Molecular and Cellular Biochemistry 258: 219–222, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
219 Short communication
Cholesterol-sensor initiates M. tuberculosis entry into human macrophages D. Kaul, P.K. Anand and I. Verma Departments of Experimental Medicine and Biotechnology and Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India Received 19 February 2003; accepted 11 July 2003
Abstract Cholesterol-mediated mycobacteria entry into and survival within macrophages has added a new dimension to Tuberculosis research. The molecular mechanism through which cholesterol initiates this process is still poorly understood. The present study addressed to resolve this mechanism revealed that Mycobacterium tuberculosis possesses cholesterol-specific Receptor ‘Ck’like molecule responsible for mycobacterial entry into macrophages. Further human Receptor-Ck was found to regulate transcriptional expression of a gene that codes for Tryptophan-Aspartate containing coat (TACO) protein responsible for survival of mycobacteria within cells. Based upon these results, we propose that interaction of Receptor-Ck with cholesterol-rich membrane domains helps to create a ‘Synaptic-junction’ between mycobacteria and macrophage resulting in signalling events that are responsible for mycobacterium entry into and survival within macrophages. (Mol Cell Biochem 258: 219–222, 2004) Key words: mycobacterial entry, cholesterol, Receptor-Ck, TACO gene
Introduction Worldwide concern about tuberculosis has resulted in lot of investigations to understand why Mycobacterium tuberculosis, that can survive and replicate extracellularly, has evolved multiple mechanisms to gain entry to the intracellular environment within the macrophages that is equipped with reactive oxygen and nitrogen intermediates that are meant to kill it [1, 2]. The route of entry of mycobacteria into the macrophages is still unclear, although several cellular receptors (especially complement receptors) have been implicated [1] and several bacterial determinants suggested [3]. A new dimension was added recently to the research on tuberculosis [2, 4, 5] by the findings: (a) cholesterol plays crucial role not only in entry of mycobacteria into macrophages but also mediates the phagosomal association of TACO, a coat protein that prevents degradation of mycobacteria in lysosomes; (b) cholesterol is not required for complement receptor function; (c) unlike other bacteria M. tuberculosis possess a high affinity
binding site for cholesterol; (d) M. tuberculosis does display a sterol-biosynthetic pathway. The fact that cholesterol is a hydrophobic molecule which resides in lipoproteins and cell membranes, raised two fundamental questions: (a) how does cell sense the level of cholesterol? (b) how is this cholesterolspecific signalling transmitted to the nucleus for the regulation of various genes? Recent studies directed to resolve these questions, led to the discovery of a novel cell surface cholesterol-sensor designated as Receptor-Ck which was not only shown to be ubiquitously present in various human organs but also regulated various genes involved in cholesterol homeostasis; Cell growth; Cell-death and cytokine production etc. [6, 7]. Receptor-Ck like molecule was also shown to be present in giardia and this receptor regulates giardia encystation process [8]. Based upon these findings the present study was addressed to understand two specific issues: (a) whether or not M. tuberculosis has ability to express Receptor-Ck like molecule? (b) If yes, how blockage of this receptor molecule influences entry of M. tuberculosis into human macrophages?
Address for offprints: D. Kaul, Departments of Experimental Medicine and Biotechnology and Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012, India (E-mail:
[email protected])
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Materials and methods Cells and antibodies Human macrophages, from normal subjects, were maintained at 37°C in 5% CO2 atmosphere in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with antibiotics and 10% F.C.S. The purity of macrophage preparations were checked by subjecting the cells to Giemsa stain followed by examination under the light microscope. The human promyelocytic leukemic HL-60 cell line (obtained from National Centre for Cell science, Pune) was used as archetype cellular model for this study [9, 10] because: (a) This cell line is unable to express Receptor-Ck in its native state; (b) Exposure of these cells to 12-O-tetradecanoyl phorbol-13-acetate (TPA) results in the expression of Receptor-Ck which is accompanied by differentiation of these cells into monocytes/ macrophages [10]. HL-60 cells were exposed to DMEM enriched with and without 32 nM of ‘TPA’ as well as TPA (32 nM) + Ab*-RCk for 24 h at 37°C in 5% CO2-atmosphere to understand the relationship between Receptor-Ck and TACO-gene expression. Polyclonal monospecific antibodies against purified human Receptor-Ck protein was raised as described previously [7].
Bacterial-labelling and immunodetection Mycobacterium tuberculosis H37Ra strain was labelled in Kreb-Ringer buffer containing 14C-Leucine. The cells were subsequently washed thrice with PBS to remove any nonspecific activity. In order to explore whether or not M. tuberculosis has the inherent ability to express Receptor-Ck like molecule on its surface, the protein extract (from both H37Rv and H37Ra) in equal amount was subjected to SDS-PAGE followed by Western-blotting. The blots were subjected to standard immunodetection procedure using specific antibody against human Receptor-Ck [10].
H37Ra was added to each well. After 1 h incubation at 37°C, the cells were washed free of any non-specific adhesion and subsequently processed for liquid-scintillation counting.
TACO-gene transcription RNA was extracted from HL-60 cells (with and without treatment with TPA as well as TPA + Ab*-RCk) and cDNA was synthesized as described previously [8]. Using specific primers for TACO gene (5′-CCAGTGCTATGAGGATGTGCGCG-3′ and 5′-GACACGACTCGCTTGTCACGGC-3′) and invariant control β2-Microglobulin gene (5′-GAATTGCTATGTGTCTGGGT-3′ and 5′-CATCTTCAAACCTCCATGATG3′), these genes were amplified by PCR using following programmes: For TACO: 94°C for 45 sec; 60°C for 45 sec; 72°C for 90 sec (30 cycles) For β2M: 94°C for 30 sec; 60°C for 30 sec; 72°C for 60 sec (20 cycles)
The intensity of each band was obtained by scanning the electrophoretic gels with gel-documentation equipment in order to get the quantitative expression of TACO gene with respect to invariant control β2M-gene.
