Mycolyltransferase-mediated Glycolipid Exchange in Mycobacteria

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. 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. Mycobacterium avium complex (MAC) 2The abbreviations used are: MAC, Mycobacterium avium complex; Ag85, antigen 85; GC-MS, gas chromatography-mass spectrometry; GMM, glucose 6-monomycolate; IL-2, interleukin-2; MALDI-TOF MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry; TCR, T cell receptor; TDM, trehalose 6,6′-dimycolate; TMM, trehalose 6-monomycolate; LC-MS, liquid chromatography-mass spectrometry. 2The abbreviations used are: MAC, Mycobacterium avium complex; Ag85, antigen 85; GC-MS, gas chromatography-mass spectrometry; GMM, glucose 6-monomycolate; IL-2, interleukin-2; MALDI-TOF MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry; TCR, T cell receptor; TDM, trehalose 6,6′-dimycolate; TMM, trehalose 6-monomycolate; LC-MS, liquid chromatography-mass spectrometry. includes a group of acid-fast bacteria that distribute widely in natural environments, including soil, water, aerosols, and dust (1Primm T.P. Lucero C.A. Falkinham III, J.O. Clin. Microbiol. Rev. 2004; 17: 98-106Crossref PubMed Scopus (414) Google Scholar). Although less virulent than Mycobacterium tuberculosis, these environmental mycobacteria occasionally infect humans, especially patients infected with human immunodeficiency virus type 1, where they represent a major cause of morbidity. The incidence of clinically overt MAC infection has increased significantly in recent years, and because of the multidrug resistance evolved by the microbes, MAC infection is difficult to clear with chemotherapeutic agents. Thus, M. tuberculosis and MAC are now the two major groups of mycobacteria species that require further efforts for prevention and treatment. Unlike M. tuberculosis, which transmits primarily from individuals with active disease, epidemiologic evidence suggests that such transmission pathways are unlikely for MAC. Rather, MAC infection appears to occur when susceptible individuals are exposed to environmental MAC. These observations predict that, upon infection, environmental MAC should undergo significant adaptive changes to allow its survival and replication within the host. Mycobacteria possess highly lipid-rich cell walls that are critical not simply for their acid-fast properties but also for their survival and replication. The cell wall contains mycolic acids, an α-alkyl-β-hydroxy fatty acid with extremely long carbon chains (∼C80), which are densely aligned in covalent association with the 6-position of arabinose termini of the underlying arabinogalactan sugar layer or exist as free molecules complexed to sugars, either glucose or trehalose. Arabinogalactan-linked mycolates are proposed to extend outward and interact noncovalently with carbon chains of the so-called surface-exposed glycolipids, including trehalose 6-monomycolate (TMM), trehalose 6,6′-dimycolate (TDM), and glucose 6-monomycolate (GMM), thereby forming the hydrophobic cell wall architecture that is essential for protection against chemical attack, such as reactive oxygen intermediates and hydrolytic enzymes derived from the host cells. Among the most abundant surface-exposed glycolipids is TDM that is biosynthesized from its precursor, TMM, by the mycolyltransferase activity of antigen 85 (Ag85) (2Sathyamoorthy N. Takayama K. J. Biol. Chem. 1987; 262: 13417-13423Abstract Full Text PDF PubMed Google Scholar). Many biological functions have been assigned to TDM (3Ryll R. Kumazawa Y. Yano I. Microbiol. Immunol. 2001; 45: 801-811Crossref PubMed Scopus (129) Google Scholar) that may impact on survival of mycobacteria within the host and possibly their virulence. Therefore, it is important to determine how biosynthesis of TDM and other mycolic acid-containing glycolipids is regulated by external factors. GMM exists at varied levels in the mycobacterial cell wall (4Brennan P. Ballou C.E. J. Biol. Chem. 1967; 242: 3046-3056Abstract Full Text PDF PubMed Google Scholar, 5Moody D.B. Guy M.R. Grant E. Cheng T.Y. Brenner M.B. Besra G.S. Porcelli S.A. J. Exp. Med. 2000; 192: 965-976Crossref PubMed Scopus (131) Google Scholar). In addition to its role in cell wall barrier functions, GMM is a granuloma-forming agent in mice (6Matsunaga I. Oka S. Inoue T. Yano I. FEMS Microbiol. Lett. 1990; 55: 49-53Crossref PubMed Google Scholar) as well as a CD1b presented antigen in humans (7Moody D.B. Reinhold B.B. Guy M.R. Beckman E.M. Frederique D.E. Furlong S.T. Ye S. Reinhold V.N. Sieling P.A. Modlin R.L. Besra G.S. Porcelli S.A. Science. 