Modulation of Macrophage Biology and Host Immune Response by Mycobacterial Lipids

Members of the Mycobacterium genus present a serious health threat which account for significant morbidity and mortality in humans and other species. Many pathogenic mycobacterial species maintain an intracellular life style within macrophages serving as the primary host cell. The mycobacterium's ability to modulate the macrophage function requires the expression of specific mycobacterial surface components. Moreover, a number of studies have shown that various mycobacterial surface components can promote macrophage activation. This dissertation aims to define the role of glycopeptidolipids (GPL), which are highly antigenic glycolipids, in the immune-modulation of the host and thus establish its function as an important virulence factor.

To address the role of GPL as a virulence factor, we utilized M. avium strains either naturally lacking GPL expression or genetically altered to express modified GPL. Our studies showed that the loss of GPL or altering its structure leads to an M. avium strain which induces increased macrophage activation compared to GPL sufficient M. avium strains. Additional analysis showed attenuated virulence of GPL-deficient/modified M. avium strains in a mouse infection model.

To further understand the mechanism of action of GPL, we investigated the trafficking of GPL in primary macrophages infected with M. avium. Our studies revealed significant release and trafficking of GPL from the mycobacteria containing phagosome to a distinct endocytic compartment called the multivesicular bodies (MVB). Additional analysis showed that mycobacterial components including GPL are trafficked outside the infected cells on 'exosomes.' Further, we determined that exosomes purified from the culture supernatants of macrophages infected with M. avium, M. tuberculosis H37Rv or M. bovis BCG stimulate a pro-inflammatory response in uninfected macrophages.

Our studies are first to establish GPL's importance as a virulence factor in the M. avium pathogenesis. The data demonstrate a significant shedding of glycopeptidolipids in M. avium infected macrophages and its transport to the neighboring 'bystander cells'. This transport of mycobacterial components occur through release of exosomes extracellularly which can also act to stimulate uninfected macrophages both in vitro and in vivo. Together, our studies put forward a novel mechanism of intercellular communication mediated by exosomes which can carry mycobacterial components to neighboring cells.