liver. Likewise, Trypanosoma brucei is resistant to complete RPIB gene removal and mice infected with sKO mutants showed prolonged survival upon infection. Taken together our results genetically validate RPIB as a potential drug target in trypanosomatids.

More than just exosomes: distinct Leishmania infantum extracellular products potentiate the establishment of infection

“…CD11c+ MHC-II+ cells, considered as BMDC and determined by flow cytometry, represented more than 75% of the culture. Bone marrow-derived macrophages (BMMØ) were obtained from non-adhered bone marrow cells collected after an initial overnight incubation (37°C and 5% CO 2 ) in complete Dulbecco’s modified Eagle’s medium (DMEM) (Lonza, Switzerland) as previously described [27]. Non-adherent cells were recovered, counted and distributed in 96 well culture plates at 1 x 10 5   cells/well in 100 µl of complete DMEM supplemented with 5% of L-929 cell conditioned medium (LCCM).…”

Potential Drug Targets in the Pentose Phosphate Pathway of Trypanosomatids

“…The same analysis detected HGPRT (PTS-1), aldolase (PTS-2), as well as some enzymes of PPP that are upstream RPIB, namely putative G6PD (PTS-1), putative 6PGDH (non-identified), or enzymes downstream RPIB, such as TKL (PTS-1), putative RPE (PTS-1) and putative TAL (nonidentified signal peptide), or other related proteins like putative ribokinase (PTS-2 signal peptide). In both T. brucei and L. infantum RPIB shows a dual localization despite preferentially located in the cytosol compared to the glycosomes [261,263]. The amount of RPBI that is in the cytosol or the glycosomes, as well as whether the distribution changes throughout the parasite life cycle needs further investigation.…”

“…An adverse phenotype was observed in E. coli [258] and also humans [259] upon RPI deficiency, suggesting a critical conserved role through evolution. Trypanosomatids possess a type B RPI, which is absent from humans that instead have a structurally unrelated type A and therefore possibly an ideal drug target [260][261][262][263][264][265]. Recombinant enzymes from L. donovani [262], L. infantum [261], L. major [261], T. brucei [263] and T. cruzi [263,264] have been formally demonstrated to have in vitro isomerase activity by catalysing the interconversion of R5P and Ru5P.…”

“…Trypanosomatids possess a type B RPI, which is absent from humans that instead have a structurally unrelated type A and therefore possibly an ideal drug target [260][261][262][263][264][265]. Recombinant enzymes from L. donovani [262], L. infantum [261], L. major [261], T. brucei [263] and T. cruzi [263,264] have been formally demonstrated to have in vitro isomerase activity by catalysing the interconversion of R5P and Ru5P. The Km values for both R5P and Ru5P were similar between Leishmania and trypanosomes enzymes, however, Kcat values are considerably higher for Leishmania, in both direct and inverse reactions [261][262][263][264].…”

“…Recombinant enzymes from L. donovani [262], L. infantum [261], L. major [261], T. brucei [263] and T. cruzi [263,264] have been formally demonstrated to have in vitro isomerase activity by catalysing the interconversion of R5P and Ru5P. The Km values for both R5P and Ru5P were similar between Leishmania and trypanosomes enzymes, however, Kcat values are considerably higher for Leishmania, in both direct and inverse reactions [261][262][263][264]. A decrease in Km and an increase in Kcat were consistently observed for Ru5P in comparison to R5P, suggesting that the conversion of Ru5P into R5P is favoured, which might be explained by the important role of R5P as a building block for nucleic acid synthesis.…”