Laboratoire des Mécanismes Moléculaires de la Pathogénie Microbienne, INSERM U447, Institut Pasteur de Lille/IBL, 1 rue du Pr. Calmette, BP245-59019 Lille Cedex, France1
Laboratoire de Glycobiologie Structurale et Fonctionnelle, CNRS UMR8576, Université des Sciences et Technologies de Lille, F-59655 Villeneuve dAscq Cedex, France2
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK3
Author for correspondence: Laurent Kremer. Tel: +33 3 20 87 11 54. Fax: +33 3 20 87 11 58. e-mail: laurent.kremer{at}ibl.fr
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ABSTRACT |
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Keywords: mycobacteria, fatty acid synthase II, mycolic acid, KasA, temperature
Abbreviations: AcpM, mycobacterial acyl carrier protein; AG, arabinogalactan; FAME, fatty acid methyl ester; FAS, fatty acid synthase; KasA, ß-ketoacyl AcpM synthase A; LAM, lipoarabinomannan; LM, lipomannan; MAME, mycolic acid methyl ester; PIM, phosphatidylinositol mannoside; PPM, polyprenol monophosphoryl mannose; TDM, trehalose dimycolate
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INTRODUCTION |
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The mycobacterial cell envelope differs substantially from the canonical cell-wall structures of Gram-negative and Gram-positive bacteria (Brennan & Nikaido, 1995 ). In addition to the typical cell-membrane bilayer and peptidoglycan layers found in other bacteria, the mycobacterial cell envelope contains mycolic acids covalently attached to arabinogalactan (AG) and a wide range of non-covalently attached complex lipids and glycolipids (Minnikin, 1982
). Some of these lipids also exhibit potent biological activities and thus may be considered as important virulence factors (Cox et al., 1999
; Camacho et al., 2001
). Interestingly, some of them are exported and can be found within the cytoplasm of host macrophages, dissociated from the intact bacilli (Beatty et al., 2000
). It has been proposed that the chemical diversity of lipids and glycolipids found in the mycobacterial cell envelope mediates specific interactions with host ligands or membranes rather than being solely responsible for the hydrophobicity and rigidity of the mycobacterial cell wall.
The capacity of pathogenic mycobacteria to survive and replicate within the hostile environment of the host macrophage depends on the existence of adaptive mechanisms of bacterial cell physiology. Although environmental adaptation of the mycobacterial cell wall and its components remains largely unknown, it is very likely that they participate in the adaptive process. The first evidence for this kind of regulatory process came from work conducted by Davidson et al. (1982) in Mycobacterium microti, a close relative of Mycobacterium tuberculosis. It was shown that young growing cultures harvested from mouse lungs contained high proportions of ketomycolates, whereas stationary phase cultures had roughly equal proportions of keto- and methoxymycolates. The proportion of
-mycolates increased slightly with the age of the culture, but was always less than one-third of the total mycolate content. More recently, Yuan et al. (1998)
reported that ketomycolates were produced more predominantly during intracellular growth of M. tuberculosis within THP-1 macrophages. These authors also demonstrated that the ratio of methoxymycolates to ketomycolates was altered by oxygen tension and that the ratio produced under low oxygen tension was identical to that of organisms grown in vivo.
In this report, we describe the effect of growth temperature on mycolic acid, fatty acid and complex lipid composition in Mycobacterium thermoresistibile. The rationale of the use of this strain is that it survives and replicates at elevated temperatures (up to 55 °C) compared to most mycobacterial species, a property that facilitates the analysis of cell-wall component changes induced by growth at high temperature.
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METHODS |
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Mycolic acid and fatty acid profiles of cells grown at high temperature.
[1,2-14C]Acetate [5062 mCi mmol-1 (1·852·29 GBq mmol-1); Amersham] was added at 1 µCi ml-1 (37 kBq ml-1) to mycobacterial cultures grown at mid-exponential phase, followed by a further 6 h incubation at either 37 °C or 55 °C. 14C-Labelled cells were harvested by centrifugation at 2000 g and washed once with PBS. Washed cells were then subjected to alkaline hydrolysis using 2 ml 15% tetrabutylammonium hydroxide (TBAH) at 100 °C overnight and mixed with 4 ml CH2Cl2, 300 µl CH3I and 2 ml H2O for 1 h. The upper aqueous phase was discarded and the lower organic phase was washed twice with water and dried. The lipids were extracted using diethyl ether, re-dried and resuspended in 200 µl CH2Cl2. An aliquot of the resultant mixture of fatty acid methyl esters (FAMEs) and mycolic acid methyl esters (MAMEs) was then subjected to TLC using silica gel plates (5735 silica gel 60F254; Merck) developed in petroleum ether/acetone (19:1, v/v). The FAMEs were separated by reverse-phase TLC on C18-silica gel plates (Sigma) using chloroform/methanol (2:3, v/v).
