Mycolic Acid Modification by the mmaA4 Gene of M. tuberculosis Modulates IL-12 Production

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Abstract
Mycobacterium tuberculosis has evolved many strategies to evade elimination by the host immune system, including the selective repression of macrophage IL-12p40 production. To identify the M. tuberculosis genes responsible for this aspect of immune evasion, we used a macrophage cell line expressing a reporter for IL-12p40 transcription to screen a transposon library of M. tuberculosis for mutants that lacked this function. This approach led to the identification of the mmaA4 gene, which encodes a methyl transferase required for introducing the distal oxygen-containing modifications of mycolic acids, as a key locus involved in the repression of IL-12p40. Mutants in which mmaA4 (hma) was inactivated stimulated macrophages to produce significantly more IL-12p40 and TNF-α than wild-type M. tuberculosis and were attenuated for virulence. This attenuation was not seen in IL-12p40-deficient mice, consistent with a direct linkage between enhanced stimulation of IL-12p40 by the mutant and its reduced virulence. Treatment of macrophages with trehalose dimycolate (TDM) purified from the ΔmmaA4 mutant stimulated increased IL-12p40, similar to the increase observed from ΔmmaA4 mutant-infected macrophages. In contrast, purified TDM isolated from wild-type M. tuberculosis inhibited production of IL-12p40 by macrophages. These findings strongly suggest that M. tuberculosis has evolved mmaA4-derived mycolic acids, including those incorporated into TDM to manipulate IL-12-mediated immunity and virulence. Currently, one-third of the world's population has tuberculosis (TB). TB, an ancient foe, has reemerged to become a threat to global public health. A central problem in TB research is to investigate why the host immune system cannot sterilize the infection caused by the bacterium Mycobacterium tuberculosis. Interleukin-12 (IL-12), a molecule produced by macrophages in response to pathogens, plays an important role in orchestrating sterilizing immunity. However, M. tuberculosis has evolved mechanisms that block IL-12 production and thereby assist the bacterium in establishing chronic infection. We discovered that mutation of the mycobacterial mmaA4 gene, which controls the chemical modification of complex lipids of M. tuberculosis called mycolic acids, renders the bacterium unable to block IL-12 production. Mycolic acids incorporated into a secreted bacterial molecule called trehalose dimycolate (TDM) from M. tuberculosis had the ability on their own to suppress the production of IL-12 by activated macrophages; we also showed that TDM from the mmaA4 mutant of M. tuberculosis is attenuated for suppression. Our results identify a critical part of the genetic basis and mechanism for an important immune evasion function in M. tuberculosis, and should contribute to the design of future vaccines and immunotherapies for disease caused by this pathogen.