Department of Microbiology and Immunology1 and Department of Chemistry2, University of Newcastle, Newcastle upon Tyne, UK
MRC Laboratories, Fajara, Banjul, The Gambia, West Africa2
Author for correspondence: Natalie J. Garton. Tel: +44 116 252 2955. Fax: +44 116 252 5030. e-mail: njg17{at}le.ac.uk
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ABSTRACT |
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Keywords: mycobacterial lipids, triacylglycerols, storage lipids, nitrogen and carbon limitation, cytochemistry
Abbreviations: DAG, diacylglycerol; FAME, fatty acid methyl ester; ILI, intracellular lipophilic inclusion; MB, Middlebrook 7H9 broth with ADC enrichment; PHB, poly-ß-hydroxybutyrate; TAG, triacylglycerol; YB, Youmans broth
a Present address: Microbiology and Immunology Department, Leicester University, PO Box 138, Medical Sciences Building, University Road, Leicester LE1 9HN, UK.
b Present address: School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
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INTRODUCTION |
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Mycobacterial intracellular inclusion bodies were reported by Burdon (1946) and have subsequently been observed by others using both light and electron microscopy (Table 1
). There has been little recent interest in these structures. Most studies have concentrated on ultrastructural issues, particularly on whether the inclusions are membrane-bound, and little is known about their natural history.
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Here we report our studies on ILIs in Mycobacterium smegmatis and preliminary work on these structures in Mycobacterium tuberculosis. We have investigated the factors affecting the occurrence of ILIs, whether they may be formed by direct fatty acid uptake or biosynthetically using de novo synthesized fatty acids, and also the corresponding ILI lipid composition. We also present evidence that ILIs are present in M. tuberculosis cells in human sputum. We suggest that our results have important implications for the understanding of lipid physiology in mycobacteria, and that they are relevant to the metabolism of pathogenic mycobacteria in vivo and consequently present a potential target for treatment of mycobacterial diseases.
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METHODS |
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Labelling of cells, recording and presentation of images.
Labelling of cultures with Nile red, microscopy and deconvolution of fluorescence images were performed as detailed by Christensen et al. (1999).
Comparison of growth on Middlebrook 7H10 and Youmans agar.
Colonial growth of M. smegmatis on Middlebrook 7H10 with OADC enrichment (MA, Becton Dickinson) or Youmans agar (YA) was removed daily and labelled.
Comparison of growth on Middlebrook 7H9 and Youmans broth.
Fifty-millilitre volumes of Middlebrook 7H9 with ADC enrichment (MB) or Youmans broth (YB) were inoculated at 4% with a suspension (approx. 108 cells ml-1) of M. smegmatis from a freshly grown lawn (2 days). The cultures were incubated at 37 °C with shaking (140 r.p.m.), and samples were removed as required and labelled with Nile red.
Growth of M. smegmatis in variants of Youmans medium.
For a comparison of glycerol and glucose as major carbon sources, cultures were made as described for YB (4%, w/v, glycerol) with 4% (w/v) glucose substituted for glycerol. Duplicate samples were removed daily and labelled with Nile red. To supplement the medium with fatty acid, the method of preparation was adapted from Weir et al. (1972) . Oleic acid (C18:1, 200 µl) or palmitic acid (C16:0, 198 mg) was added to 100 ml BSA solution (fraction V, 5%, w/v). The mixture was emulsified by sonicating in a sonic bath (Decon FS 100b, mean power output 100 W) for 3x20 min. The emulsion was then filter-sterilized and stored at 4 °C until required. Cultures were prepared in 45 ml YB as described and after 23 h, fatty acid-BSA supplement (5 ml) was added. Incubation was resumed and duplicate samples were removed and labelled as required. Cultures were also prepared as for YB, in low-carbon YB and low-nitrogen YB. The former contained 0·4% (w/v) glycerol while the latter contained 1·25 g L-asparagine l-1. Duplicate samples were removed daily and labelled with Nile red.
Preparation of samples of M. smegmatis for lipid extraction.
