Mycolactone Disrupts Endothelial Glycocalyx and Basement Membrane Integrity: A Novel Mechanism of Tissue Necrosis in Buruli Ulcer Disease

Kernkonzepte

Mycolactone, the toxin produced by Mycobacterium ulcerans, causes Buruli ulcer disease by inhibiting Sec61, leading to the depletion of proteins crucial for endothelial glycocalyx and basement membrane integrity, ultimately contributing to tissue necrosis.

Zusammenfassung

• Bibliographic Information: Please note that the provided content lacks complete bibliographic information. For accurate citation, please refer to the original source.
• Research Objective: This study investigates the molecular mechanisms underlying mycolactone-induced endothelial cell dysfunction in the pathogenesis of Buruli ulcer disease.
• Methodology: The researchers used a combination of in vitro and in vivo approaches, including time-lapse imaging of primary human dermal microvascular endothelial cells (HDMECs) exposed to mycolactone, scratch assays to assess cell migration, immunostaining of mouse footpad tissue, total membrane proteomics analysis, flow cytometry, immunoblotting, and transmission electron microscopy.
• Key Findings:
  • Mycolactone induces morphological changes in endothelial cells, leading to cell elongation, rounding, and detachment.
  • These phenotypic changes are dependent on mycolactone's action at the Sec61 translocon, as structurally distinct Sec61 inhibitors phenocopied the observed effects.
  • Proteomic analysis revealed that mycolactone predominantly targets proteins involved in glycosylation, matrix organization, adhesion, and cell migration.
  • Mycolactone disrupts the endothelial glycocalyx by inhibiting the synthesis of glycosaminoglycans (GAGs) and depleting proteoglycans.
  • Knockdown of galactosyltransferase II (B3GALT6), a key enzyme in GAG biosynthesis, recapitulated the mycolactone-induced changes in cell morphology and monolayer permeability.
  • Mycolactone also depletes basement membrane components, including laminins, collagens, and nidogen 1, leading to basement membrane disruption observed both in vitro and in vivo.
  • Exogenous addition of laminin-511, a major basement membrane component, ameliorated mycolactone-driven cell detachment and impaired migration.
• Main Conclusions:
  • Mycolactone disrupts endothelial cell function by inhibiting Sec61-dependent protein translocation, leading to the depletion of proteins essential for glycocalyx and basement membrane integrity.
  • This disruption of the endothelial barrier contributes to the pathogenesis of Buruli ulcer disease by promoting vascular leakage, fibrin deposition, and tissue necrosis.
  • Targeting the mycolactone-induced endothelial dysfunction, potentially through supplementation of basement membrane components like laminin-511, represents a promising therapeutic avenue for improving wound healing in Buruli ulcer patients.
• Significance: This study provides novel insights into the pathogenesis of Buruli ulcer disease, highlighting the critical role of endothelial cell dysfunction in disease progression. The findings suggest potential therapeutic targets for improving wound healing and reducing the debilitating effects of this neglected tropical disease.
• Limitations and Future Research: Further research is needed to fully elucidate the downstream effects of mycolactone-induced endothelial dysfunction and to explore the therapeutic potential of targeting these pathways in vivo.