1997; 278: 283-286Crossref PubMed Scopus (381) Google Scholar). Here we identify Ag85A as an enzyme that produces GMM by transfer of mycolate to glucose. Furthermore, mechanistic studies show that glucose present in its growth environment regulates the spectrum of mycolylglycolipids made by MAC, and glucose from the host influences GMM production in vivo during infection of mice. Mechanistic studies showed that glucose and trehalose compete as substrates for Ag85A, linking the biosynthesis pathways of GMM and TDM. Reagents and Bacteria—Chemical reagents were purchased from Nacalai Tesque (Kyoto, Japan) unless otherwise indicated. M. avium ATCC 35767 (serovar 4) was obtained from American Type Culture Collection (Manassas, VA). The bacteria were maintained on a plate of Middlebrook 7H10 media supplemented with 10% oleic acid/albumin/dextrose/catalase (BD Biosciences). For extraction of the total lipid fraction, the bacteria were cultured in Middlebrook 7H9 broth media (containing 0.05% Tween 80 but not glycerol) supplemented with 10% albumin/dextrose/catalase (BD Biosciences). The log phase culture was diluted with 20 volumes of 7H9 media containing various concentrations of glucose, and the culture was continued for another 5–7 days until the absorbance at 600 nm reached ∼1. In some experiments, bacteria were grown in media containing either 0.01 or 0.1% glucose, and the media were replaced every day with fresh media containing the same concentrations of glucose. After 5 days, the bacteria were harvested for lipid extraction. To monitor early GMM production, bacteria were grown either in 7H9 media containing 0.01 or 0.1% glucose or in human serum and were harvested after 2, 4, 8, 18, and 24 h of culture. Preparation of Mycolylglycolipids from MAC—Total lipids from mycobacteria were prepared as described previously (8Matsunaga I. Bhatt A. Young D.C. Cheng T.Y. Eyles S.J. Besra G.S. Briken V. Porcelli S.A. Costello C.E. Jacobs Jr., W.R. Moody D.B. J. Exp. Med. 2004; 200: 1559-1569Crossref PubMed Scopus (147) Google Scholar). The total lipids were then dissolved in chloroform/methanol (C/M, 2:1, v/v), and 20 volumes of ice-cold acetone were added. After 30 min of incubation on ice, the suspension was subjected to centrifugation at 1,500 × g for 15 min at 1 °C, and the supernatant was carefully removed. The pellet was then washed with ice-cold acetone, and the residue was dissolved in C/M (2:1) and fractionated by TLC using an Analtech TLC plate (Newark, DE) with a solvent system of chloroform/methanol/acetone/acetic acid (90:10:10:1, v/v). GMM, TDM and TMM fractions were extracted with C/M (2:1) from the silica gels. For GMM and TDM purification, the fractions were further fractionated by TLC with a solvent system of chloroform/acetone/methanol/water (50:60:2.5:0.6, v/v). Finally, the GMM, TDM and TMM fractions were extracted with C/M (2:1), dried, and rinsed several times with methanol at room temperature to remove any residual contamination of glycopeptidolipids and phospholipids. Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS)—MALDI-TOF MS analyses of glycolipids were carried out according to the method described previously (9Enomoto Y. Sugita M. Matsunaga I. Naka T. Sato A. Kawashima T. Shimizu K. Takahashi H. Norose Y. Yano I. Biochem. Biophys. Res. Commun. 2005; 337: 452-456Crossref PubMed Scopus (10) Google Scholar). Briefly, MALDI-TOF MS spectra were acquired on a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems) with a pulse laser emitting at 337 nm. Samples were analyzed in the reflectron mode with an accelerating voltage operating in positive ion mode of 20 kV. As the matrix, 2,5-dihydroxybenzoic acid was used. Gas Chromatography-Mass Spectrometry (GC-MS)—GC-MS analysis of the sugar moiety of GMM was carried out according to the method described previously (9Enomoto Y. Sugita M. Matsunaga I. Naka T. Sato A. Kawashima T. Shimizu K. Takahashi H. Norose Y. Yano I. Biochem. Biophys. Res. Commun. 