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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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Related in: MedlinePlus

Altered proteins upon mycolactone treatment.Figure shows the proteins identified for each spot (numbers according to figure 3 and figure S1) clustered into five categories. (1) Expression levels show decreased (red), increased (green) or no changes (black) in spot intensities upon mycolactone treatment. (2) MS and MS/MS parameters are presented, including the number of specific matched peptides (P), the sequence coverage (SC) in percentage, as well as the MS scores. Phosphoproteins (*) and putative protein fragments (#) are indicated. Full MS and MS/MS data in Supplementary Information.
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pntd-0003066-g004: Altered proteins upon mycolactone treatment.Figure shows the proteins identified for each spot (numbers according to figure 3 and figure S1) clustered into five categories. (1) Expression levels show decreased (red), increased (green) or no changes (black) in spot intensities upon mycolactone treatment. (2) MS and MS/MS parameters are presented, including the number of specific matched peptides (P), the sequence coverage (SC) in percentage, as well as the MS scores. Phosphoproteins (*) and putative protein fragments (#) are indicated. Full MS and MS/MS data in Supplementary Information.

Mentions: The comparison of control cells proteome at different time-points did not reveal any significantly changed spots, showing that ethanol (vehicle) represents a suitable control. In contrast, the comparison between the proteome of control and mycolactone-treated cells at each time-point revealed a time-dependent increase in the number of changed spots, with 4 spots changed at 24 h, 10 at 48 h and 20 at 48 h+48 h. All 20 spots were identified by mass spectrometry and found to correspond to 18 proteins comprising 5 up- and 13 down-regulated (figure 3 and figure 4).


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)

Altered proteins upon mycolactone treatment.Figure shows the proteins identified for each spot (numbers according to figure 3 and figure S1) clustered into five categories. (1) Expression levels show decreased (red), increased (green) or no changes (black) in spot intensities upon mycolactone treatment. (2) MS and MS/MS parameters are presented, including the number of specific matched peptides (P), the sequence coverage (SC) in percentage, as well as the MS scores. Phosphoproteins (*) and putative protein fragments (#) are indicated. Full MS and MS/MS data in Supplementary Information.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003066-g004: Altered proteins upon mycolactone treatment.Figure shows the proteins identified for each spot (numbers according to figure 3 and figure S1) clustered into five categories. (1) Expression levels show decreased (red), increased (green) or no changes (black) in spot intensities upon mycolactone treatment. (2) MS and MS/MS parameters are presented, including the number of specific matched peptides (P), the sequence coverage (SC) in percentage, as well as the MS scores. Phosphoproteins (*) and putative protein fragments (#) are indicated. Full MS and MS/MS data in Supplementary Information.
Mentions: The comparison of control cells proteome at different time-points did not reveal any significantly changed spots, showing that ethanol (vehicle) represents a suitable control. In contrast, the comparison between the proteome of control and mycolactone-treated cells at each time-point revealed a time-dependent increase in the number of changed spots, with 4 spots changed at 24 h, 10 at 48 h and 20 at 48 h+48 h. All 20 spots were identified by mass spectrometry and found to correspond to 18 proteins comprising 5 up- and 13 down-regulated (figure 3 and figure 4).

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