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

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.

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Heterogeneity of Mycolactones Produced by Clinical Isolates of Mycobacterium ulcerans: Implications for Virulence

Infection and Immunity ◽

10.1128/iai.71.2.774-783.2003 ◽

2003 ◽

Vol 71 (2) ◽

pp. 774-783 ◽

Cited By ~ 123

Author(s):

Armand Mve-Obiang ◽

Richard E. Lee ◽

Françoise Portaels ◽

P. L. C. Small

Keyword(s):

Clinical Evidence ◽

Buruli Ulcer ◽

Mycobacterium Ulcerans ◽

Biologically Active ◽

In Vivo Studies ◽

Tissue Destruction ◽

The Core ◽

A Minor ◽

Layer Chromatography

ABSTRACT Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe necrotizing skin disease endemic in tropical countries. Clinical evidence suggests that M. ulcerans isolates from Asia, Mexico, and Australia may be less virulent than isolates from Africa. In vivo studies suggest that mycolactone, a polyketide-derived macrolide toxin, plays a major role in the tissue destruction and immune suppression which occur in cases of Buruli ulcer. Mycolactones were extracted from 34 isolates of M. ulcerans representing strains from Africa, Malaysia, Asia, Australia, and Mexico. Thin-layer chromatography, mass spectroscopic analysis, and cytopathic assays of partially purified mycolactones from these isolates revealed that M. ulcerans produces a heterogeneous mixture of mycolactone variants. Mycolactone A/B, the most biologically active mycolactone species, was identified by mass spectroscopy as [M+Na]+ at m/z 765.5 in all cytotoxic isolates except for those from Mexico. Mycolactone C [M+Na]+ at m/z 726.3 was the dominant mycolactone species in eight Australian isolates, and mycolactone D [M+Na]+ m/z 781.2 was characteristic of two Asian strains. Mycolactone species are conserved within specific geographic areas, suggesting that there may be a correlation between mycolactone profile and virulence. In addition, the core lactone, [M+Na]+ m/z 447.4, was identified as a minor species, supporting the hypothesis that mycolactones are synthesized by two polyketide synthases. A cytopathic assay of the core lactone showed that this molecule is sufficient for cytotoxicity, although it is much less potent than the complete mycolactone.

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Experimental pieces of evidence for Mycobacterium ulcerans dormancy

10.1101/2020.08.27.269712 ◽

2020 ◽

Author(s):

A. Loukil ◽

R. Lalaoui ◽

H. Bogreau ◽

S. Regoui ◽

M. Drancourt ◽

...

Keyword(s):

Physiological State ◽

Nile Red ◽

Buruli Ulcer ◽

Ambient Air ◽

Opportunistic Pathogen ◽

Oxygen Depletion ◽

Mycobacterium Ulcerans ◽

Ecological Niches ◽

Intracellular Lipid ◽

First Time

ABSTRACTBackgroundWhether Mycobacterium ulcerans, the etiological agent of the neglected Buruli ulcer in numerous tropical countries, would exist in a dormant state as reported for closely related Mycobacterium species, is not established.MethodologySix M. ulcerans strains were exposed to a progressive depletion in oxygen for two months, using a previously described Wayne model of dormancy; and further examined by microscopy using DDD staining, microcalorimetry and subculture in the presence of dead and replicative M. ulcerans as controls.Principal Findings/ConclusionsM. ulcerans CU001 strain died during the progressive oxygen depletion and four of five remaining strains exhibited Nile Red-stained intracellular lipid droplets after DDD staining and a 14-20-day regrowth when exposed to ambient air, diagnosing dormancy. A fifth M. ulcerans 19423 strain stained negative in DDD and slowly regrew in 27 days. Three tested M. ulcerans strains yielded microcalorimetric pattern similar to that of the negative (dead) homologous controls, differing from that of the homologous positive (replicative) controls. The relevance of these experimental observations, suggesting a previously unreported dormancy state of M. ulcerans, needs to be investigated in the natural ecological niches where M. ulcerans thrive and in Buruli ulcer lesions.Author summaryMycobacterium ulcerans is an environmental opportunistic pathogen of mammals and humans, causing a subcutaneous necrotizing infection named Buruli ulcer. Molecular detection of M. ulcerans DNA revealed different ecological niches where M. ulcerans may thrive, but the molecular biology approach does not catch the physiological state of M. ulcerans in these different ecological niches. Thus, the reservoir and the mode of transmission of M. ulcerans remain elusive. Here, we investigated experimental dormancy of M. ulcerans by using a previously described Wayne model of dormancy coupled with microscopy using DDD staining, microcalorimetry and subculture. Our findings demonstrate for the first time that some M. ulcerans strains exhibit a physiological state of dormancy; potentially limiting isolation and culture of M. ulcerans from environmental niches.