2005; 337: 452-456Crossref PubMed Scopus (10) Google Scholar). Briefly, GMM was hydrolyzed with 2 m trifluoroacetic acid at 120 °C for 2 h. The aqueous phase was dried, reduced with 10 mg/ml solution of NaBD4 (1 m NH4OH/C2H5OH, 1:1, v/v) at room temperature for 2 h, and then acetylated with acetic anhydride/pyridine (1:1, v/v) at 100 °C for 1 h. The resulting alditol acetate derivatives were analyzed by GC-MS with GCMS-QP2010 plus (Shimazu Co., Ltd., Kyoto, Japan), using a fused silica capillary 30 m × was at °C for and then the temperature was increased to °C at a of The temperature was then at °C for of gas was of the from MAC, Preparation of the and was from the MAC using the according to the Ltd., The that the Ag85A the was by using a specific as in which the is and in which the is was carried out using a Ltd., Japan), and the for were as °C, 2 by 30 of °C, 20 and °C, and a of °C, The were with and and to a The of the Ag85A were for was with the Ag85A in and of expression was according to a method of Besra G.S. Lett. Microbiol. PubMed Scopus Google Scholar). The bacteria the Ag85A were harvested and by in ice-cold 20 containing m and The was at × g for 30 min at °C to remove and then the supernatant was a with the at After the with the the Ag85A was with 20 containing m and m The was and against containing 10% at concentration of the Ag85A was by the of the was by and were carried out by of a method of Besra G.S. Lett. Microbiol. PubMed Scopus Google Scholar). of TMM was by in of in the or of concentration of The was by the addition of of the enzyme containing After 1 h of incubation at °C, the was by the addition of 2 of C/M (2:1) and of The lipids were extracted by the method of Besra G.S. Lett. Microbiol. PubMed Scopus Google Scholar) and analyzed by silica The lipids on the TLC plate were by acid and GMM in were carried out the with M. tuberculosis with mice after days of were with and at × g for 30 min at room The pellet was with to with m acid in and at × g for 30 min to were extracted from with in C/M 1:1, and to and in These lipids were further fractionated by acetone to for lipids that were analyzed by phase on a was and solvent was 0.1% acid and 0.05% was used at solvent for to solvent 5 at solvent for to solvent at solvent for to solvent and at solvent for the mass for GMM were at after min of these The mass was carried out with an Mass in the positive mode with an liquid system. was carried out with a mass spectrometer with in with GMM derived from Mycobacterium T.Y. M. I. Young D.C. Besra G.S. Briken V. Porcelli S. Moody D.B. J. PubMed Scopus Google Scholar). T T cell by with have been described previously M. S. Modlin R.L. Porcelli S.A. Brenner M.B. J. Exp. Med. PubMed Scopus Google Scholar). The T × were in with the human (1 × either with CD1b or with M. Porcelli S.A. Brenner M.B. J. Immunol. 1997; Google Scholar) in the of and concentrations of lipid In some experiments, were used as cells. After 20 h, of the culture were and the of the was by the (BD Biosciences). of TDM and GMM by MAC in to is an essential to which is as a not for production but also for biosynthesis of of cellular Unlike other sugars, glucose is maintained at levels in the and of mammalian hosts. Therefore, we that, upon infection the MAC grown in undergo significant alterations in glycolipid biosynthesis by to host-derived glucose. To the impact of glucose on glycolipid in we monitored glycolipid production by M. avium (serovar 4) that was harvested after in liquid media supplemented with concentrations of glucose. The total lipid was obtained by with and The lipids were then analyzed on a TLC plate with a solvent system for of glycolipid species grown in the of a of glucose mycobacteria produced levels of TDM and TMM 2, with As the glucose concentrations present in media were TDM production the of TMM an in a lipid species with a than that of TDM was with an To