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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.

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Mycolactone inhibits co-translational translocation of proteins into the ER via a mechanism that does not disrupt the structural integrity of the ER.A–E. In vitro translation (IVT) reactions of different capped transcripts were performed as described +/− mycolactone (MYC; 200 ng/ml unless indicated otherwise), Eeyarestatin 1 (ES1; 250 µM) or DMSO (-; 0.02%). Data representative of 3 independent experiments. A. IVT of luciferase and TNF mRNAs detected by 35S incorporation and Western blot (∼26 kDa) respectively. B. IVT of TNF mRNA was performed in the presence of semi-permeabilised RAW264.7 cells then incubated with no addition (-), or Proteinase K (PK) +/−0.1% Triton-x-100 (PKT) for 1 hr at 4°C before stopping the reaction. C. Dose dependence of loss of the PK protected band. Signal intensity was quantified by ImageJ analysis of non-saturated blots and the protected band (PK) was normalised to total pro-TNF (-). D. IVT of prepro-α Factor (PPAF) and β-lactamase (LACTB) mRNAs in the absence or presence of canine pancreatic microsomal membranes (CPMM). Black arrowhead, glycosylated forms of α Factor; *, signal peptide-cleaved LACTB. E. IVT of PPAF, Sec61β and cytochrome B5 (Cyt-B5) mRNAs in the presence CPMM. After labelling with 35S as described, membranes were isolated by centrifugation through a sucrose cushion [31]. Endoglycosidase H (EndoH) is used to confirm glycosylation of proteins (black arrowhead), the thin line represents where an empty lane was removed from the image for ease of interpretation. F. RAW264.7 cells were incubated +/−125 ng/ml mycolactone (MYC), washed and incubated without mycolactone for various periods (recovery time) before stimulating with LPS for 4 hrs. Supernatant TNF levels were measured by ELISA (mean±SEM of triplicate assays).
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ppat-1004061-g004: Mycolactone inhibits co-translational translocation of proteins into the ER via a mechanism that does not disrupt the structural integrity of the ER.A–E. In vitro translation (IVT) reactions of different capped transcripts were performed as described +/− mycolactone (MYC; 200 ng/ml unless indicated otherwise), Eeyarestatin 1 (ES1; 250 µM) or DMSO (-; 0.02%). Data representative of 3 independent experiments. A. IVT of luciferase and TNF mRNAs detected by 35S incorporation and Western blot (∼26 kDa) respectively. B. IVT of TNF mRNA was performed in the presence of semi-permeabilised RAW264.7 cells then incubated with no addition (-), or Proteinase K (PK) +/−0.1% Triton-x-100 (PKT) for 1 hr at 4°C before stopping the reaction. C. Dose dependence of loss of the PK protected band. Signal intensity was quantified by ImageJ analysis of non-saturated blots and the protected band (PK) was normalised to total pro-TNF (-). D. IVT of prepro-α Factor (PPAF) and β-lactamase (LACTB) mRNAs in the absence or presence of canine pancreatic microsomal membranes (CPMM). Black arrowhead, glycosylated forms of α Factor; *, signal peptide-cleaved LACTB. E. IVT of PPAF, Sec61β and cytochrome B5 (Cyt-B5) mRNAs in the presence CPMM. After labelling with 35S as described, membranes were isolated by centrifugation through a sucrose cushion [31]. Endoglycosidase H (EndoH) is used to confirm glycosylation of proteins (black arrowhead), the thin line represents where an empty lane was removed from the image for ease of interpretation. F. RAW264.7 cells were incubated +/−125 ng/ml mycolactone (MYC), washed and incubated without mycolactone for various periods (recovery time) before stimulating with LPS for 4 hrs. Supernatant TNF levels were measured by ELISA (mean±SEM of triplicate assays).

Mentions: The lack of glycosylation of Cox-2 and the failure of PSI treatment to cause TNF secretion (Fig. 3A), suggested the restored proteins could not gain access to the ER. Indeed, after digitonin permeabilisation of PSI-treated cells, unglycosylated Cox-2 and pro-TNF were predominantly found in the cytosolic fraction of mycolactone treated cells (Fig. 3D, lane 3 [closed arrow]), in contrast to the LPS-induced proteins that were glycosylated and membrane associated (Figure 3D lane 6). Unglycosylated Cox-2 was found in the membrane fraction after TUN treatment showing that lack of N-glycosylation alone was insufficient to explain its localisation in mycolactone-treated cells (Fig. 3D lane 8). Inhibition of the Sec61 translocon by small molecule inhibitors, rather than causing accumulation of proteins intended for export in the cytoplasm, tends to trigger rapid degradation of the mislocalised proteins [29], [30], similarly to mycolactone. We therefore assessed whether the degradative loss we observe could be due to a blockade of translocation, using assays in which different mRNAs (transcribed and capped in vitro) undergo in vitro translation (IVT) in the absence or presence of ER containing cellular membrane preparations (Fig. 4). When such membranes are absent, no modifications of the proteins are possible. However, when membranes are present, the nascent proteins produced can undergo co-translational translocation into the ER via the Sec61 translocon and can consequently be either glycosylated or processed to remove the signal peptide sequence. In agreement with our other findings, mycolactone had a minimal direct effect on the synthesis of TNF in both the absence (Fig. 4A; TNF and luciferase mRNAs) and presence (Fig. 4B, TNF compare lanes 1 and 4) of membranes provided by semi-permeabilised RAW264.7 cell extracts.


