Institut de pharmacologie et de biologie structurale, UMR 5089, CNRS, 205 route de Narbonne, 31077 Toulouse Cedex, France1
Unité de Génétique Mycobactérienne, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France2
Departamento de Microbiología, Medicina Preventiva y Salud Pública, Universidad de Zaragoza, C/Domingo Miral sn., 50009 Zaragoza, Spain3
Author for correspondence: Germain Puzo. Tel: +33 5 61 17 55 04. Fax: +33 5 61 17 55 05. e-mail: Germain.Puzo{at}ipbs.fr
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ABSTRACT |
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Keywords: lipoglycans, acylation state, two-component systems, adaptation
Abbreviations: APTS, 1-aminopyrene-3,6,8-trisulfonate; CE, capillary electrophoresis; HMQC, heteronuclear multiple quantum correlation spectroscopy; HOHAHA, homonuclear HartmannHahn spectroscopy; LM, lipomannan; MALDI, matrix assisted laser desorption ionization; ManLAM, mannosylated lipoarabinomannan; Me2SO-d6, deuterated dimethylsulfoxide; MPI, mannosyl-phosphatidyl-myo-inositol; TOF, time of flight; Manp, mannopyranosyl unit; Araf, arabinofuranosyl unit; myo-Ins, myo-inositol; t, terminal; 1-D, one-dimensional; 2-D, two-dimensional
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INTRODUCTION |
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Two-component systems, composed of a membrane-associated sensor kinase that uses energy released from ATP hydrolysis to modulate a cytoplasmic transcriptional regulator activity (Stock et al., 2000 ), exhibit multiple cellular implications. For instance, the two-component systems of some bacterial pathogens have been shown to regulate virulence factor biosynthesis pathways (Bernardini et al., 1990
; Engleberg et al., 2001
; Kinnear et al., 2001
; Miller et al., 1989
; Sola-Landa et al., 1998
; Yarwood et al., 2001
), modulating the response to immune defence of the infected host. This was particularly evidenced with the PhoP/PhoQ system in Salmonella typhimurium which regulates genes implicated in the biosynthesis of LPS (Guo et al., 1997
), resulting in the addition of aminoarabinose and 2-hydroxy-myristate to lipid A. Consequently, this structurally modified lipid A altered expression of the adhesion molecule E-selectin by endothelial cells, and tumour necrosis factor-
expression by adherent monocytes, and thus represents a potential mechanism for bacteria to gain advantage within host tissues (Guo et al., 1997
).
Recently, Pérez et al. (2001) have disrupted the phoP gene from M. tuberculosis MT103, which encodes a putative transcription regulator factor of the two-component system PhoP/PhoR. Interestingly, the phoP mutant strain exhibited impaired multiplication in vitro, brought into evidence when cultured in mouse bone-marrow-derived macrophages, and also in vivo as the viability was attenuated in a mouse infection model (Perez et al., 2001
). These results suggest that the phoP gene is required for intracellular growth of M. tuberculosis but open up the question of a hypothetical modification of mycobacterial surface antigens.
Mannosylated lipoarabinomannans (ManLAMs) are lipoglycans ubiquitously found in the mycobacterial cell wall (Brennan & Nikaido, 1995 ; Vercellone et al., 1998
) and play an important role in tuberculosis immunopathogenesis (Chatterjee et al., 1992
; Roach et al., 1993
; Vercellone et al., 1998
). Indeed, ManLAMs were found in vitro to inhibit IL-12 production by human dendritic cells (Nigou et al., 2001
) or human mononuclear phagocytes (Knutson et al., 1998
), to block the transcriptional activation of interferon-
(Riedel & Kaufmann, 1997
) and to neutralize the potentially cytotoxic oxygen free radicals (Chan et al., 1991
). All these activities require the acylation of the mannosyl-phosphatidyl-myo-inositol (MPI) anchor. Moreover, the degree of MPI acylation was found to control IL-12 inhibitory activity of ManLAMs (Nigou et al., 2001
). In addition, the manno-oligosaccharide caps mediate the ManLAM binding to the mannose receptor (Schlesinger et al., 1994
, 1996
; Venisse et al., 1995
) involved in ManLAM signalling; consequently, ManLAM devoid of the mannooligosaccharide caps failed to inhibit IL-12 production by human dendritic cells (Nigou et al., 2001
).
In the present study, the structure of the two major functional domains of ManLAM from phoP mutant and wild-type M. tuberculosis MT103 were investigated as follows (i) the cap motifs by capillary electrophoresis and (ii) the MPI anchor by NMR spectroscopy. These structural findings will be discussed so as to evaluate their potential implication in the different growth rates observed between phoP mutant and wild-type M. tuberculosis MT103.
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METHODS |
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ManLAM extraction and purification.
ManLAMs from phoP mutant and wild-type M. tuberculosis MT103 cells were purified as described (Nigou et al., 1997 ). Briefly, bacterial cells were delipidated by five extractions using CHCl3/CH3OH (1:1, v/v), the resulting cells were washed, weakened in ice by sonication, disrupted using a cell disrupter (one-step lysis at a pressure maintained at 2·6 kPa) and extracted (refluxing three times at 65 °C) for 8 h with ethanol/water (1:1, v/v). Most of the cellular proteins contained in this extract were removed by a hot 80% (w/w) aqueous phenol biphasic wash. Then each aqueous extract was treated to remove DNA, RNA and glucose leading to glycanic- and lipoglycanic-rich extracts.
Hydrophobic interaction chromatography of lipoglycans.
Hydrophobic interaction chromatography was used to separate glycans, mainly arabinomannan from lipoglycans [ManLAMs and lipomannans (LMs)]. Sample (5·7 mg) was reconstituted in 0·05 M ammonium acetate solution containing 5% (v/v) propan-2-ol and loaded on an Octyl-Sepharose CL-4B (Pharmacia) column (20x1·5 cm) pre-equilibrated in the same buffer. The column was then eluted with 3 column vols 5% then 50% (v/v) propan-2-ol containing 0·05 M ammonium acetate at a flow rate of 3 ml h-1. Lipoarabinomannans and LMs were only recovered in the 50% (v/v) propan-2-ol/0·05 M ammonium acetate fractions, as observed by SDS-PAGE (Venisse et al., 1993 ), and were dialysed and separated by gel filtration as previously described (Nigou et al., 1997
).
Capillary electrophoresis.
Analyses were performed on a P/ACE 5000 Capillary zone electrophoresis system (Beckman Instruments) operating in reverse mode and monitored on-column with a Beckman laser-induced fluorescence detection system. The electropherograms were acquired and stored on a Dell XPS 60 computer using the System Gold software package (Beckman Instruments).
Dried (3060 pmol) mild hydrolysed (0·1 M HCl for 20 min at 110 °C) ManLAMs were derivatized with 1-aminopyrene-3,6,8-trisulfonate (APTS; Interchim) (Guttman et al., 1996 ) in the presence of mannoheptose (100 pmol) as the internal standard. The reaction was performed for 90 min at 55 °C and was quenched by adding 20 µl water. Separations were performed as previously described (Nigou et al., 2000
).
MALDI-TOF mass spectrometry experiments.
MALDI-TOF/MS (matrix-assisted laser desorption ionization/time of flight mass spectroscopy) analyses were carried out on a Voyager DE-STR (Applied Biosystems) using the linear mode. Lipoglycan mass spectra were recorded in the negative mode using a 300 ns time delay with a grid voltage of 94% of full acceleration voltage (20 kV) and a guide-wire voltage of 0·1% of full acceleration. All samples were prepared for MALDI-TOF/MS analysis using the on-probe sample cleanup procedure with -form cation-exchange resin (Ludwiczak et al., 2001
) and the so-called Super DHB matrix [2,5-dihydroxybenzoic acid/2-hydroxy-5-methoxysalicylic acid (9:1, w/w) in a mixture of acetonitrile/water (7:3, v/v)].
Fatty acid quantification.
phoP mutant and wild-type M. tuberculosis MT103 ManLAM (300 µg) were deacylated by alkalinolysis in the presence of pentadecanoic acid (8 nmol) as the internal standard and were then extracted three times with 400 µl chloroform, dried and methylated with three drops 10% (w/v) BF3 in methanol (Fluka) at 60 °C for 5 min. Fatty acid methyl esters were extracted three times with 400 µl chloroform and analysed as trimethylsylilated methyl esters by routine GC and GC/mass spectrometry (GC/MS).
GC and GC/MS analysis.
GC was performed on a Girdel series 30 chromatograph equipped with an OV1 capillary column (0·22 mmx25 m) using helium gas with a flow rate of 2·5 ml min-1 and a flame-ionization detector at 310 °C. The injector temperature was 260 °C and the temperature separation program was from 100 to 290 °C, rising at 3 °C min-1. GC/MS analysis were performed on a Hewlett Packard 5889X mass spectrometer (electron energy, 70 eV) working in electron impact coupled to a Hewlett Packard 5890 gas chromatography series II fitted with a similar OV1 column (0·30 mmx12 m).
NMR analysis.
NMR spectra were recorded with an Avance DMX500 spectrometer (Bruker) equipped with an Origin 200 SGI using Xwinnmr 2.6. Samples were exchanged in 2H2O with intermediate freeze-drying, then dissolved in Me2SO-d6 (deuterated dimethylsulfoxide) and analysed in 200x5 mm 535-PP NMR tubes at 343 K. Proton chemical shifts are expressed in p.p.m. downfield from the signal of the methyl of Me2SO-d6 (H/TMS 2·52 and
C/TMS 40·98). The one-dimensional (1-D) phosphorus (31P) spectra were measured at 202 MHz with phosphoric acid (85%) as the external standard (
p 0·0). All the details concerning NMR sequences used and experimental procedures were detailed in previous studies (Gilleron et al., 1999
, 2000
; Nigou et al., 1999
)
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RESULTS AND DISCUSSION |
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General structural features
ManLAMs arising from both phoP mutant and wild-type M. tuberculosis MT103 strains were analysed by linear MALDI-TOF/MS in negative mode (Fig. 1). The phoP mutant ManLAM mass spectrum (Fig. 1a
) shows one intense broad signal, approximately centred around m/z 19000 assigned to pseudomolecular ions revealing a molecular mass of 19 kDa with an evaluated molecular heterogeneity of 89 kDa. Likewise, the wild-type ManLAM mass spectrum shows a similar signal centred at the same value (Fig. 1b
). These experiments reveal that the phoP mutation appears to affect neither ManLAM molecular mass nor the observed molecular heterogeneity for these lipoglycans.
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The phosphatidyl-myo-Ins (phosphatidyl-myo-inositol) anchor structure of wild-type and phoP mutant ManLAM were consequently investigated from 1-D and 2-D 31P NMR experiments. We have recently shown that the multiacylated structures of M. bovis BCG ManLAMs and LMs can be efficiently displayed through 1-D 31P NMR experiments by the use of Me2SO-d6 as a solvent system (Gilleron et al., 1999 ; Nigou et al., 1999
). 1-D 31P spectra of both phoP mutant and wild-type ManLAMs dissolved in Me2SO-d6 were thus recorded. The 1-D 31P spectrum of the phoP mutant ManLAM (Fig. 4a
) showed three signals, at 1·65 p.p.m., 1·83 p.p.m. and 3·54 p.p.m., attributed from their chemical shifts to P1, P3 and P5 and respectively assigned to ManLAM containing at least a tri-, di- and mono-acylated MPI anchor in agreement with our previous results (Gilleron et al., 2000
; Nigou et al., 1999
). Similarly, the 1-D 31P spectrum obtained for the wild-type ManLAM (Fig. 4b
) exhibited the same three resonance signals P1, P3 and P5 with almost identical chemical shifts (
1·67,
1·83 and
3·52 p.p.m., respectively). From these 1-D 31P experiments it can be assumed that both phoP mutant and wild-type ManLAMs share the same acyl-form structures assigned to at least tri-, di- and mono-acylated MPI anchor. Nevertheless, the relative abundance of the different acyl forms, determined by their 31P signal integration, was found to be different between the ManLAMs of the two strains. Indeed, whereas the abundance P1, 36%, P3, 55%, P5, 9% was observed for the wild-type ManLAM acyl forms (Fig. 4b
), the determined ratio for the phoP mutant ManLAM acyl forms was P1, 25%, P3, 52%, P5, 23% (Fig. 4a
). These data highlighted a decrease of the triacylated form and an increase in the monoacylated form, respectively typified by the P1 and the P5 resonances, for the phoP mutant compared to those observed for the wild-type strain.
