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Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis
Villela, Anne Drumond; Rodrigues-Junior, Valnês da Silva; Pinto, Antônio Frederico Michel; Wink, Priscila Lamb; Sánchez-Quitian, Zilpa Adriana; Petersen, Guilherme Oliveira; Campos, Maria Martha; Basso, Luiz Augusto & Santos, Diógenes Santiago
Abstract
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.
Keywords
iunH gene; nucleoside hydrolase; gene knockout; Mycobacterium tuberculosis
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