In some instances, the individual MAMEs were resolved through two-dimensional silver ion argentation TLC by immersing 80% of a square silica gel TLC plate into a 10% (w/v) aqueous silver nitrate solution. Following air-drying, plates were activated at 100 °C for 1 h. 14C-Labelled samples were run in the first dimension along the narrow strip without silver impregnation by developing twice with hexane/ethyl acetate (19:1, v/v). The plates were then dried, turned 90 degrees and run into the silver layer by developing three times with petroleum ether/diethyl ether (17:3, v/v). Autoradiograms were obtained by exposure to Kodak X-Omat AR film to reveal [14C]FAMEs and [14C]MAMEs.
Systematic analysis of complex mycobacterial lipids.
The lipid composition was analysed using [1,2-14C]acetate-labelled M. thermoresistibile cells. Lipids were extracted by adding 2 ml CH3OH/0·3% NaCl (10:1, v/v) and 1 ml petroleum ether to pelleted cells. After centrifugation, the upper petroleum ether layer was removed and 1 ml petroleum ether was added. The combined petroleum ether extracts were then evaporated under nitrogen to yield apolar lipids that were resuspended in CH2Cl2 prior to TLC analysis. For extraction of polar lipids, the methanolic saline extract was heated at 65 °C for 5 min and 2·3 ml CHCl3/CH3OH/0·3% NaCl (9:10:3, by vol.) was added to the extract and mixed on a tube rotator for 10 min. The solvent extract was then separated from the biomass by centrifugation and the supernatant retained. The pellet was further extracted with 0·75 ml CHCl3/CH3OH/0·3% NaCl (5:10:4, by vol.) for 10 min. The combined solvent extracts were mixed with 1·3 ml CHCl3 and 1·3 ml 0·3% NaCl for 5 min. After centrifugation the lower organic layer was collected and evaporated to dryness to yield the polar lipids which were resuspended in CHCl3/CH3OH/H2O (10:10:3, by vol.) prior to TLC analysis.
The complex lipids were analysed by two-dimensional TLC using a variety of solvent systems. Five systems were necessary to cover the polarity range of both non-polar and polar mycobacterial lipids (Besra, 1998 ): briefly, system A, first dimension, petroleum ether/ethyl acetate 98:2, v/v (x3); second dimension, petroleum ether/acetone (49:1, v/v); system B, first dimension, petroleum ether/acetone 23:2, v/v (x3); second dimension, toluene/acetone (19:1, v/v); system C, first dimension, CHCl3/CH3OH (24:1, v/v); second dimension, toluene/acetone (4:1, v/v); system D, first dimension, CHCl3/CH3OH/H2O (100:14:0·8 by vol.); second dimension, CHCl3/acetone/CH3OH/H2O (50:60:2·5:3 by vol.); and system E, first dimension, CHCl3/CH3OH/H2O (10:5:1, by vol.); second dimension, CHCl3/acetic acid/CH3OH/H2O (40:25:3:6, by vol.). Lipids were detected by exposure of the TLCs to Kodak X-Omat AR film and charred at 110 °C following spraying with
-naphthol or 5% molybdophosphoric acid.
Anti-KasA immune serum.
Recombinant His-tagged ß-ketoacyl mycobacterial ACP (AcpM) synthase (KasA) protein was produced in E. coli carrying pET28a-kasA and purified by affinity chromatography using a His-Trap column (Pharmacia) as described previously (Kremer et al., 2002a ). Purified KasA was used to prepare an anti-KasA immune serum. A rat was injected four times with 25 µg KasA mixed with an equal volume of adjuvant (monophosphoryl lipid A+trehalose dicorynomycolate; Sigma) at 1, 15, 30 and 60 days. The serum was collected at different time points after the last injection and the specificity of the antibodies was tested by Western blotting.