Statistiken

After 24 hours exposure to mycolactone, approximately half the cells (51.63±2.89%) had an elongated phenotype.
The average ratio of cell length to width doubled in 16 hours, and quadrupled after 24 hours exposure to mycolactone.
A small proportion (9.73±4.01%) of cells acquired a rounded appearance after 24 hours of mycolactone exposure.
Proteomic analysis identified 482 proteins significantly downregulated and 220 upregulated by mycolactone (> 2-Fold change, p < 0.05).
84.6% of the downregulated proteins were trafficked via the secretory/endolysosomal pathways that primarily depend on the Sec61 translocon.
CS fluorescence intensity was 60% lower in chondroitinase ABC-treated cells compared to untreated cells.
Mycolactone-exposed cells showed even lower CS fluorescence intensity than chondroitinase ABC-treated cells.
Surface HS expression was significantly reduced in mycolactone-exposed cells (14.11±7.40%) compared to DMSO solvent control (105.30±9.79%, p = 0.0002).
B3GALT6 siRNA-treated cells showed a comparable reduction in B3GALT6 protein expression to that caused by mycolactone (∼80%).
Mycolactone exposure increased HUVEC monolayer permeability to 23.13±7.38%, comparable to the effect of 100 ng/mL IL-1β (21.30±3.48%).
B3GALT6 knockdown in HUVECs also led to increased monolayer permeability (10.08±4.37% and 15.47±1.27% of the values seen in empty wells, p = 0.2371 and 0.0367, for two different oligonucleotides).
Perlecan and glypican-1 levels were significantly reduced in mycolactone-treated cells (10.8±4.8% and 28.8±9.0% of untreated control, respectively).
Biglycan levels were partly reduced (43.7±6.8% of untreated control) in mycolactone-treated cells.
A ∼50% reduction in perlecan was evident after only 2 hours of mycolactone treatment.
Integrin β1, integrin β4, and laminin α5 levels were reduced to 45.0±6.2%, 27.3±7.7%, and 15.6±5.4% of control levels, respectively, after 24 hours of mycolactone exposure.
Fibronectin levels decreased rapidly, showing >75% depletion after 4 hours of mycolactone exposure (p<0.01).
Integrin α5 levels decreased more slowly, reaching ∼50% of control levels at 24 hours of mycolactone exposure (p<0.01).

Zitate

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Wichtige Erkenntnisse aus

Mycolactone causes destructive Sec61-dependent loss of the endothelial glycocalyx and vessel basement membrane: a new indirect mechanism driving tissue necrosis in Mycobacterium ulcerans infection

by Hsieh,L. T.-... um www.biorxiv.org 02-21-2023

https://www.biorxiv.org/content/10.1101/2023.02.21.529382v2

Tiefere Fragen

How can the findings of this study be translated into effective therapies for Buruli ulcer, considering the challenges associated with drug delivery and the complexity of wound healing in vivo?

This study highlights the potential of targeting mycolactone-induced endothelial dysfunction as a therapeutic strategy for Buruli ulcer. However, translating these findings into effective therapies presents several challenges:
Drug Delivery:

Mycolactone Diffusion: Mycolactone is a lipid-soluble toxin that diffuses rapidly through tissues, making it difficult to neutralize with systemically administered drugs.

Potential Solutions:

Localized Drug Delivery: Direct injection of therapeutic agents into the lesion site could achieve higher local concentrations and minimize systemic side effects. This could involve sustained-release formulations or biocompatible implants.
Nanoparticle-Based Delivery: Encapsulating drugs in nanoparticles could improve their delivery to the target site and enhance their stability and penetration into affected tissues.
Complexity of Wound Healing:

Multifaceted Impairment: Mycolactone disrupts multiple aspects of wound healing, including angiogenesis (new blood vessel formation), ECM deposition, and immune cell recruitment.

Potential Solutions:

Combination Therapies: A multi-pronged approach targeting different aspects of wound healing might be necessary. This could involve combining:

Antimicrobials: To eliminate the source of mycolactone production.
Sec61 Modulators: To counteract the effects of residual mycolactone.
Pro-angiogenic Factors: To stimulate blood vessel growth.
ECM Components: To promote tissue regeneration (e.g., laminin-511 as demonstrated in the study).
Immunomodulators: To enhance the immune response and prevent secondary infections.

Chronic Inflammation: Prolonged mycolactone exposure can lead to chronic inflammation, further hindering wound healing.

Potential Solutions:

Anti-inflammatory Agents: Incorporating anti-inflammatory drugs, such as corticosteroids or specific cytokine inhibitors, could help dampen the inflammatory response and promote tissue repair.
Further Considerations:

Timing of Intervention: Early diagnosis and treatment are crucial to minimize tissue damage and improve wound healing outcomes.
Patient Variability: Treatment strategies may need to be tailored to individual patients based on factors such as lesion size, location, and immune status.

