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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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PGL-1 production impairs the initiation of innate immune responses.(A) Activation of the NF-κB pathway, as evaluated by the THP-1 Blue cell line and assay of SEAP activity, following infection with WT BCG (black bars) or r-BCG PGL-1 (white bars) at various MOI. The mean OD630nm values for WT BCG were 1.13, 0.905, and 0.246 at MOI 10∶1, 1∶1 and 1∶10 respectively whereas the value obtained for r-BCG PGL-1 were 0.87, 0.368 and 0.073 at the same MOI. The value used to normalize at 100% was 1.13 units. (B) TNF-α production by hMDM after 2 hours of post-infection. One hundred percent corresponded to 10538±1579 pg/ml. (C) TNF-α production by hMDM pre-incubated with mAbs directed against CR3 or with irrelevant isotype control and infected during 2 hours at a MOI of 10∶1. One hundred percent corresponded to 4101±551 pg/ml. (D) Expression of maturation markers at the surface of infected hDC, as analyzed by flow cytometry. Data are presented as the percentage of SEAP activity, TNF-α production or MFI with respect to the WT BCG (100%). The values are means ± SEM of 2 (panels A and C) and 4 independent experiments (panels B and D). In panels C and D, grey bars corresponded to uninfected controls. Differences between BCG control and r-BCG PGL-1 were statistically evaluated: *, p<0.05 ;**, p<0.01; ***, p<0.001; n.s., not significant.
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ppat-1001159-g006: PGL-1 production impairs the initiation of innate immune responses.(A) Activation of the NF-κB pathway, as evaluated by the THP-1 Blue cell line and assay of SEAP activity, following infection with WT BCG (black bars) or r-BCG PGL-1 (white bars) at various MOI. The mean OD630nm values for WT BCG were 1.13, 0.905, and 0.246 at MOI 10∶1, 1∶1 and 1∶10 respectively whereas the value obtained for r-BCG PGL-1 were 0.87, 0.368 and 0.073 at the same MOI. The value used to normalize at 100% was 1.13 units. (B) TNF-α production by hMDM after 2 hours of post-infection. One hundred percent corresponded to 10538±1579 pg/ml. (C) TNF-α production by hMDM pre-incubated with mAbs directed against CR3 or with irrelevant isotype control and infected during 2 hours at a MOI of 10∶1. One hundred percent corresponded to 4101±551 pg/ml. (D) Expression of maturation markers at the surface of infected hDC, as analyzed by flow cytometry. Data are presented as the percentage of SEAP activity, TNF-α production or MFI with respect to the WT BCG (100%). The values are means ± SEM of 2 (panels A and C) and 4 independent experiments (panels B and D). In panels C and D, grey bars corresponded to uninfected controls. Differences between BCG control and r-BCG PGL-1 were statistically evaluated: *, p<0.05 ;**, p<0.01; ***, p<0.001; n.s., not significant.

Mentions: We next examined whether PGL-1 production influenced the innate responses of human phagocytes to mycobacterial infection. This was first investigated by monitoring the effect of PGL-1 on the activity of the transcription factor NF-κB, which controls the expression of multiple inflammatory genes in hMDM and hDC. We used a THP-1 cell line transfected with a reporter system under the control of a promoter inducible by NF-κB. Infection of THP-1 cells with WT BCG induced strong expression of the reporter gene, indicative of potent activation of the NF-κB pathway. After 16 h of incubation in the detection medium, the mean OD630nm values obtained for the WT BCG were 23%, 60%, 70% higher than for r-BCG PGL-1 at MOI 10∶1, 1∶1 and 1∶10, respectively (Figure 6A). Therefore, the NF-κB response triggered by r-BCG PGL-1 was significantly lower (p<0.01) than that of WT BCG, whatever the MOI considered. Accordingly, hMDM infected with r-BCG PGL-1 produced lower amounts of the inflammatory cytokine TNF-α than BCG-infected controls after 2 hours of infection, even though bacteria expressing PGL-1 were more efficiently internalized than the WT controls (Figure 6B). Other cytokines, IL-12 (p40 and p70) and IL-10, were also assayed. Both WT BCG and r-BCG PGL-1 induced the production of poor levels of these cytokines both at 2h and 24h post-infection, and no difference was observed between WT-BCG and r-BCG PGL-1 infected hMDM (Figure S3).