determine the of the it was and subjected to TLC and MS The lipid was as on a TLC plate with a solvent system of C/M v/v) MALDI-TOF MS analysis that the mass of were with of of monomycolate the of of the method of the in of the of the the of mass of species of mycolate and the mass the major which for by in carbon and of groups III, C.E. K. Y. Res. PubMed Scopus Google Scholar). For to the mass of of monomycolate with fatty acid and a group on the GC-MS analysis of an alditol acetate of the sugar moiety derived from the lipid glucose as the group to mycolates The on a TLC plate were to represent two of mycolates as described previously D.B. Guy M.R. Grant E. Cheng T.Y. Brenner M.B. Besra G.S. Porcelli S.A. J. Exp. Med. 2000; 192: 965-976Crossref PubMed Scopus (131) Google Scholar, Y. Oka S. K. Y. Yano I. Immunol. 1990; PubMed Scopus Google Scholar). Finally, the production of GMM in response to glucose is on the of mycobacteria to abundant at D.B. Guy M.R. Grant E. Cheng T.Y. Brenner M.B. Besra G.S. Porcelli S.A. J. Exp. Med. 2000; 192: 965-976Crossref PubMed Scopus (131) Google Scholar). These results a reciprocal production of TDM and GMM by MAC in response to glucose alterations in the levels of carried out in the that compete for for GMM known also as Ag85, the of TDM using TMM as a models of the that two molecules of TMM are in the two of the and the group of the TMM substrate in is to the other TMM substrate in the other resulting in of of TDM and of trehalose (2Sathyamoorthy N. Takayama K. J. Biol. Chem. 1987; 262: 13417-13423Abstract Full Text PDF PubMed Google Scholar, J. Biol. 2001; PubMed Scopus Google Scholar). Although GMM an abundant in the cell wall and functions to T and its mechanism of was We that GMM biosynthesis by glucose, of TMM, the To we made Ag85A enzyme from the M. avium (serovar and we in enzymatic To we carried out from the of the MAC as a and the Ag85A that the the of the that were as with the previously Ag85A derived from M. avium 1 N. K. R. A. H. T. PubMed Google but the acid were in We then an expression in which the was at the and the a was in at the of the Ag85A The enzyme was in E. and by The was as a with an mass of on a with its the Ag85A of TMM in in the of enzyme in of TDM the mycolyltransferase activity by the addition of glucose to in TDM production in a which was with an in GMM GMM was when enzyme was used further that GMM was produced by the mycolyltransferase activity of Ag85A but not as a of These results that Ag85A of In we that TMM and glucose compete for to the of the Ag85A, and the enzyme biosynthesis of GMM, than TDM, when glucose is readily available The substrate selection by the mycolyltransferase provide a for the in cultured by MAC Ag85A and a were on a for the and are indicated. enzymatic were at °C at and the lipids were extracted from the by analysis on a TLC 1, Ag85A and TMM with glucose h of 2, °C, Ag85A and TMM with glucose 1 h of Ag85A and TMM either with glucose glucose and glucose or glucose 1 h of GMM at a observations made have an enzymatic pathway for GMM production in mycobacteria that are grown in the of levels of glucose. it to mycobacteria GMM physiological concentrations of glucose present in mammalian which is maintained at To we GMM production by mycobacteria cultured in liquid media with a glucose concentration with that in the host. The MAC culture was in the of either 0.01 or 0.1% glucose, and every 24 h, the culture media were replaced with fresh media to the glucose concentrations at After 5 days of the bacteria were and the total lipids were lipids were from these total by on TLC Although TDM production was readily in GMM production was in the of 0.1% glucose but not in the of glucose was also by T in which T specific GMM in the of CD1b molecules were used. of the T with in the of the total lipids from the 0.1% culture in production by the T levels of when growing at physiological glucose concentrations The specific response was not when were used as that the response was We then how of GMM production after to 0.1% glucose. MAC was cultured either in liquid media containing or 0.1% or in human serum and the bacteria were harvested