Biosynthesis and Regulation of Sulfomenaquinone, a Metabolite Associated with Virulence in <i>Mycobacterium tuberculosis</i>

Sulfomenaquinone (SMK) is a recently identified metabolite that is unique to the <i>Mycobacterium tuberculosis</i> (<i>M. tuberculosis</i>) complex and is shown to modulate its virulence. Here, we report the identification of the SMK biosynthetic operon that, in addition to a previously identified sulfotransferase <i>stf3</i>, includes a putative cytochrome P450 gene (<i>cyp128</i>) and a gene of unknown function, <i>rv2269c</i>. We demonstrate that <i>cyp128</i> and <i>stf3</i> are sufficient for the biosynthesis of SMK from menaquinone and <i>rv2269c</i> exhibits promoter activity in <i>M. tuberculosis</i>. Loss of Stf3 expression, but not that of Cyp128, is correlated with elevated levels of menaquinone-9, an essential component in the electron-transport chain in <i>M. tuberculosis</i>. Finally, we showed in a mouse model of infection that the loss of <i>cyp128</i> exhibits a hypervirulent phenotype similar to that in previous studies of the <i>stf3</i> mutant. These findings provide a platform for defining the molecular basis of SMK’s role in <i>M. tuberculosis</i> pathogenesis

Probing the Mycobacterial Trehalome with Bioorthogonal Chemistry

Mycobacteria, including the pathogen <i>Mycobacterium tuberculosis</i>, use the non-mammalian disaccharide trehalose as a precursor for essential cell-wall glycolipids and other metabolites. Here we describe a strategy for exploiting trehalose metabolic pathways to label glycolipids in mycobacteria with azide-modified trehalose (TreAz) analogues. Subsequent bioorthogonal ligation with alkyne-functionalized probes enabled detection and visualization of cell-surface glycolipids. Characterization of the metabolic fates of four TreAz analogues revealed unique labeling routes that can be harnessed for pathway-targeted investigation of the mycobacterial trehalome

Sulfolipid-1 Biosynthesis Restricts <i>Mycobacterium tuberculosis</i> Growth in Human Macrophages

<i>Mycobacterium tuberculosis</i> (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host–pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a <i>stf0</i>-deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide <i>in vitro</i>, despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the <i>stf0</i> mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages