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Mycobacterium leprae phenolglycolipid-1 expressed by engineered M. bovis BCG modulates early interaction with human phagocytes.

Tabouret G, Astarie-Dequeker C, Demangel C, Malaga W, Constant P, Ray A, Honoré N, Bello NF, Perez E, Daffé M, Guilhot C - PLoS Pathog. (2010)

Bottom Line: We found that PGL-1 production endowed recombinant BCG with an increased capacity to exploit complement receptor 3 (CR3) for efficient invasion of human macrophages and evasion of inflammatory responses.PGL-1 production also promoted bacterial uptake by human dendritic cells and dampened their infection-induced maturation.Our results therefore suggest that M. leprae produces PGL-1 for immune-silent invasion of host phagocytic cells.

View Article: PubMed Central - PubMed

Affiliation: CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France.

ABSTRACT
The species-specific phenolic glycolipid 1 (PGL-1) is suspected to play a critical role in the pathogenesis of leprosy, a chronic disease of the skin and peripheral nerves caused by Mycobacterium leprae. Based on studies using the purified compound, PGL-1 was proposed to mediate the tropism of M. leprae for the nervous system and to modulate host immune responses. However, deciphering the biological function of this glycolipid has been hampered by the inability to grow M. leprae in vitro and to genetically engineer this bacterium. Here, we identified the M. leprae genes required for the biosynthesis of the species-specific saccharidic domain of PGL-1 and reprogrammed seven enzymatic steps in M. bovis BCG to make it synthesize and display PGL-1 in the context of an M. leprae-like cell envelope. This recombinant strain provides us with a unique tool to address the key questions of the contribution of PGL-1 in the infection process and to study the underlying molecular mechanisms. We found that PGL-1 production endowed recombinant BCG with an increased capacity to exploit complement receptor 3 (CR3) for efficient invasion of human macrophages and evasion of inflammatory responses. PGL-1 production also promoted bacterial uptake by human dendritic cells and dampened their infection-induced maturation. Our results therefore suggest that M. leprae produces PGL-1 for immune-silent invasion of host phagocytic cells.

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Construction of a recombinant BCG producing the PGL-1 of M. leprae.(A) TLC analysis of lipids extracted from various recombinant BCG strains. Lipid extracts dissolved in CHCl3 were run in CHCl3/CH3OH (95∶5, v/v). Glycolipids were visualized by spraying the plates with 0.2% anthrone (w/v) in concentrated H2SO4. The various glycolipids analyzed by mass spectrometry and 1H NMR are numbered. (B) MALDI-TOF mass spectrum of purified PGL-1 produced by r-BCG PGL-1. (C) 1H NMR spectra of native PGL-1 (black spectrum) from M. leprae and recombinant PGL-1 (red spectrum) from r-BCG PGL-1. Inserts correspond to enlargement of parts of spectra that are relevant for structure determination. (D) Structures of the glycolipids produced by the various recombinant BCG strains after deletion of Rv2959c gene and transfer of 3 or 6 M. leprae genes.
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ppat-1001159-g002: Construction of a recombinant BCG producing the PGL-1 of M. leprae.(A) TLC analysis of lipids extracted from various recombinant BCG strains. Lipid extracts dissolved in CHCl3 were run in CHCl3/CH3OH (95∶5, v/v). Glycolipids were visualized by spraying the plates with 0.2% anthrone (w/v) in concentrated H2SO4. The various glycolipids analyzed by mass spectrometry and 1H NMR are numbered. (B) MALDI-TOF mass spectrum of purified PGL-1 produced by r-BCG PGL-1. (C) 1H NMR spectra of native PGL-1 (black spectrum) from M. leprae and recombinant PGL-1 (red spectrum) from r-BCG PGL-1. Inserts correspond to enlargement of parts of spectra that are relevant for structure determination. (D) Structures of the glycolipids produced by the various recombinant BCG strains after deletion of Rv2959c gene and transfer of 3 or 6 M. leprae genes.

Mentions: To reprogram the PGL biosynthesis pathway in M. bovis BCG, we first disrupted the Rv2959c ortholog by allelic exchange [21]. One clone exhibiting the expected PCR profile for a BCG ΔRv2959c::km mutant was retained for further studies (Figure S1). The kanamycin cassette used in this construct, flanked by two res sites from transposon γδ, was removed after transient expression of the transposon γδ resolvase from plasmid pWM19 [22] to generate the unmarked BCG ΔRv2959c (Figure S1). The lipids produced by this mutant strain were analyzed by thin layer chromatography (TLC) (Figure 2A). As expected, the spot corresponding to PGL-bovis was no longer detectable and a new, more polar, glycolipid (product 1) was observed. Matrix-assisted laser desorption-ionisation time-of-flight (MALDI-TOF) mass spectrometry analyses of purified product 1 gave a series of pseudomolecular ions (M+Na)+ with a major peak at 1516 amu, i.e. 14 mass units lower than those of the usual PGL from wild-type (WT) M. bovis BCG [23]. Therefore, we concluded that this compound corresponded to the expected unmethylated rhamnosyl-phenolphthiocerol dimycocerosates.