Electrophoresis and Western blot analyses.
M. thermoresistibile cells were harvested, resuspended in PBS and disrupted by sonication. Protein concentration was determined on total lysates using the BCA Protein Assay Reagent kit (Pierce) according to the manufacturers instructions. Twenty micrograms of total protein was subjected to SDS-PAGE as described by Laemmli (1970) with 12% acrylamide gels on a MiniProtean II system (Bio-Rad). Proteins were either stained with Coomassie blue R350 (Amersham Pharmacia) or transferred to nitrocellulose membranes for Western blot analysis (Kremer et al., 1995
). For detection of KasA, membranes were incubated overnight with the rat anti-serum directed against KasA at a dilution of 1/500, washed and subsequently incubated with anti-rat antibodies conjugated to alkaline phosphatase (Promega) used at a 1/7000 dilution. Detection of the antigen 85 (Ag85) complex was done by probing the membranes with a mixture of monoclonal antibodies 17/4 and 32/4 diluted 1/10 (Huygen et al., 1994
). Membranes were then incubated with anti-mouse antibodies coupled to alkaline phosphatase (Promega) and used at a 1/7000 dilution.
Incorporation of radiolabelled mannose (Man) from GDP-[14C]Man into membrane lipids.
The enzymically active membrane fraction of M. thermoresistible was prepared as previously described (Besra et al., 1997 ). To mannosylate endogenous polyprenol monophosphate lipid substrates, the membrane fractions were incubated in buffer A [50 mM MOPS (adjusted to pH 7·9 with KOH), 5 mM ß-mercaptoethanol, 10 mM MgCl2] containing 0·1 mM DTT, 20 mM NaF, 2·4 µM [0·125 µCi (4·62 kBq)] GDP-[U-14C]mannose [321·4 mCi mmol-1 (11·9 GBq) DuPont NEN] and 62·5 µM ATP in a total volume of 50 µl at 37 °C for 30 min. The reaction was terminated by the addition of 4 ml CHCl3/CH3OH/0·8 M NaOH (10:10:3, by vol.) and was followed by incubation at 55 °C for 15 min. The reaction mixture was cooled and 1·75 ml CHCl3 and 0·75 ml H2O were added. After centrifugation the upper aqueous phase was discarded. The lower phase was washed three times with 2 ml CHCl3/CH3OH/H2O (3:47:48, by vol.) to yield an organic fraction, which contained exclusively the mild-alkali stable family of polyprenol monophosphoryl mannoses (PPMs). This fraction was then dried under a stream of nitrogen and resuspended in 200 µl CHCl3/CH3OH (2:1, v/v). PP[14C]Ms were quantified by scintillation counting and equal aliquots (4000 c.p.m.) were resolved by TLC on silica gel plates using CHCl3/CH3OH/H2O (65:25:4, by vol.). Autoradiograms were obtained by exposing the chromatograms to Kodak X-Omat AR films for 23 days.
To assess the transfer of [14C]Man from GDP-[14C]Man to the phosphatidylinositol mannosides (PIMs), membrane fractions were incubated in buffer A containing 0·1 mM DTT, 20 mM NaF, 62·5 µM ATP, 10 mM CaCl2 and 2·5 µg amphomycin (a lipopeptide antibiotic that specifically inhibits polyprenyl-phosphate-requiring translocases and the synthesis of C35/C50-P-Man) in a total volume of 50 µl. Reactions were incubated for 10 min at 37 °C prior to the addition of 2·4 µM (0·125 µCi) GDP-[U-14C]Man, and held at 37 °C for a further 50 min. The reaction was stopped by the addition of 4 ml CHCl3/CH3OH/H2O (10:10:3, by vol.) and incubation at room temperature for 30 min, followed by the addition of 1·75 ml CHCl3 and 0·75 ml H2O. The lower organic layer of the biphasic mixture was washed three times with 2 ml CHCl3/CH3OH/H2O (3:47:48, by vol.), dried under a stream of nitrogen and resuspended in 200 µl CHCl3/CH3OH (2:1, v/v). The transfer of [14C]Man from GDP-[14C]Man to the PIMs was quantified by scintillation counting and the material was analysed by TLC-autoradiography as described previously (Besra et al., 1997 ).