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Analysis of Mycobacterium Species for the Presence of a Macrolide Toxin, Mycolactone

Infection and Immunity ◽

10.1128/iai.72.1.123-132.2004 ◽

2004 ◽

Vol 72 (1) ◽

pp. 123-132 ◽

Cited By ~ 14

Author(s):

Alexa K. Daniel ◽

Richard E. Lee ◽

Francoise Portaels ◽

P. L. C. Small

Keyword(s):

Cultured Cells ◽

Cell Envelope ◽

Buruli Ulcer ◽

Fibroblast Cell ◽

Mycobacterium Ulcerans ◽

Mycobacterial Species ◽

Murine Fibroblast ◽

A Cell ◽

Layer Chromatography ◽

Slow Growing

ABSTRACT Mycobacterium ulcerans is an environmental organism which is responsible for the disease Buruli ulcer, a necrotizing skin disease emerging in west Africa. M. ulcerans produces the polyketide-derived macrolide mycolactone, which is required for the immunosuppression and tissue damage which characterizes Buruli ulcer. We have extracted lipids from the cell envelope and culture filtrate from 52 isolates of Mycobacterium species, analyzed them with thin-layer chromatography, and tested them in a murine fibroblast cell line (L929) cytotoxicity assay to investigate whether these mycobacterial species produce mycolactone. For these studies chloroform-methanol (2:1, vol/vol) extracts were prepared from representative fast- and slow-growing mycobacterial species. Isolates tested included 16 uncharacterized, slow-growing, environmental mycobacterial species isolated from areas in which M. ulcerans infection is endemic. Although several strains of mycobacteria studied produced cytopathic lipids, none of these produced a phenotype on cultured cells consistent with that produced by mycolactone. Two mycobacterial species, M. scrofulaceum and M. kansasii, and eight of the environmental mycobacterial isolates contained cell-associated lipids cytopathic to fibroblasts at concentrations of 33 to 1,000 μg/ml. In contrast, mycolactone produces cytotoxicity at less than 2 ng/ml. Analysis of 16S rRNA sequences from the eight environmental isolates suggests that these are novel mycobacterial species. Results from these studies suggest that, although production of cytopathic lipids is relatively common among mycobacterial species, the production of mycolactone as a cell-associated or secreted molecule appears so far to be restricted to M. ulcerans.

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Mycobacterium ulcerans Population Genomics To Inform on the Spread of Buruli Ulcer across Central Africa

mSphere ◽

10.1128/msphere.00472-18 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 6

Author(s):

Koen Vandelannoote ◽

Delphin Mavinga Phanzu ◽

Kapay Kibadi ◽

Miriam Eddyani ◽

Conor J. Meehan ◽

...

