Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis

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.

Comprehensive exploration of the enzymes catalysing oxygen‐involved reactions and COGs relevant to bacterial oxygen utilization

“…To identify pathways or genes relevant to specific phenotypic traits, researchers can employ experimental methods, such as gene knockout and knockdown technology. Gene knockout/knockdown technology is widely used to infer the function of one sequenced gene by inactivation or decreasing the expression of the gene and then observing subsequent phenotype variations, as conducted in Escherichia coli (Sun et al ., ) and Mycobacterium tuberculosis (Villela et al ., ). High‐throughput technologies, such as microarray analysis (Gupta et al ., ) and RNA‐seq (Xiong et al ., ), reveal extensive numbers of differentially expressed genes under varied conditions and can identify many genes relevant to a certain individual phenotype.…”

“…Mycobacterium tuberculosis (Villela et al, 2017). Highthroughput technologies, such as microarray analysis (Gupta et al, 2015) and RNA-seq (Xiong et al, 2017), reveal extensive numbers of differentially expressed genes under varied conditions and can identify many genes relevant to a certain individual phenotype.…”

Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages

“…M. tuberculosis H37Rv strain was transformed by electroporation with ~ 2 μg of pPR27 xylE :: cdd kan plasmid. Possible KO clones were selected in two steps, as described previously ( Villela et al 2017 ). Genomic DNA was isolated and PCRs were carried out using gene-specific screening primers forward (5’-gt- gtctttgcggctgtagtc-3’) and reverse (5’-gggcagttcatctcc- gtca-3’) to determine whether the WT or the KO strain was present in the targeted chromosomal region ( Fig.…”

“…To obtain the CP strain, a fragment containing the cdd gene, its upstream (183 bp) region containing the natural promoter, and 101 bp downstream to cdd was amplified by PCR from M. tuberculosis H37Rv genomic DNA using primers forward (5’-gggg tctaga ttgtcgccgttgtattcacc-3’) and reverse (5’-gggg tctaga gtcggtataggccttgacga-3’), both containing XbaI restriction sites (in bold). Next, the amplicon was cloned into XbaI linearised pNIP40/b(pNIP40::cdd), a mycobacteriophage Ms6-derived integrative vector ( Freitas-Vieira et al 1998 ), and the KO strain was transformed by electroporation with pNIP40::cdd, as described previously ( Villela et al 2017 ). The stability of the mutation introduced by gene replacement in M. tuberculosis was evaluated by plating KO and CP strains on media with and without antibiotics.…”

“…To examine whether the cdd gene was important for invasion and growth in phagocytic cells, we determined the bacterial loads of the WT, KO, and CP strains by using the macrophage model of infection. RAW 264.7 macrophage cell line was cultured and infected with WT, KO or CP M. tuberculosis strains as described previously ( Villela et al 2017 ), with minor modifications. Briefly, infection of macrophages was performed at a multiplicity of infection of 1:1 (bacteria/macrophage) at 37°C with 5% CO 2 .…”