Results Both virulent M. tuberculosis H37Rv and attenuated strain H37Ra possess a molecule with epitopes similar to human cholesterol specific Receptor-Ck (Fig. 1). Blockage of Recep-
Mycobacterial uptake experiments Human macrophages were exposed to DMEM containing 10% F.C.S + 14C-labelled H37Ra or 14C-labelled E. coli + antibody RCk (0–1.6 µg/ml) for 1 h at 37°C in 5% CO2-atmosphere. After the incubation period, the macrophages were washed free of any non-specific adhesion and subsequently lysed. The lysate was counted for 14C-isotope using liquidscintillation counter in order to calculate the number of bacteria per macrophage. In another set of experiments, HL-60 cells were exposed to DMEM containing 10% F.C.S + either no stimulus or TPA + Ab*-RCk or TPA for 24 h at 37°C in 5% CO2 atmosphere. After this incubation period, 14C-labelled
Fig. 1. Representative Western-blot showing expression of Receptor-Cklike molecule (having identical molecular weight of 69 kDa as found in human cells) in both virulent mycobacterium tuberculosis H37Rv and attenuated strain H37Ra. Receptor-Ck expression was not observed in E. coli. Same amount of protein was loaded in each lane before subjecting to SDS-PAGE followed by Western blotting.
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A
(HL-60+TPA) exposed to specific antibody to Receptor-Ck (Fig. 3).
Discussion
B
Fig. 2. (A) Uptake of mycobacterium and E. coli by macrophages in presence of increasing concentration of antibody specific for human ReceptorCk. Each value represents mean of experiments done in triplicate. (B) Uptake of mycobacterium by HL-60 cells pre-exposed to either no stimulus or TPA or TPA + Ab*-RCk for 24 h at 37°C. Each value represents mean of experiments done in triplicate.
tor-Ck with its specific antibody resulted in dose-dependent inhibition of mycobacterium uptake by macrophages whereas E-coli uptake remained unaffected (Fig. 2A). Mycobacterium uptake was found to be identical (Fig. 2B) in Receptor-Ck (+ive) cells (HL-60 + TPA) and Receptor-Ck (-ive) cells (HL60 cells in their native state). However Mycobacterium uptake was reduced by 50% in Receptor-Ck (+ive) cells pre-exposed to specific antibody to Receptor-Ck (Fig. 2B). Since gene coding for tryphophan-aspartate containing coat protein (TACO) is responsible for intracellular survival of mycobacterium tuberculosis and this protein is recruited to phagosomal membrane by cholesterol, an attempt was made to explore whether or not human Receptor-Ck has the ability to regulate TACO gene transcription. Receptor-Ck (+ive) cells (induced in HL-60 cells by exposure to TPA) exhibited 60% increase in TACO-gene transcription (Fig. 3) than Receptor-Ck (-ive) cells (HL-60 in their native state) or Receptor-Ck (+ive) cells
A crucial link between cholesterol-molecule and tuberculosis was established by the finding [2] which revealed that mycobacterium entry and survival within macrophages is dependent upon plasma membrane-cholesterol which also facilitates phagosomal association of TACO, a coat protein that prevents degradation of mycobacteria in lysosomes. This study raised several questions especially: (a) Does mycobacterium possess a cholesterol-sensor on its surface? (b) If yes, what is the role of this sensor in the entry of mycobacteria into macrophage? (c) How does this sensor regulate TACO gene? It is in this context the results reported here assume importance. The presence of Receptor-Ck-like molecule on the surface of mycobacterium (Fig. 1) explains as to why unlike other bacteria (e.g. E. coli; S. typhimurium etc.) mycobacterium tuberculosis possesses high affinity binding site for cholesterol as well as cholesterol biosynthetic pathway [2, 5]. Keeping in view role of cholesterol-specific Receptor-Ck in mycobacterium entry (Fig. 2A) and TACO gene expression (Fig. 3), it is not unlikely that interaction of Receptor-‘Ck’-like molecule (present on the surface of mycobacterium) with cholesterolrich membrane domains of macrophages may help in the creation of synaptic-junction that gives rise to signalling events responsible for mycobacterium entry into and survival within macrophages. However, the presence of Receptor-’Ck’-like molecule in M. tuberculosis may help its entry into cells that do not express Receptor-Ck (Fig. 2B). Further, host response to M. tuberculosis includes granuloma formation at the site of infection. Cytokines have been identified in granulomas induced by BCG-infection and are released by mononuclear
Fig. 3. TACO gene expression in Receptor-Ck (+ive) and Receptor-Ck (ive) cells. β2-microglobulin gene expression was used as invariant control to get exact expression of TACO gene. HL-60, without treatment, represent Receptor-Ck (-ive) cells and HL-60 exposed to TPA represent Receptor-Ck (+ive) cells.
222 phagocytes upon stimulation by mycobacterial proteins [11]. Receptor-Ck dependent signalling has the capacity to regulate IL-6 gene [6] and it is interesting to note that M. tuberculosis has been shown to activate interleukin 6 (IL-6) gene in monocytes [11]. In this regard, Receptor-Ck mediated activation of IL-6 gene by M. tuberculosis may play a role in the clinical manifestations and pathological events of tuberculosis infection. However more data is required to characterise the gene coding for Receptor-Ck-like molecule present on the surface of M. tuberculosis.
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