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Title: Understanding the Significance of Biochemistry in the Storage, Handling, Purification, and Sampling of Amphiphilic Mycolactone

Abstract

Mycolactone, the amphiphilic macrolide toxin secreted by Mycobacterium ulcerans, plays a significant role in the pathology and manifestations of Buruli ulcer (BU). Consequently, it follows that the toxin is a suitable target for the development of diagnostics and therapeutics for this disease. Yet, several challenges have deterred such development. For one, the lipophilic nature of the toxin makes it difficult to handle and store and contributes to variability associated with laboratory experimentation and purification yields. In this manuscript, we have attempted to incorporate our understanding of the lipophilicity of mycolactone in order to define the optimal methods for the storage, handling, and purification of this toxin. We present a systematic correlation of variability associated with measurement techniques (thin-layer chromatography (TLC), mass spectrometry (MS), and UV-Vis spectrometry), storage conditions, choice of solvents, as well as the impact of each of these on toxin function as assessed by cellular cytotoxicity. We also compared natural mycolactone extracted from bacterial culture with synthesized toxins in laboratory (solvents, buffers) and physiologically relevant (serum) matrices. Our results point to the greater stability of mycolactone in organic, as well as detergent-containing, solvents, regardless of the container material (plastic, glass, or silanized tubes). They also highlight the presencemore » of toxin in samples that may be undetectable by any one technique, suggesting that each detection approach captures different configurations of the molecule with varying specificity and sensitivity. Most importantly, our results demonstrate for the very first time that amphiphilic mycolactone associates with host lipoproteins in serum, and that this association will likely impact our ability to study, diagnose, and treat Buruli ulcers in patients.« less

Authors:
ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [1]; ORCiD logo [1];  [2]; ORCiD logo [1];  [2];  [1]; ORCiD logo [1]
+ Show Author Affiliations
  1. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  2. Johns Hopkins Univ. Center for Tuberculosis Research, Baltimore, MD (United States)
Publication Date:
Research Org.:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
National Institutes of Health (NIH); USDOE
OSTI Identifier:
1511262
Report Number(s):
LA-UR-19-20386
Journal ID: ISSN 2072-6651; TOXIB7
Grant/Contract Number:  
89233218CNA000001
Resource Type:
Accepted Manuscript
Journal Name:
Toxins
Additional Journal Information:
Journal Volume: 11; Journal Issue: 4; Journal ID: ISSN 2072-6651
Publisher:
MDPI
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Biological Science; mycolactone; storage and handling; amphiphilic; lipoproteins

Citation Formats

Kubicek-Sutherland, Jessica Zofie, Vu, Dung M., Anderson, Aaron S., Sanchez, Timothy Charles, Converse, Paul J., Martí-Arbona, Ricardo, Nuermberger, Eric L., Swanson, Basil Ian, and Mukundan, Harshini. Understanding the Significance of Biochemistry in the Storage, Handling, Purification, and Sampling of Amphiphilic Mycolactone. United States: N. p., 2019. Web. doi:10.3390/toxins11040202.
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Kubicek-Sutherland, Jessica Zofie, Vu, Dung M., Anderson, Aaron S., Sanchez, Timothy Charles, Converse, Paul J., Martí-Arbona, Ricardo, Nuermberger, Eric L., Swanson, Basil Ian, & Mukundan, Harshini. Understanding the Significance of Biochemistry in the Storage, Handling, Purification, and Sampling of Amphiphilic Mycolactone. United States. https://doi.org/10.3390/toxins11040202
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Kubicek-Sutherland, Jessica Zofie, Vu, Dung M., Anderson, Aaron S., Sanchez, Timothy Charles, Converse, Paul J., Martí-Arbona, Ricardo, Nuermberger, Eric L., Swanson, Basil Ian, and Mukundan, Harshini. Thu . "Understanding the Significance of Biochemistry in the Storage, Handling, Purification, and Sampling of Amphiphilic Mycolactone". United States. https://doi.org/10.3390/toxins11040202. https://www.osti.gov/servlets/purl/1511262.
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@article{osti_1511262,
title = {Understanding the Significance of Biochemistry in the Storage, Handling, Purification, and Sampling of Amphiphilic Mycolactone},
author = {Kubicek-Sutherland, Jessica Zofie and Vu, Dung M. and Anderson, Aaron S. and Sanchez, Timothy Charles and Converse, Paul J. and Martí-Arbona, Ricardo and Nuermberger, Eric L. and Swanson, Basil Ian and Mukundan, Harshini},
abstractNote = {Mycolactone, the amphiphilic macrolide toxin secreted by Mycobacterium ulcerans, plays a significant role in the pathology and manifestations of Buruli ulcer (BU). Consequently, it follows that the toxin is a suitable target for the development of diagnostics and therapeutics for this disease. Yet, several challenges have deterred such development. For one, the lipophilic nature of the toxin makes it difficult to handle and store and contributes to variability associated with laboratory experimentation and purification yields. In this manuscript, we have attempted to incorporate our understanding of the lipophilicity of mycolactone in order to define the optimal methods for the storage, handling, and purification of this toxin. We present a systematic correlation of variability associated with measurement techniques (thin-layer chromatography (TLC), mass spectrometry (MS), and UV-Vis spectrometry), storage conditions, choice of solvents, as well as the impact of each of these on toxin function as assessed by cellular cytotoxicity. We also compared natural mycolactone extracted from bacterial culture with synthesized toxins in laboratory (solvents, buffers) and physiologically relevant (serum) matrices. Our results point to the greater stability of mycolactone in organic, as well as detergent-containing, solvents, regardless of the container material (plastic, glass, or silanized tubes). They also highlight the presence of toxin in samples that may be undetectable by any one technique, suggesting that each detection approach captures different configurations of the molecule with varying specificity and sensitivity. Most importantly, our results demonstrate for the very first time that amphiphilic mycolactone associates with host lipoproteins in serum, and that this association will likely impact our ability to study, diagnose, and treat Buruli ulcers in patients.},
doi = {10.3390/toxins11040202},
journal = {Toxins},
number = 4,
volume = 11,
place = {United States},
year = {2019},
month = {4}
}
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Journal Article:
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Publisher's Version of Record
https://doi.org/10.3390/toxins11040202
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Cited by: 3 works
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Figures / Tables:

Table 1 Table 1: Characterization of natural mycolactone A/B from Mycobacterium ulcerans lipid extracts.
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    Works referencing / citing this record:

    Could Mycolactone Inspire New Potent Analgesics? Perspectives and Pitfalls
    journal, September 2019

    • Reynaert, Marie-Line; Dupoiron, Denis; Yeramian, Edouard
    • Toxins, Vol. 11, Issue 9
    • DOI: 10.3390/toxins11090516
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