title: Validation of Mycobacterium tuberculosis dihydroneopterin aldolase as a molecular target for anti-tuberculosis drug development.
abstract: An early step of target validation in antimicrobial drug discovery is to prove that a gene coding for a putative target is essential for pathogen's viability. However, little attention has been paid to demonstrate the causal links between gene essentiality and a particular protein function that will be the focus of a drug discovery effort. This should be considered an important step in target validation since a growing number of proteins are found to exhibit multiple and unrelated tasks. Here, we show that the Mycobacterium tuberculosis (Mtb) folB gene is essential and that this essentiality depends on the dihydroneopterin aldolase/epimerase activities of its protein product, the FolB protein from the folate biosynthesis pathway. The wild-type (WT) MtFolB and point mutants K99A and Y54F were cloned, expressed, purified and monitored for the aldolase, epimerase and oxygenase activities using HPLC. In contrast to the WT MtFolB, both mutants have neither aldolase nor epimerase activities in the conditions assayed. We then performed gene knockout experiments and showed that folB gene is essential for Mtb survival under the conditions tested. Moreover, only the WT folB sequence could be used as a rescue copy in gene complementation studies. When the sequences of mutants K99A or Y54F were used for complementation, no viable colonies were obtained, indicating that aldolase and/or epimerase activities are crucial for Mtb survival. These results provide a solid basis for further work aiming to develop new anti-TB agents acting as inhibitors of the aldolase/epimerase activities of MtFolB.
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular (PUCRS), Brazil.
authors.forename: V C A
authors.initials: VC
authors.lastname: Falcão
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Brazil; Programa de Pós-Graduação em Medicina e Ciências da Saúde (PUCRS), Porto Alegre, RS, Brazil.
authors.forename: A D
authors.initials: AD
authors.lastname: Villela
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Brazil; Programa de Pós-Graduação em Medicina e Ciências da Saúde (PUCRS), Porto Alegre, RS, Brazil.
authors.forename: V S
authors.initials: VS
authors.lastname: Rodrigues-Junior
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Brazil.
authors.forename: K
authors.initials: K
authors.lastname: Pissinate
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Brazil.
authors.forename: P
authors.initials: P
authors.lastname: Eichler
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Brazil.
authors.forename: A F M
authors.initials: AF
authors.lastname: Pinto
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular (PUCRS), Brazil; Programa de Pós-Graduação em Medicina e Ciências da Saúde (PUCRS), Porto Alegre, RS, Brazil.
authors.forename: L A
authors.initials: LA
authors.lastname: Basso
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular (PUCRS), Brazil.
authors.forename: D S
authors.initials: DS
authors.lastname: Santos
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular (PUCRS), Brazil. Electronic address: cristiano.bizarro@pucrs.br.
authors.forename: C V
authors.initials: CV
authors.lastname: Bizarro
mesh_terms: D000446:Aldehyde-Lyases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D055808:Drug Discovery / Q000379:methods*; D020043:Genes, Essential / Q000235:genetics; D005816:Genetic Complementation Test / Q000379:methods; D006801:Humans; D050296:Microbial Viability / Q000187:drug effects / Q000235:genetics; D058990:Molecular Targeted Therapy / Q000379:methods; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D015203:Reproducibility of Results; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D014376:Tuberculosis / Q000188:drug therapy / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D000446:Aldehyde-Lyases; C020044:dihydroneopterin aldolase
keywords: Essentiality evaluation; Gene knockout; Site-directed mutagenesis; TB drug discovery; Target validation