Summary The lipidic envelope of Mycobacterium tuberculosis promotes virulence in many ways, so we developed a lipidomics platform for broad survey of cell walls. Here we report two new databases (MycoMass, MycoMap), 30 lipid fine maps and mass spectrometry datasets that comprise a static lipidome. Further, by rapidly regenerating lipidomic datasets during biological processes, comparative lipidomics provides statistically valid, organism-wide comparisons that broadly assess lipid changes during infection or among clinical strains of mycobacteria. Using stringent data filters, we tracked more than 5,000 molecular features in parallel with few or no false positive molecular discoveries. The low error rates allowed the first chemotaxonomic analyses of mycobacteria, which describe the extent of chemical change in each strain and identified particular strain-specific molecules for use as biomarkers.

Cytochrome P450 isolated from Bacillus subtilis (P450 BS␤ ; molecular mass, 48 kDa) catalyzes the hydroxylation of a long-chain fatty acid (e.g. myristic acid) at the ␣-and ␤-positions using hydrogen peroxide as an oxidant. We report here on the crystal structure of ferric P450 BS␤ in the substrate-bound form, determined at a resolution of 2.1 Å. P450 BS␤ exhibits a typical P450 fold. The substrate binds to a specific channel in the enzyme and is stabilized through hydrophobic interactions of its alkyl side chain with some hydrophobic residues on the enzyme as well as by electrostatic interaction of its terminal carboxylate with the Arg 242 guanidium group. These interactions are responsible for the site specificity of the hydroxylation site in which the ␣-and ␤-positions of the fatty acid come into close proximity to the heme iron sixth site. The fatty acid carboxylate group interacts with Arg 242 in the same fashion as has been reported for the active site of chloroperoxidase, His 105 -Glu 183 , which is an acid-base catalyst in the peroxidation reactions. On the basis of these observations, a possible mechanism for the hydroxylation reaction catalyzed by P450 BS␤ is proposed in which the carboxylate of the bound-substrate fatty acid assists in the cleavage of the peroxide O-O bond.Two bacterial cytochrome P450s isolated from Sphingomonas paucimobilis and Bacillus subtilis, P450 SP␣ 1 and P450 BS␤ , respectively, are heme-containing enzymes that catalyze the hydroxylation reaction of long chain fatty acids (e.g. myristic acid) using hydrogen peroxide (H 2 O 2 ) as an oxidant to produce hydroxylated (-OH) fatty acids (1, 2). In the enzymatic reactions, an oxygen atom derived from H 2 O 2 is efficiently introduced into the substrate with a high catalytic turnover (1,000 min Ϫ1 ) (2-4). P450 SP␣ produces the ␣-OH fatty acid (100%) as the product, whereas P450 BS␤ produces both the ␤-OH (60%) and the ␣-OH (40%) fatty acids (1, 2, 4, 5). The amino acid sequence of the two enzymes shares a 44% identity (2). Data base investigation has shown that P450 SP␣ and P450 BS␤ belong to the P450 superfamily and, therefore, they have been given the systematic nomenclature designations CYP152B1 and CYP152A1, respectively (6). However, when compared with reactions catalyzed by other P450s, two characteristic properties in the P450 SP␣ and P450 BS␤ reactions were found, i.e. the utilization of H 2 O 2 and the site specificity of the reaction.In typical P450 reactions an oxygen atom derived from molecular oxygen (O 2 ) is inserted into the substrates (7), and the reaction is referred to as a monooxygenation reaction. Two protons and two electrons are required in the monooxygenation reaction. The electrons are supplied from NAD(P)H through mediation by flavoproteins and iron-sulfur proteins, and the protons are probably delivered from solvent water to the active site through a specific hydrogen-bonding network (8). In the monooxygenase P450 system, H 2 O 2 is sometimes used as a surrogate for the O 2 /2e Ϫ /2H ϩ system (peroxide sh...

CD1c-mediated T cells are activated by a mycobacterial phospholipid antigen whose carbohydrate structure precisely corresponds to mammalian mannosyl β-1-phosphodolichol (MPD), but contains an unusual lipid moiety. Here, we show that this T cell antigen is a member of a family of branched, alkane lipids that vary in length (C30-34) and are produced by medically important mycobacteria such as M. tuberculosis and M. bovis Bacille-Calmette-Guerin. The alkane moiety distinguished these mycobacterial lipid antigens from mammalian MPDs and was necessary for activation of CD1c-restricted T cells, but could not be accounted for by any known lipid biosynthetic pathway. Metabolic labeling and mass spectrometric analyses suggested a mechanism for elongating lipids using alternating C2 and C3 units, rather than C5 isopentenyl pyrophosphate. Inspection of the M. tuberculosis genome identified one candidate gene, pks12, which was predicted to encode the largest protein in M. tuberculosis, consisting of 12 catalytic domains that correspond to key steps in the proposed pathway. Genetic deletion and complementation showed that Pks12 was necessary for antigen production, but did not affect synthesis of true isoprenols. These studies establish the genetic and enzymatic basis for a previously unknown type of polyketide, designated mycoketide, which contains a lipidic pathogen-associated molecular pattern.

Mycobacterium tuberculosis remains a major pathogen of worldwide importance, which releases lipid Ags that are presented to human T cells during the course of tuberculosis infections. Here we report that cellular infection with live M. tuberculosis or exposure to mycobacterial cell wall products converted CD1− myeloid precursors into competent APCs that expressed group 1 CD1 proteins (CD1a, CD1b, and CD1c). The appearance of group 1 CD1 proteins at the surface of infected or activated cells occurred via transcriptional regulation, and new CD1 protein synthesis and was accompanied by down-regulation of CD1d transcripts and protein. Isolation of CD1-inducing factors from M. tuberculosis using normal phase chromatography, as well as the use of purified natural and synthetic compounds, showed that this process involved polar lipids that signaled through TLR-2, and we found that TLR-2 was necessary for the up-regulation of CD1 protein expression. Thus, mycobacterial cell wall lipids provide two distinct signals for the activation of lipid-reactive T cells: lipid Ags that activate T cell receptors and lipid adjuvants that activate APCs through TLR-2. These dual activation signals may represent a system for selectively promoting the presentation of exogenous foreign lipids by those myeloid APCs, which come into direct contact with pathogens.