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RESULTS |
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To investigate whether the FAMEs accumulating at 55 °C were associated to the cell wall or were present as free lipids, 14C-labelled cells were delipidated to extract free lipids. FAMEs and MAMEs were then isolated and analysed by TLC and autoradiography. As shown in Fig. 3(a) (lower panel), nearly all FAMEs from cells incubated at 37 °C were removed after delipidation. In contrast, FAMEs were still present in delipidated cells and associated with isolated cell walls from bacilli grown at 55 °C (Fig. 3a
). In addition, two-dimensional argentation TLC clearly showed that both unsaturated and saturated FAMEs accumulated at 55 °C and were associated with the cell wall. Reverse-phase TLC showed these FAMEs corresponded mainly to C16C24 fatty acids (Fig. 3b
).
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Temperature effects on other complex lipids
Associated with the mycolic acid layer are a number of polar and apolar lipids which are thought to extend into the outer surface of the cell. This waxy coat of lipids may account for the limited permeability of mycobacteria as well as for their tendency to grow in large clumps and their natural resistance to toxic substances. The profound alteration of fatty acid and mycolic acid production observed at elevated growth temperature led us to investigate the cellular complex lipid profile by in vivo labelling and two-dimensional TLC analysis of the bacteria grown either at 37 °C or at 55 °C. As shown in Fig. 5, the production of many cell-wall components, including diacylated trehalose, multiacylated trehaloses and glycerol monomycolate were altered. TDM, also called cord factor (Bloch, 1950
), was found to be down-regulated at elevated temperature. This glycolipid has been widely implicated in characteristic pathogenic features of mycobacterial diseases, including granuloma formation (Bekierkunst, 1968
).
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Incorporation of GDP-[14C]Man by M. thermoresistibile membranes
Simpler PIMs (PIM1, PIM2, PIM3) originate from phosphatidyl inositol and GDP-Man, but further growth of the linear lipomannan backbone utilizes C35-/C50-P-Man and is amphomycin-sensitive (Besra et al., 1997 ). Amphomycin treatment specifically disrupts the action of a variety of translocase enzymes, by chelating polyprenyl monophosphates in the presence of Ca2+, and inhibits the transfer of a range of monomeric units to polyprenyl-monophosphate carriers (Besra et al., 1997
). Amphomycin was therefore used to measure the transfer of radiolabelled Man from GDP-[14C]Man into membrane lipids. [14C]Man-labelled products were extracted from enzymically active membrane fractions that were incubated with GDP-[14C]Man. Incorporation was quantitatively lower with membrane fractions from M. thermoresistibile grown at 55 °C compared to bacilli grown at 37 °C, suggesting decreased GDP-Man-dependent mannosyltransferase activities at 55 °C (data not shown). Analysis by TLC and autoradiography of the [14C]mannolipids formed showed that, with low protein concentrations (up to 120 µg ml-1), PIM2 was poorly synthesized by membranes prepared from cells grown at 55 °C (Fig. 6a
). In addition, synthesis of PIM1 was inhibited in membranes from cells grown at 55 °C although this product was clearly formed in membranes from cells grown at 37 °C. Altogether, these results suggest that at 55 °C mannosyltransferases that utilize GDP-Man as the mannose donor for PIM synthesis are inhibited and/or less efficient in catalysing the mannosyltransferase reactions leading to PIMs.
