Deletion of kasB in Mycobacterium tuberculosis causes loss of acid-fastness and subclinical latent tuberculosis in immunocompetent mice

Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis, has two distinguishing characteristics: its ability to stain acid-fast and its ability to cause long-term latent infections in humans. Although this distinctive staining characteristic has often been attributed to its lipid-rich cell wall, the specific dye-retaining components were not known. Here we report that targeted deletion of kasB, one of two M. tuberculosis genes encoding distinct beta-ketoacyl- acyl carrier protein synthases involved in mycolic acid synthesis, results in loss of acid-fast staining. Biochemical and structural analyses revealed that the DeltakasB mutant strain synthesized mycolates with shorter chain lengths. An additional and unexpected outcome of kasB deletion was the loss of ketomycolic acid trans-cyclopropanation and a drastic reduction in methoxymycolic acid trans-cyclopropanation, activities usually associated with the trans-cyclopropane synthase CmaA2. Although deletion of kasB also markedly altered the colony morphology and abolished classic serpentine growth (cording), the most profound effect of kasB deletion was the ability of the mutant strain to persist in infected immunocompetent mice for up to 600 days without causing disease or mortality. This long-term persistence of DeltakasB represents a model for studying latent M. tuberculosis infections and suggests that this attenuated strain may represent a valuable vaccine candidate against tuberculosis.

Fig. 1.

Generation of a M. tuberculosis kasB mutant. (A) Map of the M. tuberculosis kasB region in WT and ΔkasB strains. [α-³²P]dCTP-labeled probes were derived from ≈500-bp upstream and downstream flanking sequences that were used to construct the knockout plasmids, and they are indicated by thick lines with square ends. The PvuII-digested fragments expected in a Southern blot are indicated by gapped lines with sizes. (B) Southern blot of PvuII-digested genomic DNA from WT and ΔkasB strains. Only one representative band pattern is shown for each mutant strain of H37Rv and CDC1551; res, γδ-resolvase site; hyg, hygromycin resistance gene.

Fig. 2.

Colonies of M. tuberculosis CDC1551 parental (WT), kasB null mutant (ΔkasB), and complemented kasB mutant [ΔkasB(pMV261kasB)] strains on 7H10 plates after incubation at 37°C for 4 weeks. (Scale bar, 2 mm.)

Fig. 3.

Light microscopy of M. tuberculosis cultures grow static in 7H9 broth at 37°C for 7 days. Cultures were fixed on glass slides, and acid-fast staining was performed on the fixed smears by using the BD TB fluorescent kit-T. (Upper) Phase-contrast microscopy images of M. tuberculosis WT, ΔkasB, and ΔkasB(pMV261kasB) strains stained with TB-auramine-rhodamine and then treated with acid-alcohol decolorizer. (Lower) Fluorescent micrographs of the same fields. (Magnification, ×400.)

Fig. 4.

TLC of ¹⁴C-labeled MAMEs from M. tuberculosis WT, ΔkasB, and ΔkasB(pMV261kasB) strains. (A) Single-dimension HPTLC of MAMEs. (B–D) Two-dimensional argentation TLC of WT, ΔkasB, and ΔkasB(pMV261kasB), respectively. O, origin; I, first dimension; II+Ag, second dimension impregnated with AgNO₃; α, α-MAMEs; M, methoxy-MAMEs; K, keto-MAMEs; F, fatty acid methyl esters. The two unknown species in the ΔkasB strain, M2 and K2, are indicated by arrows.

Fig. 5.

Pathology of infected mouse lungs. (A) Lungs of C57BL/6 mice infected with M. tuberculosis WT, ΔkasB, and ΔkasB(pMV261kasB) strains, removed at different time points after aerosol infection. (B) Hematoxylin/eosin-stained lung tissue sections from mice infected with WT, ΔkasB, or ΔkasB(pMV261kasB). (Scale bar, 200 μm.)

Fig. 6.

Attenuation of ΔkasB in C57BL/6 mice. (A) Plot of M. tuberculosis cfu levels in lungs of C57BL/6 mice at different time points after aerosol infection. (B) Survival curve of C57BL/6 mice infected with WT, ΔkasB, or ΔkasB (pMV261kasB).