Mycolyltransferase-mediated glycolipid exchange in Mycobacteria

Abstract

Trehalose dimycolate (TDM), also known as cord factor, is a major surface glycolipid of the cell wall of mycobacteria. Because of its potent biological functions in models of infection, adjuvancy, and immunotherapy, it is important to determine how its biosynthesis is regulated. Here we show that glucose, a host-derived product that is not readily available in the environment, causes Mycobacterium avium to down-regulate TDM expression while up-regulating production of another major glycolipid with immunological roles in T cell activation, glucose monomycolate (GMM). In vitro, the mechanism of reciprocal regulation of TDM and GMM involves competitive substrate selection by antigen 85A. The switch from TDM to GMM biosynthesis occurs near the physiological concentration of glucose present in mammalian hosts. We further demonstrate that GMM is produced in vivo by mycobacteria growing in mouse lung. These results establish an enzymatic pathway for GMM production. More generally, these observations provide a specific enzymatic mechanism for dynamic alterations of cell wall glycolipid remodeling in response to the transition from noncellular to cellular growth environments, including factors that are monitored by the host immune system.

FIGURE 1.

A reciprocal production of TDM and GMM in MAC in response to glucose. A, MAC was cultured in media containing 0.01% (w/v, lane 2), 1% (w/v, lane 3), 2% (w/v, lane 4), 5% (w/v, lane 5), and 10% glucose (w/v, lane 6), and the total lipid fractions (50 μg each) were analyzed on a TLC plate that was developed with chloroform/methanol/acetone/acetic acid (90:10:10:1, v/v). Purified TDM (lane 1) and TMM (lane 7) were used as references. Glucose dose-dependent production of a lipid species (indicated with an arrow) was detected. B, lipid species was purified and analyzed on a silica gel TLC plate that was developed with chloroform/methanol (9:1, v/v). C, MALDI-TOF MS profiles of the purified lipid species. D, GC-MS analysis of the sugar moiety of the purified lipid species. Arrows indicate retention times for the alditol acetate derivatives of arabinose (a), mannose (b), galactose (c)/ and glucose (d). Ion chromatogram of m/z 290 is shown. The retention time of the major ion corresponded with that of a glucose alditol acetate derivative.

FIGURE 2.

Proposed scheme for TDM (A) and GMM (B) production catalyzed by mycolyltransferase. In model A, both the donor site and the acceptor site of the enzyme interact with TMM, resulting in TDM formation. In model B, a glucose substrate competes against a TMM substrate for access to the acceptor site. When glucose is readily available, glucose rather than TMM preferentially gain access to the site, resulting in production of GMM.

FIGURE 3.

TDM-GMM exchange mediated by recombinant Ag85A. A, purified MAC Ag85A (left lane) and a size marker (right lane) were resolved on a Coomassie-stained SDS-polyacrylamide gel. Positions for the 40- and 28-kDa marker proteins are indicated. B, enzymatic reactions were performed at 37 °C at conditions indicated below, and the lipids were extracted from the reaction mixtures, followed by analysis on a TLC plate. Lane 1, Ag85A and TMM with 5% glucose (w/v), 0 h of incubation; lane 2, heat-inactivated (100 °C, 3 min) Ag85A and TMM with 5% glucose (w/v), 1 h of incubation; lanes 3–6, Ag85A and TMM either with 0.2% (w/v) glucose (lane 4), 1% glucose (w/v) (lane 5), and 5% (w/v) glucose (lane 6) or without glucose (lane 3), 1 h of incubation.

FIGURE 4.

GMM production by mycobacteria cultured at a physiological glucose concentration. A, MAC was cultured in liquid media containing either 0.01 or 0.1% glucose, and the culture media were replaced with fresh media every day to maintain the glucose concentrations. After 5 days, the bacteria were harvested, and the total lipids were extracted. The methanol-insoluble fraction was then obtained from 100 μg of each total lipid preparation and analyzed by TLC. B, GMM-specific, CD1b-restricted TCR-expressing Jurkat T cells were cocultured with either C1R/CD1b or C1R/mock in the presence of different concentrations of the total lipids derived from the 0.1% glucose-containing (upper panel) and the 0.01% glucose-containing (lower panel) cultures. The T cell response was assessed by measuring IL-2 released into the media.

FIGURE 5.

GMM production by mycobacteria during early phases of culture. MAC was cultured either in liquid media containing 0.01% (A) or 0.1% (B) glucose or in human serum (C). At indicated time points, the bacteria were harvested, and the total lipids were analyzed by TLC.

FIGURE 6.

GMM production in vivo by M. tuberculosis. A, among the mixture of lipids extracted from M. tuberculosis derived from mouse lungs, lipids that copurified with a GMM standard were analyzed in the positive mode on an Accurate Mass QTOF MS. The detected mass of m/z 1317.2577 corresponds to the predicted mass of an ammonium adduct of C₇₈ GMM (m/z 1317.2613). B, positive mode CID-MS analysis in ion trapping mass spectrometry of M. fallax GMM and the lung-derived candidate GMM molecule detected as sodium adducts show a similar pattern of product ions.