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Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

Gama JB, Ohlmeier S, Martins TG, Fraga AG, Sampaio-Marques B, Carvalho MA, Proença F, Silva MT, Pedrosa J, Ludovico P - PLoS Negl Trop Dis (2014)

Bottom Line: In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone.In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes.Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Braga, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.

ABSTRACT
Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

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Mycolactone causes an up-regulation of the ubiquitin-proteasome pathway and an accumulation of autophagosomes.In A, B and C, mouse fibroblasts L929 cells were incubated for 24 or 48(−) or mycolactone (50 ng/mL, +). Additionally, an assay was performed where cells were incubated for 48 hours in the same conditions followed by a 48 hour incubation period in fresh medium (referred to as 48 h+48 h). At each time-point, total protein was extracted and Western blot was performed to assess ubiquitinated proteins (A) or LC3 processing (B). Additionally, cytospins were made to assess immunofluorescence LC3 (red). White horizontal bars represent a 10 µm scale (C). In D, mouse fibroblasts L929 cells were incubated for 48 hours either with ethanol or mycolactone (50 ng/mL) and bafilomycin A1 (10 nM, +) or DMSO (vehicle control, −) was added 2 hours prior the end of the assay. Additionally, mouse fibroblast L929 cells were incubated for 48 hours without any stimuli until 2 hours prior the end of the assay, when autophagy was induced with rapamycin (1 µM), and bafilomycin A1 (10 nM, +) or DMSO (vehicle control, −) was added. At the end of the assay, total protein was extracted and Western blot was performed to assess LC3 processing (D).
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pntd-0003066-g005: Mycolactone causes an up-regulation of the ubiquitin-proteasome pathway and an accumulation of autophagosomes.In A, B and C, mouse fibroblasts L929 cells were incubated for 24 or 48(−) or mycolactone (50 ng/mL, +). Additionally, an assay was performed where cells were incubated for 48 hours in the same conditions followed by a 48 hour incubation period in fresh medium (referred to as 48 h+48 h). At each time-point, total protein was extracted and Western blot was performed to assess ubiquitinated proteins (A) or LC3 processing (B). Additionally, cytospins were made to assess immunofluorescence LC3 (red). White horizontal bars represent a 10 µm scale (C). In D, mouse fibroblasts L929 cells were incubated for 48 hours either with ethanol or mycolactone (50 ng/mL) and bafilomycin A1 (10 nM, +) or DMSO (vehicle control, −) was added 2 hours prior the end of the assay. Additionally, mouse fibroblast L929 cells were incubated for 48 hours without any stimuli until 2 hours prior the end of the assay, when autophagy was induced with rapamycin (1 µM), and bafilomycin A1 (10 nM, +) or DMSO (vehicle control, −) was added. At the end of the assay, total protein was extracted and Western blot was performed to assess LC3 processing (D).

Mentions: Two stress response proteins (Hspa1b, Uba52) were up-regulated upon treatment with mycolactone. Spot 10 (figure 4 and figure S1) was identified as a fusion protein (Uba52) consisting of N-terminal ubiquitin and C-terminal 60S ribosomal protein L40. The detected spot position in the gel (figure 3) in comparison with the theoretical positions for the fusion protein (pI 9.87/14.7 kDa), ubiquitin (pI 6.56/8.6 kDa) and the ribosomal protein (pI 10.32/6.2 kDa) suggest the presence of ubiquitin. Indeed, all three spot-specific peptides covered amino acids 13–55 revealing that ubiquitin is present. The here detected increase of free ubiquitin after 48 h of mycolactone treatment could result from an inhibition of ubiquitin ligases or from an up-regulation of its expression. To investigate this in more detail, protein ubiquitination was studied by western blot, which revealed that mycolactone exposure results in an increase of ubiquitinated proteins, more evident at 48 h and 48 h+48 h (figure 5A). Therefore, these data show that rather than an inhibition of ubiquitin ligases, mycolactone induces an up-regulation of the ubiquitin/proteasome system (UPS).


Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

Gama JB, Ohlmeier S, Martins TG, Fraga AG, Sampaio-Marques B, Carvalho MA, Proença F, Silva MT, Pedrosa J, Ludovico P - PLoS Negl Trop Dis (2014)

Mycolactone causes an up-regulation of the ubiquitin-proteasome pathway and an accumulation of autophagosomes.In A, B and C, mouse fibroblasts L929 cells were incubated for 24 or 48(−) or mycolactone (50 ng/mL, +). Additionally, an assay was performed where cells were incubated for 48 hours in the same conditions followed by a 48 hour incubation period in fresh medium (referred to as 48 h+48 h). At each time-point, total protein was extracted and Western blot was performed to assess ubiquitinated proteins (A) or LC3 processing (B). Additionally, cytospins were made to assess immunofluorescence LC3 (red). White horizontal bars represent a 10 µm scale (C). In D, mouse fibroblasts L929 cells were incubated for 48 hours either with ethanol or mycolactone (50 ng/mL) and bafilomycin A1 (10 nM, +) or DMSO (vehicle control, −) was added 2 hours prior the end of the assay. Additionally, mouse fibroblast L929 cells were incubated for 48 hours without any stimuli until 2 hours prior the end of the assay, when autophagy was induced with rapamycin (1 µM), and bafilomycin A1 (10 nM, +) or DMSO (vehicle control, −) was added. At the end of the assay, total protein was extracted and Western blot was performed to assess LC3 processing (D).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4125307&req=5

pntd-0003066-g005: Mycolactone causes an up-regulation of the ubiquitin-proteasome pathway and an accumulation of autophagosomes.In A, B and C, mouse fibroblasts L929 cells were incubated for 24 or 48(−) or mycolactone (50 ng/mL, +). Additionally, an assay was performed where cells were incubated for 48 hours in the same conditions followed by a 48 hour incubation period in fresh medium (referred to as 48 h+48 h). At each time-point, total protein was extracted and Western blot was performed to assess ubiquitinated proteins (A) or LC3 processing (B). Additionally, cytospins were made to assess immunofluorescence LC3 (red). White horizontal bars represent a 10 µm scale (C). In D, mouse fibroblasts L929 cells were incubated for 48 hours either with ethanol or mycolactone (50 ng/mL) and bafilomycin A1 (10 nM, +) or DMSO (vehicle control, −) was added 2 hours prior the end of the assay. Additionally, mouse fibroblast L929 cells were incubated for 48 hours without any stimuli until 2 hours prior the end of the assay, when autophagy was induced with rapamycin (1 µM), and bafilomycin A1 (10 nM, +) or DMSO (vehicle control, −) was added. At the end of the assay, total protein was extracted and Western blot was performed to assess LC3 processing (D).
Mentions: Two stress response proteins (Hspa1b, Uba52) were up-regulated upon treatment with mycolactone. Spot 10 (figure 4 and figure S1) was identified as a fusion protein (Uba52) consisting of N-terminal ubiquitin and C-terminal 60S ribosomal protein L40. The detected spot position in the gel (figure 3) in comparison with the theoretical positions for the fusion protein (pI 9.87/14.7 kDa), ubiquitin (pI 6.56/8.6 kDa) and the ribosomal protein (pI 10.32/6.2 kDa) suggest the presence of ubiquitin. Indeed, all three spot-specific peptides covered amino acids 13–55 revealing that ubiquitin is present. The here detected increase of free ubiquitin after 48 h of mycolactone treatment could result from an inhibition of ubiquitin ligases or from an up-regulation of its expression. To investigate this in more detail, protein ubiquitination was studied by western blot, which revealed that mycolactone exposure results in an increase of ubiquitinated proteins, more evident at 48 h and 48 h+48 h (figure 5A). Therefore, these data show that rather than an inhibition of ubiquitin ligases, mycolactone induces an up-regulation of the ubiquitin/proteasome system (UPS).

Bottom Line: In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone.In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes.Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Braga, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.

ABSTRACT
Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

Show MeSH
Related in: MedlinePlus