Attenuation of Mycobacterium tuberculosis by disruption of a mas-like gene or a chalcone synthase-like gene, which causes deficiency in dimycocerosyl phthiocerol synthesis

Abstract

Tuberculosis is one of the leading preventable causes of death. Emergence of drug-resistant tuberculosis makes the discovery of new targets for antimycobacterial drugs critical. The unique mycobacterial cell wall lipids are known to play an important role in pathogenesis, and therefore the genes responsible for their biosynthesis offer potential new targets. To assess the possible role of some of the genes potentially involved in cell wall lipid synthesis, we disrupted a mas-like gene, msl7, and a chalcone synthase-like gene, pks10, with phage-mediated delivery of the disruption construct, in which the target gene was disrupted by replacement of an internal segment with the hygromycin resistance gene (hyg). Gene disruption by allelic exchange in the case of each disruptant was confirmed by PCR and Southern blot analyses. Neither msl7 nor pks10 mutants could produce dimycocerosyl phthiocerol, although both could produce mycocerosic acids. Thus, it is concluded that these gene products are involved in the biosynthesis of phthiocerol. Both mutants were found to be attenuated in a murine model, supporting the hypothesis that dimycocerosyl phthiocerol is a virulence factor and thus the many steps involved in its biosynthesis offer potential novel targets for antimycobacterial therapy.

FIG. 1.

Structure of dimycocerosyl esters of glycosylated (Gl) phenolphthiocerol (mycoside) and phthiocerol (DIM).

FIG. 2.

Disruption strategy for msl7 and evidence for gene replacement in the M. tuberculosis H37Rv mutant used for making the disruption construct. (A) Schematic representation of the construct used for disruption. Hatched, coding sequence; checkered, internal segment that was replaced with hyg gene cassette (black box); unshaded, regions of the gene outside those used to make the disruption construct. Primers A and B were used to amplify the DNA segment used to generate the disruption construct. WT PstI, PstI fragment from the wild type, expected to hybridize with probe P₁, representing the fragment deleted in making the construct. M PstI, PstI fragment form the msl7 mutant, expected to hybridize with probe P₂ (hyg gene). (B) PCR analysis of msl7 mutant. Lanes 1 and 2, PCR analysis of the 5′-flanking region, showing the product expected from the gene replacement mutant in lane 2 but not in the wild type (lane 1); primers C and H1. Lanes 3 and 4, PCR analysis of the 3′-flanking region, showing the expected product from the msl7 mutant (lane 4) but not from the wild type (lane 3); primers H2 and D. Lanes 5 and 6 show the PCR product representing the internal segment replaced by hyg from the wild type (lane 5) but not from the msl7 mutant (lane 6); primers E and F. (C) Southern blot analysis of M. tuberculosis H37Rv and the msl7 mutant. Genomic DNA was digested with PstI and hybridized with probe P₁ (left) or P₂ (right). WT, wild type; M, mutant. (D) Reverse transcription-PCR showing the presence of transcripts containing the pks1 segment of msl7 in M. tuberculosis H37Rv. For lanes 1, 2, and 3, primers E and F were used. Lane 1, wild type; lane 2, wild type without reverse transcriptase; lane 3, msl7 mutant. In all panels, sizes are shown in kilobases.

FIG. 3.

Disruption strategy for pks10 and evidence for pks10 gene replacement in M. tuberculosis H37Rv. (A) Schematic representation of the disruption construct. Hatched and checkered regions represent the region used to make the disruption construct (amplified with primers A and B). The checkered segment was replaced with the hyg gene cassette (black box). Primer pairs C/H1, D/H2, and E/F were used for PCR analysis of homologous recombination as described in the text. WT PstI, WT BamHI, M PstI, and M BamHI are PstI and BamHI fragments from the wild-type (WT) and pks10 mutant (M) that are expected to hybridize with probes P1, representing the fragment deleted in making the construct, and P2, the hygromycin resistance gene cassette, respectively. (B) PCR analysis of 5′-flanking (lanes 1 and 2, primers C and H₁), 3′-flanking (lanes 3 and 4, primers H₂ and D), and the segment replaced by hyg (lane 5 and 6, primers E and F), demonstrating homologous recombination. Lanes 1, 3, and 5, wild type; lanes 2, 4, and 6, mutant. (C) Reverse transcription-PCR analysis showing expression of pks10 in the wild type (right) but not in the pks10 mutant (left). Primers, E and F. (D) Southern blot analysis of M. tuberculosis H37Rv and pks10 mutants. Genomic DNA was digested with PstI and BamHI. Left, hybridized with pks10 segment replaced by hyg (probe P₁); right, probed with hyg gene (probe P₂). WT, wild type; M₁ and M₂, mutants.

FIG. 4.

Autoradiogram of thin-layer chromatogram of lipids derived from sodium [1-¹⁴C]propionate in M. tuberculosis H37Rv (wild type [WT]), msl7 mutant, and the pks10 mutant. Total lipids were subjected to TLC on silica gel G with 10% ethyl ether in n-hexane as the solvent. A similar amount of radioactivity was used in each case. DIM, dimycocerosyl phthiocerol.

FIG. 5.

Radio-GC analysis of total fatty acid methyl esters derived from sodium [1-¹⁴C]propionate in M. tuberculosis H37Rv (wild type), the msl7 mutant, and the pks10 mutant. Retention time ranges for branched short-chain fatty acids (A), mycolipanoic and mycolipenic acids (B), mycocerosic acids (C), and phthioceranic acids (D) are indicated.

FIG. 6.

Growth of intranasally administered M. tuberculosis H37Rv and its msl7 and pks10 gene-disrupted mutants in the lungs of C57BL/6J mice. The experimental details are in the text.

FIG. 7.

Major stages involved in the biosynthesis of phenolphthiocerol and phthiocerol. CoA, coenzyme A.