Two M. smegmatis cultures (200 ml) were prepared in MB and were incubated for 27 h. The cultures were harvested and cells washed with Youmans broth containing no glycerol and with ammonium chloride (2 g l-1) in place of L-asparagine (no-carbon YB, 2x20 ml). The cell pellet from one culture was stored at -20 °C prior to lipid analysis. The other cell pellet was resuspended in no-carbon YB (2x10 ml) and used to inoculate two 1 l flasks containing no-carbon YB (190 ml). After 7 days incubation, the cultures were harvested and washed with PBS (2x20 ml). The cell pellet from one culture was stored at -20 °C prior to lipid analysis. The other cell pellet was resuspended in YB (2x10 ml) and used to inoculate a 500 ml flask containing YB with oleic acid supplement (90 ml) and a 500 ml flask containing low-nitrogen YB (90 ml). The oleic acid YB culture was incubated for 1 h then harvested, washed and stored as described previously. The low-nitrogen YB culture was incubated with shaking for 9 days then harvested, washed and stored as described previously. Samples from each culture were labelled with Nile red prior to storage at -20 °C.
Lipid extraction of M. smegmatis.
Samples of M. smegmatis grown under various conditions were lyophilized prior to extraction. The non-covalently bound non-polar lipids of the samples were extracted and analysed by TLC according to the methods of Dobson et al. (1985) .
Purification of TAG components.
The tentatively identified TAG components of each non-polar extract (60 mg) from the samples of M. smegmatis and a sample of triolein (60 mg) were purified by preparative TLC using plastic TLC plates (Merck, silica gel 60, F254, layer thickness 0·2 mm). Plates were developed with petroleum ether (b.p. 4060 °C)/acetone (95:5, v/v) and TAG was visualized under UV after treating the plate with rhodamine 6G (0·001%, w/v, in acetone). The TAG was removed from the plates and extracted into diethyl ether (3x10 ml) which after evaporation under nitrogen left approximately 7 mg of purified TAG. The purified TAG and triolein (2 mg) were analysed by proton NMR (Brucker WP 200 spectrometer) and GC-MS.
GC-MS.
The purified triolein and TAG samples from M. smegmatis were analysed by GC-MS on a Fisons 8060 GC, linked to a VG/Fisons Trio 1000 quadrupole mass spectrometer (electron voltage 70 eV, filament current 4·2 A, source current 75 µA, source temperature 270 °C, multiplier voltage, 500 V, interface temperature 350 °C). The full scan mode acquisition (50950 a.m.u. s-1) was controlled by an Intel 486 computer running VG Masslab software. The sample (1 µl) in dichloromethane was injected using the cold column injection method. After the solvent peak had passed through the mass spectrometer (5 min) the GC temperature programme and data acquisition commenced. Separation was performed on a J&W fused silica capillary column (15 mx0·25 mm i.d.) coated (0·10 µm) with DB5-HT phase, with helium as the carrier gas (flow 1 ml min-1, pressure 30 kPa). The temperature programme of the GC ran from 50 to 200 °C at 10 °C min-1 and then from 200 to 370 °C at 5 °C min-1, and the final temperature was then held for 30 min. The acquisition data were stored on DAT tape for later data processing, integration and printing.
GC analysis of fatty acid methyl esters.
Fatty acid methyl esters (FAMEs) were prepared from purified TAG (2 mg) according to the method of Walker et al. (1970) . The FAMEs were analysed by GC performed on a PYE Unicam series 104 chromatograph equipped with a 6 ftx
inch o.d. (approx. 1·8 mx6 mm o.d.) stainless steel Silar 5CP column. FAMEs were chromatographed from 100 °C to 240 °C at 8 °C min-1, with a carrier gas flow rate of 50 ml nitrogen min-1. Authentic FAME standards were run for comparison. The FAMEs of the TAG samples were identified by their relative retention times and by co-chromatography with the standard samples.
Combined Auramine-Nile red labelling of human sputum samples.
Sputum samples from patients with clinical tuberculosis were spread evenly on the surface of glass slides, heat-fixed and labelled for 15 min in Auramine O/phenol (Sommers & Good, 1985 ). The stain was differentiated in acid methanol for 15 min, then the slides were washed briefly in tap water followed by labelling with Nile red (10 µg ml-1 in ethanol) for 10 min. After further washing, the smear was treated with KMnO4 (0·1% w/v) for 1 min, washed and mounted in PBS. Preparations were observed by fluorescence microscopy using a Leica DMLB microscope equipped with 50 W Hg epifluorescence illumination. Images were captured using an integrating chip CCD Sony camera controlled by microcomputer (Optivision, UK).
