Anne Drumond Villela1, Valnês da Silva Rodrigues-Junior1,2,3, Antônio Frederico Michel Pinto1, Priscila Lamb Wink1,4, Zilpa Adriana Sánchez-Quitian1,4, Guilherme Oliveira Petersen1, Maria Martha Campos2,3,4, Luiz Augusto Basso1,2,4, Diógenes Santiago Santos1,4,+
1Pontifícia Universidade Católica do Rio Grande do Sul, Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose, Porto Alegre, RS, Brasil
2Pontifícia Universidade Católica do Rio Grande do Sul, Programa de Pós-Graduação em Medicina e Ciências da Saúde, Porto Alegre, RS, Brasil
3Pontifícia Universidade Católica do Rio Grande do Sul, Instituto de Toxicologia e Farmacologia, Porto Alegre, RS, Brasil
4Pontifícia Universidade Católica do Rio Grande do Sul, Programa de Pós-Graduação em Biologia Celular e Molecular, Porto Alegre, RS, Brasil
BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role.
OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection.
METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains.
FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells.
MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.
Financial support: CNPq (304051/1975-06, 520182/99-5, 163507/2014-7, 304156/2014-0), BNDES (14.2.0914.1), FAPERGS-CAPES (DOCFIX, 05/2013), Decit/SCTIE/MS-MCT-CNPq-FNDCT-CAPES to INCT-TB, CNPq (441720/2014-5), FAPERGS-CNPq-PRONEX-2009.
Received 18 October 2016
Accepted 2 December 2016