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. 2012 Jun;80(6):1958-68.
doi: 10.1128/IAI.00021-12. Epub 2012 Mar 19.

Mycobacterium tuberculosis lacking all mycolic acid cyclopropanation is viable but highly attenuated and hyperinflammatory in mice

Daniel Barkan  1 , Dorsaf Hedhli, Han-Guang Yan, Kris Huygen, Michael S Glickman
Affiliations

Mycobacterium tuberculosis lacking all mycolic acid cyclopropanation is viable but highly attenuated and hyperinflammatory in mice

Daniel Barkan et al. Infect Immun. 2012 Jun.

Abstract

Mycolic acids, the major lipid of the Mycobacterium tuberculosis cell wall, are modified by cyclopropane rings, methyl branches, and oxygenation through the action of eight S-adenosylmethionine (SAM)-dependent mycolic acid methyltransferases (MAMTs), encoded at four genetic loci. Mycolic acid modification has been shown to be important for M. tuberculosis pathogenesis, in part through effects on the inflammatory activity of trehalose dimycolate (cord factor). Studies using the MAMT inhibitor dioctylamine have suggested that the MAMT enzyme class is essential for M. tuberculosis viability. However, it is unknown whether a cyclopropane-deficient strain of M. tuberculosis would be viable and what the effect of cyclopropane deficiency on virulence would be. We addressed these questions by creating and characterizing M. tuberculosis strains lacking all functional MAMTs. Our results show that M. tuberculosis is viable either without cyclopropanation or without cyclopropanation and any oxygenated mycolates. Characterization of these strains revealed that MAMTs are required for acid fastness and resistance to detergent stress. Complete lack of cyclopropanation confers severe attenuation during the first week after aerosol infection of the mouse, whereas complete loss of MAMTs confers attenuation in the second week of infection. Characterization of immune responses to the cyclopropane- and MAMT-deficient strains indicated that the net effect of mycolate cyclopropanation is to dampen host immunity. Taken together, our findings establish the immunomodulatory function of the mycolic acid modification pathway in pathogenesis and buttress this enzyme class as an attractive target for antimycobacterial drug development.

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Figures

Fig 1
Fig 1
Generation of M. tuberculosis strains lacking all cyclopropanation or all mycolic acid methyltransferases. (A) Deletion of mmaA1-mmaA2-mmaA3-mmaA4 locus from a merodiploid strain of M. tuberculosis Erdman with attB::mmaA1-4. When digested with EcoRI and probed with the 1,000-bp piece immediately upstream of mmaA1, the WT gives a 6.4-kb band, whereas the strain with the mmaA1-4 deletion gives a 3.1-kb band. The strain marked with an asterisk was chosen for further manipulation and was named MGM1953. (B) Deletion of pcaA/umaA1 from Δmma1-4::loxP attB::mmaA1-4 strain. The Southern blot confirms the deletion of pcaA and umaA1 from a strain previously deleted of mmaA1-4 (after removal of the loxP-hyg-loxP marker with Cre recombinase). When digested with SmaI and probed with the 600-bp piece immediately upstream of umaA1, the WT produces a 1.8-kb band, whereas the pcaA/umaA1 deletion strain produces a 4.2-kb band. The strain marked with an asterisk was designated MGM1960 and used for further genetic manipulation. (C) Deletion of cmaA2 from ΔmmaA1-4::loxp ΔpcaA/umaA1::Hyg attB::mmaA3-4 strain (MGM1961) and ΔmmaA1-4::loxp ΔpcaA/umaA1::Hyg attB::Strepr strain (MGM1962) via transduction of ΔcmaA2::zeo (phDB10). The Southern blot confirms the deletion of cmaA2 from MGM1961 or MGM1962. When digested with HindIII and probed with the 600-bp piece immediately upstream of cmaA2, the WT produces a 16.4-kb band, whereas the ΔcmaA2 strain produces a 3.5-kb band. The strains marked with asterisks were designated MGM1990 and MGM1991 and were characterized further. (D) Two-dimensional TLC of mycolic acid methyl esters of the cell wall of MGM1990. All three mycolic acid classes are present (black arrowhead, alpha class; black dashed arrow, methoxy class; and black arrow, keto class), and all are fully unsaturated. Essentially no mature cyclopropanated mycolic acids are made, as shown by the trivial amounts marked by the gray arrows (methoxy and keto classes) or gray arrowhead (alpha class). (E) Two-dimensional TLC of mycolic acid methyl esters of the cell wall of MGM1991. No oxygenated mycolates are present (no methoxy- or ketomycolates), and the remaining alpha mycolate is almost fully unsaturated. Small amounts of alpha mycolate with a single cyclopropane ring are visible (gray arrow).
Fig 2
Fig 2
(A) Macroscopic morphology of colonies. The images shown are of representative colonies of the indicated strains on 7H10 agar. The wild type and MGM1990 have similar morphologies, whereas the colonies of MGM1991 are small and smooth and have a “donut” shape. (B) Acid fastness (modified Kinyoun stain) of M. tuberculosis Erdman (WT) compared to MGM1990 and MGM1991.
Fig 3
Fig 3
Loss of cyclopropanation affects detergent susceptibility. Equivalent bacterial numbers of the WT, MGM1990, and MGM1991 strains were plated on 7H10 agar plates supplemented with the detergent tyloxapol and allowed to grow for 4 weeks. The numbers on the left give the concentrations of tyloxapol in the plates. Both mutants were sensitive to tyloxapol, MGM1990 more so than MGM1991.
Fig 4
Fig 4
(A) Mice were infected with either WT (circles) or MGM1990 (triangles) bacteria. CFU in both lungs were determined on days 1, 8, 21, 56, and 126 after infection. The combined results of two experiments are shown. (B) In vivo loss of mmaA3-4. The percentage of MGM1990 bacteria retaining the attB-integrated mmaA3-mmaA4 cassette during mouse infection was determined by plating of bacteria on plates with and without streptomycin at the indicated time points. MGM1985 is wild-type M. tuberculosis with an empty attB-integrating streptomycin-selected cassette. (C) Mice were infected with either MGM1985 (WT; circles), MGM1995 (unsaturated alpha mycolates, no oxygenated mycolates; squares), or MGM1998 (all three mycolate classes present, no cyclopropanes; triangles). CFU in both lungs were determined on days 1, 8, 21, 56, and 126 after infection. Each time point represents the mean for 4 or 5 mice, and error bars show standard deviations (SD). (D) Bacterial loads on days 8 and 21 after infection are shown for the experiment in panel C.
Fig 5
Fig 5
RT-qPCR expression profiling of the lungs of C57BL/6 mice (n = 4/group) infected intratracheally 3 weeks before with 104 CFU of MGM1985 (WT), MGM1990, or MGM1991 or of the lungs of naïve, uninfected mice. Data are expressed as normalized fold expression levels with respect to reference housekeeping genes. Results are representative of 2 separate experiments. *, P < 0.05 (Mann-Whitney test).
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