Disclosing Ribose-5-Phosphate Isomerase B Essentiality in Trypanosomatids.

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.We would like to thank Professor Ana Tomás from the Institute for Molecular and Cell Biology, University of Porto, Portugal, for providing LimTXNPx antibody; Dr. Paul Michels from Université Catholique de Louvain, Belgium, for providing Tbenolase antibody; Professor Graham Coombs, Strathclyde University, Glasgow, for LmCS antibody; Professor Buddy Ullman, School of Medicine, Oregan Health and Science University, USA, for LdHGPRT antibody; Dr. Christine Clayton, Zentrum fur Molekulare Biologie der Universitat Heidelberg, Germany, for TbAldolase antibody. We would also like to thank Professor Jeremy Mottram, University of Glasgow, for pGL345HYG and Professor Marc Ouellette, Centre de Recherche en Infectiologie, of Laval University, Canada, for pSPαNEOα and pSPαBLASTα. We would also like to thank Dr. Jane MacDougall from Photeomix, France, for proofreading the English of the manuscript. The research leading to these results has received funding from the European Community’s Seventh Framework Programme under grant agreement No. 602773 (Project KINDRED).’ The COST Action CM1307: Targeted chemotherapy towards diseases caused by endoparasites has also contributed for this work. We would like to acknowledge Fundação para a Ciência e Tecnologia (FTC) for supporting Joana Faria (SFRH/BD/79712/2011) and Inês Loureiro (SFRH/BD/64528/2009). Inês Loureiro was also supported by the European Community’s Seventh Framework Programme (KINDRED-PR300102-BD). JT is an Investigator FCT funded by National funds through FCT and co-funded through European Social Fund within the Human Potential Operating Programme. Nuno Santarem and Pedro Cecílio are supported by fellowships from the European Community’s Seventh Framework Programme under grant agreements No. 602773 (Project KINDRED) and No. 603181 (Project MuLeVaClin), respectively

Characterization of fructose 1,6-bisphosphatase and sedoheptulose 1,7-bisphosphatase from the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus

Stolzenberger J, Lindner S, Persicke M, Brautaset T, Wendisch VF. Characterization of fructose 1,6-bisphosphatase and sedoheptulose 1,7-bisphosphatase from the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus. Journal of Bacteriology. 2013;195(22):5112-5122