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 inhibits co-translational translocation of proteins into the ER via a mechanism that does not disrupt the structural integrity of the ER.A–E. In vitro translation (IVT) reactions of different capped transcripts were performed as described +/− mycolactone (MYC; 200 ng/ml unless indicated otherwise), Eeyarestatin 1 (ES1; 250 µM) or DMSO (-; 0.02%). Data representative of 3 independent experiments. A. IVT of luciferase and TNF mRNAs detected by 35S incorporation and Western blot (∼26 kDa) respectively. B. IVT of TNF mRNA was performed in the presence of semi-permeabilised RAW264.7 cells then incubated with no addition (-), or Proteinase K (PK) +/−0.1% Triton-x-100 (PKT) for 1 hr at 4°C before stopping the reaction. C. Dose dependence of loss of the PK protected band. Signal intensity was quantified by ImageJ analysis of non-saturated blots and the protected band (PK) was normalised to total pro-TNF (-). D. IVT of prepro-α Factor (PPAF) and β-lactamase (LACTB) mRNAs in the absence or presence of canine pancreatic microsomal membranes (CPMM). Black arrowhead, glycosylated forms of α Factor; *, signal peptide-cleaved LACTB. E. IVT of PPAF, Sec61β and cytochrome B5 (Cyt-B5) mRNAs in the presence CPMM. After labelling with 35S as described, membranes were isolated by centrifugation through a sucrose cushion [31]. Endoglycosidase H (EndoH) is used to confirm glycosylation of proteins (black arrowhead), the thin line represents where an empty lane was removed from the image for ease of interpretation. F. RAW264.7 cells were incubated +/−125 ng/ml mycolactone (MYC), washed and incubated without mycolactone for various periods (recovery time) before stimulating with LPS for 4 hrs. Supernatant TNF levels were measured by ELISA (mean±SEM of triplicate assays).
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Related In: Results  -  Collection

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ppat-1004061-g004: Mycolactone inhibits co-translational translocation of proteins into the ER via a mechanism that does not disrupt the structural integrity of the ER.A–E. In vitro translation (IVT) reactions of different capped transcripts were performed as described +/− mycolactone (MYC; 200 ng/ml unless indicated otherwise), Eeyarestatin 1 (ES1; 250 µM) or DMSO (-; 0.02%). Data representative of 3 independent experiments. A. IVT of luciferase and TNF mRNAs detected by 35S incorporation and Western blot (∼26 kDa) respectively. B. IVT of TNF mRNA was performed in the presence of semi-permeabilised RAW264.7 cells then incubated with no addition (-), or Proteinase K (PK) +/−0.1% Triton-x-100 (PKT) for 1 hr at 4°C before stopping the reaction. C. Dose dependence of loss of the PK protected band. Signal intensity was quantified by ImageJ analysis of non-saturated blots and the protected band (PK) was normalised to total pro-TNF (-). D. IVT of prepro-α Factor (PPAF) and β-lactamase (LACTB) mRNAs in the absence or presence of canine pancreatic microsomal membranes (CPMM). Black arrowhead, glycosylated forms of α Factor; *, signal peptide-cleaved LACTB. E. IVT of PPAF, Sec61β and cytochrome B5 (Cyt-B5) mRNAs in the presence CPMM. After labelling with 35S as described, membranes were isolated by centrifugation through a sucrose cushion [31]. Endoglycosidase H (EndoH) is used to confirm glycosylation of proteins (black arrowhead), the thin line represents where an empty lane was removed from the image for ease of interpretation. F. RAW264.7 cells were incubated +/−125 ng/ml mycolactone (MYC), washed and incubated without mycolactone for various periods (recovery time) before stimulating with LPS for 4 hrs. Supernatant TNF levels were measured by ELISA (mean±SEM of triplicate assays).
Mentions: The lack of glycosylation of Cox-2 and the failure of PSI treatment to cause TNF secretion (Fig. 3A), suggested the restored proteins could not gain access to the ER. Indeed, after digitonin permeabilisation of PSI-treated cells, unglycosylated Cox-2 and pro-TNF were predominantly found in the cytosolic fraction of mycolactone treated cells (Fig. 3D, lane 3 [closed arrow]), in contrast to the LPS-induced proteins that were glycosylated and membrane associated (Figure 3D lane 6). Unglycosylated Cox-2 was found in the membrane fraction after TUN treatment showing that lack of N-glycosylation alone was insufficient to explain its localisation in mycolactone-treated cells (Fig. 3D lane 8). Inhibition of the Sec61 translocon by small molecule inhibitors, rather than causing accumulation of proteins intended for export in the cytoplasm, tends to trigger rapid degradation of the mislocalised proteins [29], [30], similarly to mycolactone. We therefore assessed whether the degradative loss we observe could be due to a blockade of translocation, using assays in which different mRNAs (transcribed and capped in vitro) undergo in vitro translation (IVT) in the absence or presence of ER containing cellular membrane preparations (Fig. 4). When such membranes are absent, no modifications of the proteins are possible. However, when membranes are present, the nascent proteins produced can undergo co-translational translocation into the ER via the Sec61 translocon and can consequently be either glycosylated or processed to remove the signal peptide sequence. In agreement with our other findings, mycolactone had a minimal direct effect on the synthesis of TNF in both the absence (Fig. 4A; TNF and luciferase mRNAs) and presence (Fig. 4B, TNF compare lanes 1 and 4) of membranes provided by semi-permeabilised RAW264.7 cell extracts.

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