Could the disruption of the endothelial glycocalyx and basement membrane by mycolactone have implications for other aspects of Buruli ulcer pathogenesis, such as the impaired immune response and the development of chronic wounds?

Yes, the disruption of the endothelial glycocalyx and basement membrane by mycolactone likely contributes significantly to the impaired immune response and chronic wound development characteristic of Buruli ulcer:
Impaired Immune Response:

Reduced Leukocyte Adhesion and Transmigration: The glycocalyx and basement membrane are essential for leukocyte recruitment to sites of infection. Their disruption by mycolactone could:

Impair the rolling, adhesion, and transmigration of neutrophils and other immune cells across the endothelium, limiting their ability to reach the infected tissue.
Disrupt chemokine gradients, hindering the directed migration of immune cells towards the infection site.

Altered Cytokine Signaling: The glycocalyx can act as a reservoir for cytokines and growth factors involved in immune regulation. Its degradation could:

Lead to a dysregulated immune response, potentially contributing to the suppressed immune environment observed in Buruli ulcer lesions.
Chronic Wound Development:

Impaired Angiogenesis: The basement membrane provides structural support for blood vessels and regulates angiogenesis. Its disruption could:

Hinder the formation of new blood vessels, compromising nutrient and oxygen supply to the wound site and delaying healing.

Reduced ECM Deposition and Remodeling: The basement membrane serves as a scaffold for the deposition and organization of other ECM components. Its disruption could:

Impair the formation of a stable and functional extracellular matrix, hindering tissue regeneration and contributing to the development of chronic, non-healing wounds.
Overall, the disruption of the endothelial glycocalyx and basement membrane by mycolactone likely creates a vicious cycle in Buruli ulcer pathogenesis, where impaired immune responses and compromised wound healing processes exacerbate each other, leading to chronic infection and tissue destruction.

If mycolactone's impact on the endothelium is a common mechanism of action for other bacterial toxins, what broader implications might this have for understanding and treating infectious diseases?

While mycolactone's specific mechanism of action through Sec61 inhibition is unique, the broader concept of bacterial toxins targeting the endothelium and disrupting its function is relevant to many infectious diseases. This understanding has significant implications for both research and treatment:
Understanding Infectious Disease Pathogenesis:

Endothelial Dysfunction as a Common Theme: Recognizing endothelial dysfunction as a central theme in various infections could lead to:

New Diagnostic Markers: Identifying specific markers of endothelial damage could aid in early diagnosis and disease monitoring.
Unveiling Novel Therapeutic Targets: Targeting common pathways of endothelial dysfunction could lead to broad-spectrum therapies effective against multiple pathogens.

Beyond Direct Cytotoxicity: Shifting the focus from direct pathogen-mediated cytotoxicity to the role of host responses, particularly endothelial dysfunction, could:

Provide a more comprehensive understanding of disease progression and complications.
Identify new avenues for therapeutic intervention that modulate host responses rather than directly targeting the pathogen.
Treating Infectious Diseases:

Host-Directed Therapies: Developing therapies that protect or restore endothelial function could:

Complement traditional antimicrobial approaches.
Improve treatment outcomes for infections where antibiotic resistance is a concern.
Mitigate long-term complications associated with endothelial damage, such as sepsis, organ failure, and chronic inflammation.

Repurposing Existing Drugs: Drugs already approved for treating endothelial dysfunction in other contexts (e.g., cardiovascular disease) could be repurposed for infectious diseases, potentially accelerating the development of new treatments.
Examples of other bacterial toxins that target the endothelium:

Shiga toxin (produced by Shigella dysenteriae and enterohemorrhagic Escherichia coli)
Anthrax toxin (produced by Bacillus anthracis)
Streptococcal pyrogenic exotoxins (produced by Streptococcus pyogenes)
In conclusion, recognizing the widespread impact of bacterial toxins on endothelial function opens up new avenues for understanding and treating infectious diseases. By targeting the host response, particularly endothelial dysfunction, we can potentially develop more effective therapies that address both the immediate infection and its long-term consequences.