Mycobacterium leprae phenolglycolipid-1 expressed by engineered M. bovis BCG modulates early interaction with human phagocytes.

Tabouret G, Astarie-Dequeker C, Demangel C, Malaga W, Constant P, Ray A, Honoré N, Bello NF, Perez E, Daffé M, Guilhot C - PLoS Pathog. (2010)

Construction of a recombinant BCG producing the PGL-1 of M. leprae.(A) TLC analysis of lipids extracted from various recombinant BCG strains. Lipid extracts dissolved in CHCl3 were run in CHCl3/CH3OH (95∶5, v/v). Glycolipids were visualized by spraying the plates with 0.2% anthrone (w/v) in concentrated H2SO4. The various glycolipids analyzed by mass spectrometry and 1H NMR are numbered. (B) MALDI-TOF mass spectrum of purified PGL-1 produced by r-BCG PGL-1. (C) 1H NMR spectra of native PGL-1 (black spectrum) from M. leprae and recombinant PGL-1 (red spectrum) from r-BCG PGL-1. Inserts correspond to enlargement of parts of spectra that are relevant for structure determination. (D) Structures of the glycolipids produced by the various recombinant BCG strains after deletion of Rv2959c gene and transfer of 3 or 6 M. leprae genes.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958813&req=5

ppat-1001159-g002: Construction of a recombinant BCG producing the PGL-1 of M. leprae.(A) TLC analysis of lipids extracted from various recombinant BCG strains. Lipid extracts dissolved in CHCl3 were run in CHCl3/CH3OH (95∶5, v/v). Glycolipids were visualized by spraying the plates with 0.2% anthrone (w/v) in concentrated H2SO4. The various glycolipids analyzed by mass spectrometry and 1H NMR are numbered. (B) MALDI-TOF mass spectrum of purified PGL-1 produced by r-BCG PGL-1. (C) 1H NMR spectra of native PGL-1 (black spectrum) from M. leprae and recombinant PGL-1 (red spectrum) from r-BCG PGL-1. Inserts correspond to enlargement of parts of spectra that are relevant for structure determination. (D) Structures of the glycolipids produced by the various recombinant BCG strains after deletion of Rv2959c gene and transfer of 3 or 6 M. leprae genes.
Mentions: To reprogram the PGL biosynthesis pathway in M. bovis BCG, we first disrupted the Rv2959c ortholog by allelic exchange [21]. One clone exhibiting the expected PCR profile for a BCG ΔRv2959c::km mutant was retained for further studies (Figure S1). The kanamycin cassette used in this construct, flanked by two res sites from transposon γδ, was removed after transient expression of the transposon γδ resolvase from plasmid pWM19 [22] to generate the unmarked BCG ΔRv2959c (Figure S1). The lipids produced by this mutant strain were analyzed by thin layer chromatography (TLC) (Figure 2A). As expected, the spot corresponding to PGL-bovis was no longer detectable and a new, more polar, glycolipid (product 1) was observed. Matrix-assisted laser desorption-ionisation time-of-flight (MALDI-TOF) mass spectrometry analyses of purified product 1 gave a series of pseudomolecular ions (M+Na)+ with a major peak at 1516 amu, i.e. 14 mass units lower than those of the usual PGL from wild-type (WT) M. bovis BCG [23]. Therefore, we concluded that this compound corresponded to the expected unmethylated rhamnosyl-phenolphthiocerol dimycocerosates.

Bottom Line: We found that PGL-1 production endowed recombinant BCG with an increased capacity to exploit complement receptor 3 (CR3) for efficient invasion of human macrophages and evasion of inflammatory responses.PGL-1 production also promoted bacterial uptake by human dendritic cells and dampened their infection-induced maturation.Our results therefore suggest that M. leprae produces PGL-1 for immune-silent invasion of host phagocytic cells.

View Article: PubMed Central - PubMed

Affiliation: CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France.

ABSTRACT
The species-specific phenolic glycolipid 1 (PGL-1) is suspected to play a critical role in the pathogenesis of leprosy, a chronic disease of the skin and peripheral nerves caused by Mycobacterium leprae. Based on studies using the purified compound, PGL-1 was proposed to mediate the tropism of M. leprae for the nervous system and to modulate host immune responses. However, deciphering the biological function of this glycolipid has been hampered by the inability to grow M. leprae in vitro and to genetically engineer this bacterium. Here, we identified the M. leprae genes required for the biosynthesis of the species-specific saccharidic domain of PGL-1 and reprogrammed seven enzymatic steps in M. bovis BCG to make it synthesize and display PGL-1 in the context of an M. leprae-like cell envelope. This recombinant strain provides us with a unique tool to address the key questions of the contribution of PGL-1 in the infection process and to study the underlying molecular mechanisms. We found that PGL-1 production endowed recombinant BCG with an increased capacity to exploit complement receptor 3 (CR3) for efficient invasion of human macrophages and evasion of inflammatory responses. PGL-1 production also promoted bacterial uptake by human dendritic cells and dampened their infection-induced maturation. Our results therefore suggest that M. leprae produces PGL-1 for immune-silent invasion of host phagocytic cells.

Show MeSH
Related in: MedlinePlus