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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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Kinetics of mycolactone cytostatic and cytotoxic effects.Mouse fibroblasts L929 cells were incubated for 24, 48 or 72(referred to as 48 h+48 h). Cell cycle analysis (A) and annexin-V/PI assay (B) were performed for each-time point. Bars represent the mean + SD (n = 3) from one out of, at least, two independent experiments. Each condition was compared to EtOH-treated samples throughout each time-point (24 h, 48 h and 72 h) by Two-way ANOVA with Bonferroni posttest; statistical differences were represented by *** (P<0.001) for sub-G0/G1 and Annexin-V+/PI− , and by ### (P<0.001) for G0/G1 Phase and Annexin-V+/PI+. Each condition at 48 h+48 h time-point was compared with the same condition at the 48 h by Two-way ANOVA with Bonferroni posttest; statistical differences were represented by &&& (P<0.001) for sub-G0/G1 and Annexin-V+/PI−, and by §§§ (P<0.001) for G0/G1 Phase and Annexin-V+/PI+.
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pntd-0003066-g001: Kinetics of mycolactone cytostatic and cytotoxic effects.Mouse fibroblasts L929 cells were incubated for 24, 48 or 72(referred to as 48 h+48 h). Cell cycle analysis (A) and annexin-V/PI assay (B) were performed for each-time point. Bars represent the mean + SD (n = 3) from one out of, at least, two independent experiments. Each condition was compared to EtOH-treated samples throughout each time-point (24 h, 48 h and 72 h) by Two-way ANOVA with Bonferroni posttest; statistical differences were represented by *** (P<0.001) for sub-G0/G1 and Annexin-V+/PI− , and by ### (P<0.001) for G0/G1 Phase and Annexin-V+/PI+. Each condition at 48 h+48 h time-point was compared with the same condition at the 48 h by Two-way ANOVA with Bonferroni posttest; statistical differences were represented by &&& (P<0.001) for sub-G0/G1 and Annexin-V+/PI−, and by §§§ (P<0.001) for G0/G1 Phase and Annexin-V+/PI+.

Mentions: Results presented in figure 1 show that the ethanol (vehicle) equivalent (<0.002%), as well as the mycolactone concentration below the threshold (12.5 ng/mL), had no detectable cytopathic (rounding and detachment) or cytotoxic effects. Cytotoxic doses of mycolactone (>12.5 ng/mL) induced detachment, cell cycle arrest in G0/G1 phase at 48 h and 72 h of treatment and the appearance of a sub-G0/G1 population, compatible to apoptotic cells, more evident at 72 h (figure 1A). Consistent with cell cycle data, annexin-V/PI assays revealed an annexin-V+/PI− population for the highest mycolactone concentrations (25 and 50 ng/ml) at 72 h (figures 1B), indicative of apoptotic cells.


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)

Kinetics of mycolactone cytostatic and cytotoxic effects.Mouse fibroblasts L929 cells were incubated for 24, 48 or 72(referred to as 48 h+48 h). Cell cycle analysis (A) and annexin-V/PI assay (B) were performed for each-time point. Bars represent the mean + SD (n = 3) from one out of, at least, two independent experiments. Each condition was compared to EtOH-treated samples throughout each time-point (24 h, 48 h and 72 h) by Two-way ANOVA with Bonferroni posttest; statistical differences were represented by *** (P<0.001) for sub-G0/G1 and Annexin-V+/PI− , and by ### (P<0.001) for G0/G1 Phase and Annexin-V+/PI+. Each condition at 48 h+48 h time-point was compared with the same condition at the 48 h by Two-way ANOVA with Bonferroni posttest; statistical differences were represented by &&& (P<0.001) for sub-G0/G1 and Annexin-V+/PI−, and by §§§ (P<0.001) for G0/G1 Phase and Annexin-V+/PI+.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003066-g001: Kinetics of mycolactone cytostatic and cytotoxic effects.Mouse fibroblasts L929 cells were incubated for 24, 48 or 72(referred to as 48 h+48 h). Cell cycle analysis (A) and annexin-V/PI assay (B) were performed for each-time point. Bars represent the mean + SD (n = 3) from one out of, at least, two independent experiments. Each condition was compared to EtOH-treated samples throughout each time-point (24 h, 48 h and 72 h) by Two-way ANOVA with Bonferroni posttest; statistical differences were represented by *** (P<0.001) for sub-G0/G1 and Annexin-V+/PI− , and by ### (P<0.001) for G0/G1 Phase and Annexin-V+/PI+. Each condition at 48 h+48 h time-point was compared with the same condition at the 48 h by Two-way ANOVA with Bonferroni posttest; statistical differences were represented by &&& (P<0.001) for sub-G0/G1 and Annexin-V+/PI−, and by §§§ (P<0.001) for G0/G1 Phase and Annexin-V+/PI+.
Mentions: Results presented in figure 1 show that the ethanol (vehicle) equivalent (<0.002%), as well as the mycolactone concentration below the threshold (12.5 ng/mL), had no detectable cytopathic (rounding and detachment) or cytotoxic effects. Cytotoxic doses of mycolactone (>12.5 ng/mL) induced detachment, cell cycle arrest in G0/G1 phase at 48 h and 72 h of treatment and the appearance of a sub-G0/G1 population, compatible to apoptotic cells, more evident at 72 h (figure 1A). Consistent with cell cycle data, annexin-V/PI assays revealed an annexin-V+/PI− population for the highest mycolactone concentrations (25 and 50 ng/ml) at 72 h (figures 1B), indicative of apoptotic cells.

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