Limits...
The pathogenic mechanism of the Mycobacterium ulcerans virulence factor, mycolactone, depends on blockade of protein translocation into the ER.

Hall BS, Hill K, McKenna M, Ogbechi J, High S, Willis AE, Simmonds RE - PLoS Pathog. (2014)

Bottom Line: Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages.Second, restored protein is found in the cytosol, indicating an inability to translocate.Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom.

ABSTRACT
Infection with Mycobacterium ulcerans is characterised by tissue necrosis and immunosuppression due to mycolactone, the necessary and sufficient virulence factor for Buruli ulcer disease pathology. Many of its effects are known to involve down-regulation of specific proteins implicated in important cellular processes, such as immune responses and cell adhesion. We have previously shown mycolactone completely blocks the production of LPS-dependent proinflammatory mediators post-transcriptionally. Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages. Instead, it inhibits the production of these, along with nearly all other (induced and constitutive) proteins that transit through the ER. This is due to a blockade of protein translocation and subsequent degradation of aberrantly located protein. Several lines of evidence support this transformative explanation of mycolactone function. First, cellular TNF and Cox-2 can be once more detected if the action of the 26S proteasome is inhibited concurrently. Second, restored protein is found in the cytosol, indicating an inability to translocate. Third, in vitro translation assays show mycolactone prevents the translocation of TNF and other proteins into the ER. This is specific as the insertion of tail-anchored proteins into the ER is unaffected showing that the ER remains structurally intact. Fourth, metabolic labelling reveals a near-complete loss of glycosylated and secreted proteins from treated cells, whereas cytosolic proteins are unaffected. Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types. These studies provide a new mechanism underlying mycolactone's observed pathological activities both in vitro and in vivo. Mycolactone-dependent inhibition of protein translocation into the ER not only explains the deficit of innate cytokines, but also the loss of membrane receptors, adhesion molecules and T-cell cytokines that drive the aetiology of Buruli ulcer.

Show MeSH

Related in: MedlinePlus

Mycolactone does not change the polysomal association of proinflammatory mRNAs.A. RAW264.7 cells were incubated for 1+/−various concentrations of mycolactone (MYC as indicated), 0.5 µg/ml Actinomycin D (Act D) or 0.0125% DMSO then stimulated or not with LPS for 4 hr. Supernatant TNF levels were measured by ELISA (mean±SEM of triplicate assays). B–D. RAW264.7 cells were incubated for 1 hr+/−125 ng/ml mycolactone (MYC) then stimulated or not with LPS. After 4 hrs cells were harvested and lysed in the presence of CHX. B and C. Polysomes were separated on a 10–50% sucrose gradient and the profiles measured by absorbance at 254 nm. Note the increase in the 60S peak and reduced height of the polysome peaks in MYC and LPS+MYC samples. RNA was purified from gradient fractions and transcripts were detected by Northern blotting using full coding region cDNA probes for the genes indicated. D. Signal intensity was quantified by ImageJ analysis of non-saturated phosphorscreen images. Values are presented as percentage of total signal for control (dashed line), LPS (solid black line) and LPS+MYC (red line) cells. All are representative of 3 independent experiments. PABP; poly A-tract binding protein.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3974873&req=5

ppat-1004061-g001: Mycolactone does not change the polysomal association of proinflammatory mRNAs.A. RAW264.7 cells were incubated for 1+/−various concentrations of mycolactone (MYC as indicated), 0.5 µg/ml Actinomycin D (Act D) or 0.0125% DMSO then stimulated or not with LPS for 4 hr. Supernatant TNF levels were measured by ELISA (mean±SEM of triplicate assays). B–D. RAW264.7 cells were incubated for 1 hr+/−125 ng/ml mycolactone (MYC) then stimulated or not with LPS. After 4 hrs cells were harvested and lysed in the presence of CHX. B and C. Polysomes were separated on a 10–50% sucrose gradient and the profiles measured by absorbance at 254 nm. Note the increase in the 60S peak and reduced height of the polysome peaks in MYC and LPS+MYC samples. RNA was purified from gradient fractions and transcripts were detected by Northern blotting using full coding region cDNA probes for the genes indicated. D. Signal intensity was quantified by ImageJ analysis of non-saturated phosphorscreen images. Values are presented as percentage of total signal for control (dashed line), LPS (solid black line) and LPS+MYC (red line) cells. All are representative of 3 independent experiments. PABP; poly A-tract binding protein.

Mentions: In order to establish the dose of synthetic mycolactone A/B required to completely inhibit the production of TNF in RAW264.7 cells, we carried out a dose response (Fig. 1A). It was determined that the effective dose was 125 ng/ml, and this also prevented LPS-dependent Cox-2 production without affecting cell viability (Fig. S1A). This dose is marginally higher than required for inhibition of TNF production by natural mycolactone A/B in primary human macrophages (Fig. S1B), probably reflecting the known variation in sensitivity between different cell types, preparations of mycolactone (natural vs. synthetic) and/or target activities (immunosuppressive vs. cytotoxic). We then performed polysome profiling of macrophages to investigate whether mycolactone selectively inhibits the translation of inflammatory mediators. This technique allows the association of TNF, IL-6 and Cox-2 transcripts with actively translating polysomes in various experimental conditions to be assessed. RAW264.7 cells were used because, in preliminary experiments, the low mRNA yields and high RNase content of primary human monocytes and macrophages precluded the use of these cells (data not shown). The post-transcriptional mechanism of mycolactone-dependent inhibition of cytokine production observed in primary cells is conserved in this cell line (Fig. S1C, performed as a control experiment for all profiles obtained).


