Identifying vulnerable pathways in Mycobacterium tuberculosis by using a knockdown approach

Abstract

We constructed recombinant strains of Mycobacterium tuberculosis in which expression of specific genes was downregulated to identify vulnerable drug targets. Growth phenotypes in macrophages and culture were used to rank targets: the dprE1, clpP1, and fadD32 operons were the best targets and glnA1, glnE, pknL, regX3, and senX3 were poor targets.

Fig. 1.

Construction of recombinant strains by promoter replacement. (A) Chromosomal arrangement of the targeted genes. Arrows indicate the genes expressed; lines indicate the region of the target gene amplified. (B) Schematic of a recombination delivery plasmid carrying the 5′ end of the gene cloned under the control of P_tet. The TetO operator sites located within the promoters are indicated (Op). Recombination between the plasmid and the chromosomal gene results in replacement of the native promoter with the P_tet promoter. A nonfunctional fragment of the 5′ end of the gene is also incorporated.

Fig. 2.

Growth of recombinant knockdown strains in axenic culture. Strains were cultured in 3 ml liquid medium in 16-mm-diameter glass tubes containing a 12-mm magnetic stirrer and stirred at 120 rpm. Open symbols, no tetracycline; closed symbols, 200 ng/ml tetracycline. Data are the averages of three independent recombinant strains. Error bars are omitted for clarity. (A) dprE1 operon. (B) clpP1 operon and pknL. (C) glnA1 and glnE. (D) senX3 operon and regX3. OD₅₈₀, optical density at 580 nm; WT, wild type.

Fig. 3.

Growth of recombinant knockdown strains in the macrophage infection model. Strains were used to infect THP-1 cells at a multiplicity of infection of 1:1 (8). Bacteria were harvested from monocytes, and CFU were determined. Macrophages were activated by the addition of 100 units/ml of IFN-γ 24 h prior to infection. (A) Resting macrophages. (B) Activated macrophages. Open symbols, no tetracycline; closed symbols, addition of tetracycline. Data are the means and standard deviations from three independent samples.

Fig. 4.

Characterization of vulnerable cell wall targets using recombinant knockdown strains. Colony morphology of the wild-type (A) and P_tet-fadD32 operon knockdown (B) strains. Species identity was confirmed by 16S rRNA locus amplification and sequencing.