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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 induces cytoskeletal alteration, cell round up and detachment.Mouse fibroblasts L929 cells seeded on coverslips were incubated for 12, 18 or 24/mL of mycolactone. Cytoskeletal changes were visualized by immunofluorescence microscopy using rhodamine-phalloidin conjugate (red) and a tubulin-specific antibody (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The cellular shape was visualized by differential interference contrast (DIC) microscopy. White horizontal bar represents a 10 µm scale (A). Blue (nucleus) and red (actin) channels were used for 3D remodeling of the respective confocal z stacks (B).
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pntd-0003066-g002: Mycolactone induces cytoskeletal alteration, cell round up and detachment.Mouse fibroblasts L929 cells seeded on coverslips were incubated for 12, 18 or 24/mL of mycolactone. Cytoskeletal changes were visualized by immunofluorescence microscopy using rhodamine-phalloidin conjugate (red) and a tubulin-specific antibody (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The cellular shape was visualized by differential interference contrast (DIC) microscopy. White horizontal bar represents a 10 µm scale (A). Blue (nucleus) and red (actin) channels were used for 3D remodeling of the respective confocal z stacks (B).

Mentions: To further characterize our model, the kinetics of the cytopathic effects, namely cytoskeleton alterations and cell round-up and detachment, were also assessed. In cells incubated with mycolactone, we observed not only the previously described alterations for actin [26], but also changes in the tubulin cytoskeleton, which appeared bended into a microtubule hank (figure 2A). Within 12–18 h of exposure to the toxin, actin ultrastructures (stress fibers and lamellipodia) were lost, and, although still attached, most of the cells were completely round-up by 18–24 h (figures 2A and 2B). At 24 h, half of the cells were already in suspension, while the remaining cells eventually detached in the following 12 h [26]: detachment being probably a consequence of cell round-up and loss of adhesion structures.


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 induces cytoskeletal alteration, cell round up and detachment.Mouse fibroblasts L929 cells seeded on coverslips were incubated for 12, 18 or 24/mL of mycolactone. Cytoskeletal changes were visualized by immunofluorescence microscopy using rhodamine-phalloidin conjugate (red) and a tubulin-specific antibody (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The cellular shape was visualized by differential interference contrast (DIC) microscopy. White horizontal bar represents a 10 µm scale (A). Blue (nucleus) and red (actin) channels were used for 3D remodeling of the respective confocal z stacks (B).
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003066-g002: Mycolactone induces cytoskeletal alteration, cell round up and detachment.Mouse fibroblasts L929 cells seeded on coverslips were incubated for 12, 18 or 24/mL of mycolactone. Cytoskeletal changes were visualized by immunofluorescence microscopy using rhodamine-phalloidin conjugate (red) and a tubulin-specific antibody (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The cellular shape was visualized by differential interference contrast (DIC) microscopy. White horizontal bar represents a 10 µm scale (A). Blue (nucleus) and red (actin) channels were used for 3D remodeling of the respective confocal z stacks (B).
Mentions: To further characterize our model, the kinetics of the cytopathic effects, namely cytoskeleton alterations and cell round-up and detachment, were also assessed. In cells incubated with mycolactone, we observed not only the previously described alterations for actin [26], but also changes in the tubulin cytoskeleton, which appeared bended into a microtubule hank (figure 2A). Within 12–18 h of exposure to the toxin, actin ultrastructures (stress fibers and lamellipodia) were lost, and, although still attached, most of the cells were completely round-up by 18–24 h (figures 2A and 2B). At 24 h, half of the cells were already in suspension, while the remaining cells eventually detached in the following 12 h [26]: detachment being probably a consequence of cell round-up and loss of adhesion structures.

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