The pathogenic mechanism of the Mycobacterium ulcerans virulence factor, mycolactone, depends on blockade of protein translocation into the ER.

Hall BS, Hill K, McKenna M, Ogbechi J, High S, Willis AE, Simmonds RE - PLoS Pathog. (2014)

Mycolactone does not change the polysomal association of proinflammatory mRNAs.A. RAW264.7 cells were incubated for 1+/−various concentrations of mycolactone (MYC as indicated), 0.5 µg/ml Actinomycin D (Act D) or 0.0125% DMSO then stimulated or not with LPS for 4 hr. Supernatant TNF levels were measured by ELISA (mean±SEM of triplicate assays). B–D. RAW264.7 cells were incubated for 1 hr+/−125 ng/ml mycolactone (MYC) then stimulated or not with LPS. After 4 hrs cells were harvested and lysed in the presence of CHX. B and C. Polysomes were separated on a 10–50% sucrose gradient and the profiles measured by absorbance at 254 nm. Note the increase in the 60S peak and reduced height of the polysome peaks in MYC and LPS+MYC samples. RNA was purified from gradient fractions and transcripts were detected by Northern blotting using full coding region cDNA probes for the genes indicated. D. Signal intensity was quantified by ImageJ analysis of non-saturated phosphorscreen images. Values are presented as percentage of total signal for control (dashed line), LPS (solid black line) and LPS+MYC (red line) cells. All are representative of 3 independent experiments. PABP; poly A-tract binding protein.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974873&req=5

ppat-1004061-g001: Mycolactone does not change the polysomal association of proinflammatory mRNAs.A. RAW264.7 cells were incubated for 1+/−various concentrations of mycolactone (MYC as indicated), 0.5 µg/ml Actinomycin D (Act D) or 0.0125% DMSO then stimulated or not with LPS for 4 hr. Supernatant TNF levels were measured by ELISA (mean±SEM of triplicate assays). B–D. RAW264.7 cells were incubated for 1 hr+/−125 ng/ml mycolactone (MYC) then stimulated or not with LPS. After 4 hrs cells were harvested and lysed in the presence of CHX. B and C. Polysomes were separated on a 10–50% sucrose gradient and the profiles measured by absorbance at 254 nm. Note the increase in the 60S peak and reduced height of the polysome peaks in MYC and LPS+MYC samples. RNA was purified from gradient fractions and transcripts were detected by Northern blotting using full coding region cDNA probes for the genes indicated. D. Signal intensity was quantified by ImageJ analysis of non-saturated phosphorscreen images. Values are presented as percentage of total signal for control (dashed line), LPS (solid black line) and LPS+MYC (red line) cells. All are representative of 3 independent experiments. PABP; poly A-tract binding protein.
Mentions: In order to establish the dose of synthetic mycolactone A/B required to completely inhibit the production of TNF in RAW264.7 cells, we carried out a dose response (Fig. 1A). It was determined that the effective dose was 125 ng/ml, and this also prevented LPS-dependent Cox-2 production without affecting cell viability (Fig. S1A). This dose is marginally higher than required for inhibition of TNF production by natural mycolactone A/B in primary human macrophages (Fig. S1B), probably reflecting the known variation in sensitivity between different cell types, preparations of mycolactone (natural vs. synthetic) and/or target activities (immunosuppressive vs. cytotoxic). We then performed polysome profiling of macrophages to investigate whether mycolactone selectively inhibits the translation of inflammatory mediators. This technique allows the association of TNF, IL-6 and Cox-2 transcripts with actively translating polysomes in various experimental conditions to be assessed. RAW264.7 cells were used because, in preliminary experiments, the low mRNA yields and high RNase content of primary human monocytes and macrophages precluded the use of these cells (data not shown). The post-transcriptional mechanism of mycolactone-dependent inhibition of cytokine production observed in primary cells is conserved in this cell line (Fig. S1C, performed as a control experiment for all profiles obtained).

Bottom Line: Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages.Second, restored protein is found in the cytosol, indicating an inability to translocate.Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom.

ABSTRACT
Infection with Mycobacterium ulcerans is characterised by tissue necrosis and immunosuppression due to mycolactone, the necessary and sufficient virulence factor for Buruli ulcer disease pathology. Many of its effects are known to involve down-regulation of specific proteins implicated in important cellular processes, such as immune responses and cell adhesion. We have previously shown mycolactone completely blocks the production of LPS-dependent proinflammatory mediators post-transcriptionally. Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages. Instead, it inhibits the production of these, along with nearly all other (induced and constitutive) proteins that transit through the ER. This is due to a blockade of protein translocation and subsequent degradation of aberrantly located protein. Several lines of evidence support this transformative explanation of mycolactone function. First, cellular TNF and Cox-2 can be once more detected if the action of the 26S proteasome is inhibited concurrently. Second, restored protein is found in the cytosol, indicating an inability to translocate. Third, in vitro translation assays show mycolactone prevents the translocation of TNF and other proteins into the ER. This is specific as the insertion of tail-anchored proteins into the ER is unaffected showing that the ER remains structurally intact. Fourth, metabolic labelling reveals a near-complete loss of glycosylated and secreted proteins from treated cells, whereas cytosolic proteins are unaffected. Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types. These studies provide a new mechanism underlying mycolactone's observed pathological activities both in vitro and in vivo. Mycolactone-dependent inhibition of protein translocation into the ER not only explains the deficit of innate cytokines, but also the loss of membrane receptors, adhesion molecules and T-cell cytokines that drive the aetiology of Buruli ulcer.

Show MeSH
Related in: MedlinePlus