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The P1 and P3 resonances of wild-type and phoP mutant ManLAM were assigned to at least tri- and diacylated ManLAM. The glycerol spin systems of P1 and P3 acyl forms were attributed by 1H-1H HOHAHA and 1H-31P HMQC experiments. The 1H-31P HMQC-HOHAHA spectra (Fig. 5) show the downfield resonances of glycerol methine protons H-2, at
5·10 and 5·12 p.p.m. for P1 and P3 of wild-type (Fig. 5b
) and 5·11 and 5·12 p.p.m. for P1 and P3 of phoP mutant (Fig. 5a
), revealing that P1 and P3 ManLAM acyl forms share 1,2-diacyl-3-phospho-sn-glycerol units. Likewise, the myo-Ins spin systems belonging to the MPI anchor characterized by P1 and P3 were then identified. The P1 and P3 myo-Ins H-1 resonances at
4·12 and 4·02 p.p.m. of both phoP mutant and wild-type ManLAMs were attributed from 1H-31P HMQC experiments. The remaining protons were identified from 1H-31P HMQC-HOHAHA and 1H-1H HOHAHA experiments and assigned from their multiplicity and 3JH-H coupling constant values. The P3 myo-Ins spin system of the phoP mutant ManLAM was found in agreement with a non-acylated phospho-myo-Ins due to the myo-Ins proton chemical shifts (see Table 1
). Likewise, the P1 myo-Ins spin system was identified (see Table 1
). Compared to the chemical shift of the P3 myo-Ins H-3 (
3·25 p.p.m.), the deshielding of the P1 myo-Ins H-3 (
4·59 p.p.m.;
+1·34 p.p.m.) unambiguously indicated that the myo-Ins unit characterized by the P1 resonance was acylated on C-3. These data were also in agreement with our own previous work done on the tetra-acylated forms of PIM2 and ManLAM from M. bovis BCG and M. tuberculosis H37Rv ManLAMs (Gilleron et al., 1999
, 2000
; Nigou et al., 1999
). Quite similar chemical shifts were also observed for the P1 and P3 myo-Ins spin systems of the wild-type ManLAM (Table 1
). These data confirmed that P1 typified an at least triacylated ManLAM anchor comprising a diacylated glycerol and a myo-Ins acylated on C-3 while P3 characterized an at least diacylated ManLAM anchor harbouring a diacylated glycerol in both strains. It is noteworthy that the presence of a fatty acyl appendage on the Manp unit can not be excluded.
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In summary, these results demonstrate that both phoP mutant and wild-type M. tuberculosis MT103 ManLAM share the same anchor structure typified by three major resonances in 31P NMR, i.e. P1, P3 and P5 and assigned to at least tri-, di- and monoacylated ManLAM. Nevertheless their relative abundance was found to be different with an increase of the monoacylated form in the phoP mutant compared to the wild-type strain.
5-Methylthiopentose presence
The presence of 5-methylthiopentose was recently shown in M. tuberculosis H37Rv ManLAM (Treumann et al., 2002 ) and it has been postulated that these particular substituents could be implicated in some of the biological properties of the ManLAM molecules. The presence of such residues was then investigated in both phoP mutant and wild-type ManLAM. In the anomeric region of 1-D 1H NMR spectra of both strains, H-1 signals corresponding to 5-methylthiopentose at
5·24 p.p.m. and the oxidation products 5-methylsulfoxypentose at
5·28 and
5·29 p.p.m. were observed. The corresponding complete spin systems were then identified in the HOHAHA spectrum of the phoP mutant (Fig. 6
, Table 2
) and wild-type ManLAMs (Table 2
), in agreement with literature data (Treumann et al., 2002
). So, both phoP mutant and wild-type ManLAM share 5-methylthiopentose residues. So it seems that these particular monosaccharides are not implicated in the different growth rates observed between the two strains and that their biosynthesis is not under the control of the phoP system disrupted here.
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This work represents a first step in the search for a better understanding of the biological processes which allow the persistence of mycobacteria in the infected host cells. Other components of the mycobacterial envelope will be next investigated to evaluate the impact of the disruption of such an environment-sensing system.
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ACKNOWLEDGEMENTS |
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Received 2 April 2002;
revised 11 June 2002;
accepted 12 June 2002.