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RESULTS |
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Factors affecting the occurrence of ILIs
Culture medium. We first observed ILIs in cultures of M. smegmatis growing on Middlebrook 7H10 agar (MA) (Christensen et al., 1999 ) and the descriptive terms used below are defined therein (see also Fig. 1). While the ILIs seen in cells from MA cultures matured from peripheral deposits to more substantial inclusions between days 2 and 6, on Youmans agar (YA) small peripheral deposits were observed throughout. Similar observations were made on cells grown in the broth equivalents of these two media (MB and YB). In MB at 21 h (exponential phase) peripheral deposits were apparent; at 45 h (early stationary phase) there were prominent inclusions and at 141 h the cells had lost their prominent internal inclusions and showed peripheral deposits again. Growth in YB showed peripheral deposits throughout (6 days) growth.
Occurrence of ILIs in variants of Youmans medium. The clearest distinctions between ILI-rich and -poor cell populations were obtained by manipulating growth conditions on Youmans medium. The results of Nile red labelling of broth-grown cells are summarized in Fig. 1. After 7 days, low-carbon YB yielded a predominantly annular pattern of labelling and the minimum level of inclusions, while in low-nitrogen YB the annular labelling was lost and prominent inclusions were present. The use of glucose as an alternative principal carbon source to glycerol had no effect on the peripheral deposit pattern of labelling. Addition of exogenous fatty acid (oleic or palmitic) resulted in the formation of prominent inclusions within 1 h. These cells had previously shown annular or small peripheral deposit labelling patterns. The more substantial inclusions were also observed by phase-contrast as bright refractile bodies as described by Christensen et al. (1999)
. These were observed in cells cultured on MA and in low-nitrogen YB. Apparently ILIs could be formed rapidly from uptake of fatty acids or from simple carbon sources in low-nitrogen conditions. The prominent ILIs observed in oleic-acid-supplemented cultures disappeared after a further 29 h incubation (data not shown).
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Fatty acid methyl ester (FAME) analyses of purified triacylglycerols
The component FAMEs were differentiated by GC and are summarized in Fig. 3. The TAG substitution patterns were distinctive for each of the conditions analysed. While the MB and low-nitrogen YB results showed many similarities, oleic acid supplementation yielded a pattern dominated by oleate and the probable results of one and two rounds of ß-oxidation (C16:1
7, C14:1
5). Furthermore, fatty acids longer than C18 were absent from the oleic acid YB TAG profile. The chief difference between the MB and low-nitrogen YB patterns was that the ratios between saturated and unsaturated C18 components were in opposing directions, the unsaturated dominating thoughout the MB profile.
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DISCUSSION |
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The fatty acid profiles of each TAG component reflected the conditions of culture: the oleic acid YB TAG showed direct incorporation of oleic acid, and the MB TAG and low-nitrogen YB TAG contained a range of de novo-synthesized fatty acids. The metabolic pathways of TAG biosynthesis in mycobacteria remain to be elucidated. In yeast, phosphatidic acid is a common precursor for both membrane phospholipid and TAG biosynthesis (Pieringer, 1989 ). The biosynthetic pathway for the formation of mycobacterial TAG may also branch from phospholipid synthesis at this point, with dephosphorylation to form diacylglycerol (DAG) and acyl transfer to yield TAG.
Such a high proportion of C18:1 in the oleic acid YB TAG was unexpected. If TAG was formed by acyl transfer to DAG derived from endogenous phosphatidic acid, C18:1 would be expected to account for little more than one-third of the acyl substituents. The much greater proportion of C18:1 may reflect de novo synthesis of dioleoyl phosphatidic acid with exogenous oleic acid, followed by further acylation to form triolein. We were surprised by the rapid accumulation of ILIs, which were observed 15 min after transfer of the cells to oleic acid YB (the corresponding TAGs were analysed after 1 h). One explanation of the rapid accumulation of ILIs and TAG containing such a high proportion of C18:1 is that TAGs may form to detoxify free fatty acid. This role of TAG accumulation was first suggested by McCarthy (1971) and was supported by Weir et al. (1972)
in a study demonstrating the direct incorporation of [14C]oleic acid into TAG. We have observed that the rapid accumulation of ILIs in the presence of oleic acid is inhibited by azide (N.J.G., unpublished results). If the direct incorporation of host fatty acid into TAG prior to further catabolism is important in vivo, this step may represent a new chemotherapeutic target.