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DISCUSSION |
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The mycolic acid S-adenosylmethionine-dependent methyl transferases are a large family of highly homologous proteins that modify olefinic mycolic acids with cyclopropane rings and methyl branches. The three classes of mycolates, -, methoxy- and ketomycolates, are all modified with cyclopropane rings and methyl branches within their mero chain. One of these transferases, PcaA, has been shown as essential for M. tuberculosis pathogenesis, since a pcaA mutant failed to establish a chronic persistent M. tuberculosis infection in mice (Glickman et al., 2000
). PcaA is required for the synthesis of the proximal cyclopropane ring of
-mycolates. It was subsequently shown that CmaA2, another cyclopropane synthase, is required for the synthesis of the trans cyclopropane rings of both methoxy- and ketomycolates (Glickman et al., 2001
). Inactivation of cmaA2 causes accumulation of unsaturated derivatives of both methoxy- and ketomycolates (Glickman et al., 2001
). We show here that elevated growth temperatures significantly affect the distribution of the different mycolic acid subclasses. Basically, growth at 55 °C causes accumulation of unsaturated derivatives of both
- and methoxymycolates as well as loss of trans-containing olefins corresponding to oxygenated mycolic acids. It is therefore tempting to speculate that temperature-dependent inhibition of mycolic acid cyclopropane synthases results in the accumulation of unsaturated mycolic acids. In this regard, recent work has demonstrated that the cyclopropane fatty acid synthase from E. coli that catalyses cyclopropanation of fatty acids is a short-lived protein in vivo and its degradation is dependent on expression of the heat-shock regulon (Chang et al., 2000
). However, whether PcaA, CmaA2 or other mycobacterial cyclopropane synthases are regulated by temperature remains to be established. In addition, differences in the geometrical position of the centre (cis-olefin, trans-olefin or -cyclopropane) may directly affect cell-wall fluidity. Overexpression of CmaA2 in M. smegmatis was shown to be associated with an increase in the melting temperature of the cell wall (George et al., 1995
). It has therefore been suggested that cyclopropanation of the proximal double bond decreases fluidity of the cell wall. In addition, cyclopropanated fatty acids are intermediate in fluidity between the more fluid cis-olefin and the less fluid trans-olefin as measured by differential scanning calorimetry (Silvius & McElhaney, 1979
). Thus, accumulation of cis-olefins as a consequence of cyclopropanation inhibition may represent a strategy used by mycobacteria to increase cell-wall fluidity.
In addition to KasA, the expression of the mycobacterial Ag85 complex was also reduced in cells cultivated at 55 °C. In M. tuberculosis, Ag85A (FbpA), Ag85B (FbpB) and Ag85C2 (FbpC2) represent the three dominant exported proteins. By means of their fibronectin-binding activity, these proteins are supposed to be involved in the pathogenesis of tuberculosis. They have also recently been suggested to play a role in the final assembly of the mycobacterial cell wall by catalysing the transfer of mycolic acids to trehalose, to generate TDM and to the cell-wall AG (Belisle et al., 1997 ; Kremer et al., 2002b
). The decreased expression of the Ag85 complex is consistent with the observation that cells cultivated at 55 °C produce less TDM than cells grown at 37 °C, as evidenced by two-dimensional TLC analysis of complex lipids. Altogether, our results suggest that the expression level of various enzymes involved in fatty acid/mycolic acid metabolism is regulated by temperature, thus participating in the environmental adaptation of the bacilli. Consistent with this notion, we have observed that the cytosolic enzyme fraction, which contains FAS-I and FAS-II, from cells grown at 55 °C presents a distinct electrophoretic profile different from that of the corresponding fraction from cells grown at 37 °C (data not shown). Whether the proteins that are down- or up-regulated at 55 °C belong to the FAS-II system remains to be investigated.
PIMs that are known to be precursors of LM and LAM have been proposed to recruit natural killer T cells which play a primary role in the granulomatous response (Apostolou et al., 1999 ; Gilleron et al., 2001
). Moreover, a role for surface-exposed PIMs as M. tuberculosis adhesins that mediate attachment to non-phagocytic cells has also been established (Hoppe et al., 1997
). A recent study demonstrated a novel interaction between PIMs and Galectin-3, a galactoside-binding protein of macrophages (Beatty et al., 2002
), suggesting a role of these glycolipids in mycobacterial virulence. Our study shows that at elevated temperature both Ac4PIM6 and Ac3PIM6 synthesis is reduced, whereas expression of the other PIMs remains unchanged. This may be explained, at least partially, by a reduced mannosyltransferase activity as suggested by a decrease of GDP-[14C]Man incorporation into the PIM family using membranes from cells grown at elevated temperature. Although a clear function of PIM6 has not yet been assigned, its reduced expression may reflect significant changes in cell-wall fluidity and permeability. In addition, in vitro studies have shown that high temperature negatively affects PPM synthase activity responsible for C50-P-Man synthesis. This glycolipid has previously been shown to be the mannose donor for LM/LAM biosynthesis (Gurcha et al., 2002
).
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ACKNOWLEDGEMENTS |
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Received 2 May 2002;
revised 22 May 2002;
accepted 23 May 2002.