at 2, 4, 8, 18, and 24 h. GMM production was as early as h after the of the culture in 0.1% media and in human serum but not in media containing glucose. These observations that GMM production occur after to levels of glucose as a of competitive substrate selection by GMM in GMM lipids derived from Mycobacterium the that GMM is produced by mycobacteria in D.B. Guy M.R. Grant E. Cheng T.Y. Brenner M.B. Besra G.S. Porcelli S.A. J. Exp. Med. 2000; 192: 965-976Crossref PubMed Scopus (131) Google Scholar). the chemical of such mycolates not and it M. tuberculosis produces GMM during Therefore, we infected mice with M. tuberculosis and mycobacteria from the after of were from by centrifugation and with to The resulting mycobacterial lipids when analyzed by not total M. tuberculosis lipids from with an M. GMM in experiments, we analyzed the in vivo derived lipids that with the GMM Mass with an Mass of mass of 10 an ion at in lipids the and the the of an of a GMM a acid within GMM, the of ion as GMM, mycolic acid derivatives are as a of molecules that from another by mass to and the spectrum of the lipids two to the of and GMM not Finally, a carried out with an M. GMM and the showed product including with mass to the of and 120 and which represent the of and which are from the sugar These provide evidence that GMM is made in the host in vivo during an MAC a group of environmental mycobacteria that have evolved the to to In MAC and in (1Primm T.P. Lucero C.A. Falkinham III, J.O. Clin. Microbiol. Rev. 2004; 17: 98-106Crossref PubMed Scopus (414) Google where of the exist in association with the surface significant resistance to a of and chemical such as to and and is an important for the environmental mycobacteria to their in natural These environmental mycobacteria TDM on the surface of their cell wall but to GMM because of highly of glucose. that, upon the glycolipid by the host-derived glucose as a and in M. tuberculosis T. Takayama K. Besra G.S. Science. 1997; PubMed Scopus Google Scholar) of TDM from TMM in vitro, and the have been also in M. Although these enzymes have functions, their have been in mycobacteria grown Ag85A is an that is in mycobacteria PubMed Scopus Google Scholar, V. 2000; PubMed Scopus Google and its for the have an impact on functions TDM and GMM have to the cells. we that produced a of when with GMM, which with with TDM that were of not Therefore, the may in the response by the host to the of any mycolyltransferase that GMM was these results establish that Ag85A has the by the host they are then by specific T that to the acquired T are in humans and infected with and of these T is not by the major and molecules but by group 1 molecules and in I. Sugita M. Immunol. Rev. Scopus Google Scholar). These molecules are in as well as the two major cell for mycobacterial of T is in human infected with M. and the human T cell (7Moody D.B. Reinhold B.B. Guy M.R. Beckman E.M. Frederique D.E. Furlong S.T. Ye S. Reinhold V.N. Sieling P.A. Modlin R.L. Besra G.S. Porcelli S.A. Science. 1997; 278: 283-286Crossref PubMed Scopus (381) Google Scholar) and T T. Moody D.B. Grant E. Porcelli S.A. PubMed Scopus Google Scholar) that the T cell response against GMM to clear these results an that GMM as a of functions to the immune but the host with a to monitor mycobacteria and in the of the acquired may represent an of how the immune system has been during the long of to against the that GMM is produced by mycobacteria and to the well TDM, it has not been the for until is because a of GMM, as with TDM, is by growing such as M. tuberculosis and M. when cultured in the Middlebrook media the media used for of growing such as glucose, and the bacteria cultured in such a GMM and its and biological have been (6Matsunaga I. Oka S. Inoue T. Yano I. FEMS Microbiol. Lett. 1990; 55: 49-53Crossref PubMed Google Scholar, I. Oka S. N. Yano I. J. Biochem. PubMed Scopus Google Scholar). In the and of mycobacterial lipids were by using bacteria grown in but the present suggests that the that is by the bacteria grown in culture is from the lipid as a of with the host We Japan) for of the GC-MS and Cheng and for reagents and