The absence of fatty acids longer than C18 in the oleic acid YB TAG presumably reflects the carbon-restricted state of the cells prior to C18:1 uptake and confirms that TAG formed in the cells in MB is assimilated during subsequent incubation in no-carbon YB. ß-Oxidation of C18:1, forming C16:1 and C14:1, is a further consequence of the previous carbon restriction of the cells and will provide necessary energy and acetate units for the synthesis of required metabolites. C16:17 formed by the ß-oxidation of C18:1 and C16:1
9 (a possible component of the fatty acid reservoir) formed by the action of
9-desaturase would not be distinguished using this GC method.
The MB TAG FAME profile contained an increased proportion of unsaturated fatty acids compared to the low-nitrogen YB TAG profile. This may reflect the different growth phase of the cells in these conditions. Phospholipids of M. smegmatis contain a greater proportion of C18:1 and C16:1 than the corresponding saturated fatty acids (Walker et al., 1970 ). The majority of the short-chain unsaturated fatty acids within the cell would be located in phospholipid, whereas saturated fatty acids as biosynthetic precursors of mycolates, or substituents of complex lipids, would be utilized in the outer layers of the cell envelope. Turnover of phospholipid during exponential growth (MB, 27 h) might therefore enhance levels of C18:1 and C16:1 available for incorporation into intracellular TAG. There would be little or no turnover of lipids which require short-chain saturated fatty acid. The FAME profile of low-nitrogen YB TAG (after 7 days in low-nitrogen YB) indicates that the cells have recovered from the carbon restriction of no-carbon YB incubation and are again synthesizing a range of fatty acids (C14C24). As the amount of nitrogen available to the cells decreases, it is possible that there is reduced turnover of phospholipid and increased incorporation of de novo-synthesized saturated fatty acid into TAG as a result of a different growth state.
Few prokaryotes are known to utilize TAGs as energy reserves and their physiological role in mycobacteria has not been defined. Streptomyces spp. have been observed to accumulate TAG as ILIs in mycelia at the stationary phase of growth (Packter & Olukoshi, 1995 ). It has been suggested that this TAG may act as a carbon source for the production of secondary metabolites, e.g. antibiotics (Olukoshi & Packter, 1994
). Studies on the environmental isolate Rhodococcus opacus strain PD630 have demonstrated the accumulation and mobilization of TAGs (which coincided with the presence of ILIs) under different growth conditions (Alvarez et al., 1996
, 2000
; Wältermann et al., 2000
). The accumulation of TAGs has also been described for species of Actinomyces (Kovalchuk, 1973
) and Acinetobacter (Scott & Finnerty, 1976
).
The striking demonstration of ILIs by combined acid-fast and Nile red labelling of bacteria in human sputum provides strong circumstantial evidence that at least some processes observed in M. smegmatis are also a feature in the metabolism of M. tuberculosis in vivo. As we have been unable to produce large, ILI-containing cells of M. tuberculosis in vitro, we suggest that the cells observed in sputum may be in a physiological state distinct from that of cells produced in laboratory studies.
The degree to which TAG (ILI) formation, utilization and downstream processing of its components are essential to growth, viability and pathogenicity also remains to be investigated. The proposed storage role of ILIs and TAGs may be advantageous for mycobacteria in vivo. Preliminary studies have shown that M. tuberculosis H37Rv produces TAG when incubated in oleic-acid-supplemented YB (N.J.G., unpublished results). Much lipid would be available to M. tuberculosis within mammalian cells and it has been proposed that in vivo, the bacilli are lipolytic rather than lipogenic (Wheeler & Ratledge, 1994 ). Fatty acid sequestered from the host may be stored as TAG and this, or de novo synthesized TAG, might be relevant to survival in a non-replicating state.
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ACKNOWLEDGEMENTS |
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Received 21 March 2002;
revised 13 May 2002;
accepted 31 May 2002.