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)

PGL-1 production impairs the initiation of innate immune responses.(A) Activation of the NF-κB pathway, as evaluated by the THP-1 Blue cell line and assay of SEAP activity, following infection with WT BCG (black bars) or r-BCG PGL-1 (white bars) at various MOI. The mean OD630nm values for WT BCG were 1.13, 0.905, and 0.246 at MOI 10∶1, 1∶1 and 1∶10 respectively whereas the value obtained for r-BCG PGL-1 were 0.87, 0.368 and 0.073 at the same MOI. The value used to normalize at 100% was 1.13 units. (B) TNF-α production by hMDM after 2 hours of post-infection. One hundred percent corresponded to 10538±1579 pg/ml. (C) TNF-α production by hMDM pre-incubated with mAbs directed against CR3 or with irrelevant isotype control and infected during 2 hours at a MOI of 10∶1. One hundred percent corresponded to 4101±551 pg/ml. (D) Expression of maturation markers at the surface of infected hDC, as analyzed by flow cytometry. Data are presented as the percentage of SEAP activity, TNF-α production or MFI with respect to the WT BCG (100%). The values are means ± SEM of 2 (panels A and C) and 4 independent experiments (panels B and D). In panels C and D, grey bars corresponded to uninfected controls. Differences between BCG control and r-BCG PGL-1 were statistically evaluated: *, p<0.05 ;**, p<0.01; ***, p<0.001; n.s., not significant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958813&req=5

ppat-1001159-g006: PGL-1 production impairs the initiation of innate immune responses.(A) Activation of the NF-κB pathway, as evaluated by the THP-1 Blue cell line and assay of SEAP activity, following infection with WT BCG (black bars) or r-BCG PGL-1 (white bars) at various MOI. The mean OD630nm values for WT BCG were 1.13, 0.905, and 0.246 at MOI 10∶1, 1∶1 and 1∶10 respectively whereas the value obtained for r-BCG PGL-1 were 0.87, 0.368 and 0.073 at the same MOI. The value used to normalize at 100% was 1.13 units. (B) TNF-α production by hMDM after 2 hours of post-infection. One hundred percent corresponded to 10538±1579 pg/ml. (C) TNF-α production by hMDM pre-incubated with mAbs directed against CR3 or with irrelevant isotype control and infected during 2 hours at a MOI of 10∶1. One hundred percent corresponded to 4101±551 pg/ml. (D) Expression of maturation markers at the surface of infected hDC, as analyzed by flow cytometry. Data are presented as the percentage of SEAP activity, TNF-α production or MFI with respect to the WT BCG (100%). The values are means ± SEM of 2 (panels A and C) and 4 independent experiments (panels B and D). In panels C and D, grey bars corresponded to uninfected controls. Differences between BCG control and r-BCG PGL-1 were statistically evaluated: *, p<0.05 ;**, p<0.01; ***, p<0.001; n.s., not significant.
Mentions: We next examined whether PGL-1 production influenced the innate responses of human phagocytes to mycobacterial infection. This was first investigated by monitoring the effect of PGL-1 on the activity of the transcription factor NF-κB, which controls the expression of multiple inflammatory genes in hMDM and hDC. We used a THP-1 cell line transfected with a reporter system under the control of a promoter inducible by NF-κB. Infection of THP-1 cells with WT BCG induced strong expression of the reporter gene, indicative of potent activation of the NF-κB pathway. After 16 h of incubation in the detection medium, the mean OD630nm values obtained for the WT BCG were 23%, 60%, 70% higher than for r-BCG PGL-1 at MOI 10∶1, 1∶1 and 1∶10, respectively (Figure 6A). Therefore, the NF-κB response triggered by r-BCG PGL-1 was significantly lower (p<0.01) than that of WT BCG, whatever the MOI considered. Accordingly, hMDM infected with r-BCG PGL-1 produced lower amounts of the inflammatory cytokine TNF-α than BCG-infected controls after 2 hours of infection, even though bacteria expressing PGL-1 were more efficiently internalized than the WT controls (Figure 6B). Other cytokines, IL-12 (p40 and p70) and IL-10, were also assayed. Both WT BCG and r-BCG PGL-1 induced the production of poor levels of these cytokines both at 2h and 24h post-infection, and no difference was observed between WT-BCG and r-BCG PGL-1 infected hMDM (Figure S3).

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