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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 a decrease in dermal collagen fibers.Mice were infected with virulent mycolactone-secreting (MU98912: dark gray triangles) (n = 5) or avirulent mycolactone-negative (MU5114: light gray squares) (n = 4) strains of M. ulcerans, or injected with PBS (vehicle: white circles) (n = 3) as a control (A, B and C). Alternatively, mice were inoculated with 5 µg of mycolactone (dark gray hexagons) (n = 4) or ethanol (vehicle: white diamonds) (n = 4) as a control (D, E, F). Lesion progression was assessed by measurement of footpad swelling (A and D). Collagen content was assessed by a qualitative blind scoring of the amount of dermal collagen fibers (score 0 = lowest, 4 = highest) visualized in HE-stained sections by polarized light (B and E). The different groups were compared to the control groups (PBS in A and B; ethanol in C and D) by Two-way ANOVA with Bonferroni posttest; statistical differences were represented by ** (P<0.01) or *** (P<0.001). Additionally, mouse footpads exposed to the different stimuli were stained with Masson's trichrome to highlight collagen fibers (green) (C and F). On panel are depicted representative histological image each group. Black horizontal bars represent a 50 µm scale. Histological structures like dermis (D) and the stratum corneum (SC) and stratum spinosum (SS) of the epidermis (ED) are indicated on the upper-left image. Inflammatory infiltrate is indicated by an asterisk.
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pntd-0003066-g006: Mycolactone induces a decrease in dermal collagen fibers.Mice were infected with virulent mycolactone-secreting (MU98912: dark gray triangles) (n = 5) or avirulent mycolactone-negative (MU5114: light gray squares) (n = 4) strains of M. ulcerans, or injected with PBS (vehicle: white circles) (n = 3) as a control (A, B and C). Alternatively, mice were inoculated with 5 µg of mycolactone (dark gray hexagons) (n = 4) or ethanol (vehicle: white diamonds) (n = 4) as a control (D, E, F). Lesion progression was assessed by measurement of footpad swelling (A and D). Collagen content was assessed by a qualitative blind scoring of the amount of dermal collagen fibers (score 0 = lowest, 4 = highest) visualized in HE-stained sections by polarized light (B and E). The different groups were compared to the control groups (PBS in A and B; ethanol in C and D) by Two-way ANOVA with Bonferroni posttest; statistical differences were represented by ** (P<0.01) or *** (P<0.001). Additionally, mouse footpads exposed to the different stimuli were stained with Masson's trichrome to highlight collagen fibers (green) (C and F). On panel are depicted representative histological image each group. Black horizontal bars represent a 50 µm scale. Histological structures like dermis (D) and the stratum corneum (SC) and stratum spinosum (SS) of the epidermis (ED) are indicated on the upper-left image. Inflammatory infiltrate is indicated by an asterisk.

Mentions: To investigate if mycolactone was responsible for a decrease in tissue collagen, an experimental model of BU disease, the mouse footpad infection with M. ulcerans, was used. Mice were challenged with virulent mycolactone-secreting (MU98912), avirulent mycolactone-negative (MU5114) strains of M. ulcerans or PBS as a control. Pathology progression was assessed by measuring footpad swelling (figure 6A) and, at different time-points, footpads were collected for histological processing and collagen scoring (figure 6B). The results showed that, in footpads infected with mycolactone-secreting M. ulcerans (MU98912), the progressive increase of pathology (figure 6A) was associated with a decrease of the collagen score (figure 6B), preceding the breakdown of the lesion into an ulcer (by day 40 post-infection). In contrast, infection with the mycolactone-negative strain (MU5114) did not induce pathology nor did it alter the collagen content in infected footpads, similar to what was observed for the PBS-injected control group (figure 6A and 6B). These results suggest that the decrease in collagen content is not a consequence of the infection or the elicited immune response, but rather caused by mycolactone. To verify this, mice were challenged with purified toxin or ethanol equivalent as control. The results showed that mycolactone induced footpad swelling associated with a decrease of the collagen score, while the vehicle did not (figure 6D and 6E). Histological samples stained with Masson's trichrome showed a decay of collagen fibers in MU98912-infected or mycolactone-treated footpads, characterized by the disorganization and thinness of collagen fibers (figure 6C and 6F).


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 a decrease in dermal collagen fibers.Mice were infected with virulent mycolactone-secreting (MU98912: dark gray triangles) (n = 5) or avirulent mycolactone-negative (MU5114: light gray squares) (n = 4) strains of M. ulcerans, or injected with PBS (vehicle: white circles) (n = 3) as a control (A, B and C). Alternatively, mice were inoculated with 5 µg of mycolactone (dark gray hexagons) (n = 4) or ethanol (vehicle: white diamonds) (n = 4) as a control (D, E, F). Lesion progression was assessed by measurement of footpad swelling (A and D). Collagen content was assessed by a qualitative blind scoring of the amount of dermal collagen fibers (score 0 = lowest, 4 = highest) visualized in HE-stained sections by polarized light (B and E). The different groups were compared to the control groups (PBS in A and B; ethanol in C and D) by Two-way ANOVA with Bonferroni posttest; statistical differences were represented by ** (P<0.01) or *** (P<0.001). Additionally, mouse footpads exposed to the different stimuli were stained with Masson's trichrome to highlight collagen fibers (green) (C and F). On panel are depicted representative histological image each group. Black horizontal bars represent a 50 µm scale. Histological structures like dermis (D) and the stratum corneum (SC) and stratum spinosum (SS) of the epidermis (ED) are indicated on the upper-left image. Inflammatory infiltrate is indicated by an asterisk.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003066-g006: Mycolactone induces a decrease in dermal collagen fibers.Mice were infected with virulent mycolactone-secreting (MU98912: dark gray triangles) (n = 5) or avirulent mycolactone-negative (MU5114: light gray squares) (n = 4) strains of M. ulcerans, or injected with PBS (vehicle: white circles) (n = 3) as a control (A, B and C). Alternatively, mice were inoculated with 5 µg of mycolactone (dark gray hexagons) (n = 4) or ethanol (vehicle: white diamonds) (n = 4) as a control (D, E, F). Lesion progression was assessed by measurement of footpad swelling (A and D). Collagen content was assessed by a qualitative blind scoring of the amount of dermal collagen fibers (score 0 = lowest, 4 = highest) visualized in HE-stained sections by polarized light (B and E). The different groups were compared to the control groups (PBS in A and B; ethanol in C and D) by Two-way ANOVA with Bonferroni posttest; statistical differences were represented by ** (P<0.01) or *** (P<0.001). Additionally, mouse footpads exposed to the different stimuli were stained with Masson's trichrome to highlight collagen fibers (green) (C and F). On panel are depicted representative histological image each group. Black horizontal bars represent a 50 µm scale. Histological structures like dermis (D) and the stratum corneum (SC) and stratum spinosum (SS) of the epidermis (ED) are indicated on the upper-left image. Inflammatory infiltrate is indicated by an asterisk.
Mentions: To investigate if mycolactone was responsible for a decrease in tissue collagen, an experimental model of BU disease, the mouse footpad infection with M. ulcerans, was used. Mice were challenged with virulent mycolactone-secreting (MU98912), avirulent mycolactone-negative (MU5114) strains of M. ulcerans or PBS as a control. Pathology progression was assessed by measuring footpad swelling (figure 6A) and, at different time-points, footpads were collected for histological processing and collagen scoring (figure 6B). The results showed that, in footpads infected with mycolactone-secreting M. ulcerans (MU98912), the progressive increase of pathology (figure 6A) was associated with a decrease of the collagen score (figure 6B), preceding the breakdown of the lesion into an ulcer (by day 40 post-infection). In contrast, infection with the mycolactone-negative strain (MU5114) did not induce pathology nor did it alter the collagen content in infected footpads, similar to what was observed for the PBS-injected control group (figure 6A and 6B). These results suggest that the decrease in collagen content is not a consequence of the infection or the elicited immune response, but rather caused by mycolactone. To verify this, mice were challenged with purified toxin or ethanol equivalent as control. The results showed that mycolactone induced footpad swelling associated with a decrease of the collagen score, while the vehicle did not (figure 6D and 6E). Histological samples stained with Masson's trichrome showed a decay of collagen fibers in MU98912-infected or mycolactone-treated footpads, characterized by the disorganization and thinness of collagen fibers (figure 6C and 6F).

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