Keyword(s):

Disease Control ◽

Population Genomics ◽

Demographic History ◽

Buruli Ulcer ◽

Central Africa ◽

Mycobacterium Ulcerans ◽

Evolutionary Trajectory ◽

Content Type ◽

Mode Of Transmission ◽

The Impact

ABSTRACT Buruli ulcer is a neglected tropical disease of skin and subcutaneous tissue caused by infection with the pathogen Mycobacterium ulcerans. Many critical issues for disease control, such as understanding the mode of transmission and identifying source reservoirs of M. ulcerans, are still largely unknown. Here, we used genomics to reconstruct in detail the evolutionary trajectory and dynamics of M. ulcerans populations at a central African scale and at smaller geographical village scales. Whole-genome sequencing (WGS) data were analyzed from 179 M. ulcerans strains isolated from all Buruli ulcer foci in the Democratic Republic of the Congo, The Republic of Congo, and Angola that have ever yielded positive M. ulcerans cultures. We used both temporal associations and the study of the mycobacterial demographic history to estimate the contribution of humans as a reservoir in Buruli ulcer transmission. Our phylogeographic analysis revealed one almost exclusively predominant sublineage of M. ulcerans that arose in Central Africa and proliferated in its different regions of endemicity during the Age of Discovery. We observed how the best sampled endemic hot spot, the Songololo territory, became an area of endemicity while the region was being colonized by Belgium (1880s). We furthermore identified temporal parallels between the observed past population fluxes of M. ulcerans from the Songololo territory and the timing of health policy changes toward control of the Buruli ulcer epidemic in that region. These findings suggest that an intervention based on detecting and treating human cases in an area of endemicity might be sufficient to break disease transmission chains, irrespective of other reservoirs of the bacterium. IMPORTANCE Buruli ulcer is a destructive skin and soft tissue infection caused by Mycobacterium ulcerans. The disease is characterized by progressive skin ulceration, which can lead to permanent disfigurement and long-term disability. Currently, the major hurdles facing disease control are incomplete understandings of both the mode of transmission and environmental reservoirs of M. ulcerans. As decades of spasmodic environmental sampling surveys have not brought us much closer to overcoming these hurdles, the Buruli ulcer research community has recently switched to using comparative genomics. The significance of our research is in how we used both temporal associations and the study of the mycobacterial demographic history to estimate the contribution of humans as a reservoir in Buruli ulcer transmission. Our approach shows that it might be possible to use bacterial population genomics to assess the impact of health interventions, providing valuable feedback for managers of disease control programs in areas where health surveillance infrastructure is poor.

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Pathogenicity and Host Susceptibility of Penicillium spp. on Citrus

Plant Disease ◽

10.1094/pdis-02-14-0122-re ◽

2015 ◽

Vol 99 (1) ◽

pp. 21-30 ◽

Cited By ~ 13

Author(s):

Johannes Petrus Louw ◽

Lise Korsten

Keyword(s):

Citrus Fruit ◽

Storage Conditions ◽

Tissue Response ◽

Host Susceptibility ◽

The Impact ◽

First Time ◽

And Storage ◽

Fruit Pathogens ◽

Penicillium Spp ◽

Citrus Spp

Citrus fruit are exposed to numerous postharvest pathogens throughout the fresh produce supply chain. Well-known postharvest citrus fruit pathogens are Penicillium digitatum and P. italicum. Lesser-known pathogens include P. crustosum and P. expansum. This study examined pathogenicity and aggressiveness of Penicillium spp. present in fresh fruit supply chains on various Citrus spp. and cultivars. The impact of different inoculation methods and storage conditions on decay were also assessed. P. digitatum and P. italicum were the most aggressive Penicillium spp. on citrus but aggressiveness varied significantly over the evaluated citrus range. Decay and tissue-response lesions caused by P. crustosum were observed on ‘Nules Clementine’, ‘Nova’, ‘Owari Satsuma’, ‘Delta Valencia’, ‘Cambria Navel’, ‘Eureka’ seeded, and ‘Star Ruby’ for the first time. Likewise, these lesions caused by P. expansum were noted on Nules Clementine, Owari Satsuma, Delta Valencia, ‘Midknight Valencia’, and Eureka seeded for the first time. Tissue-response lesions affect fruit quality and some Penicillium spp. sporulated from the lesions, causing the inoculated species to complete their life cycle. New citrus–Penicillium spp. interactions were observed and the importance of monitoring inoculum loads of pathogens and nonhost pathogens were highlighted.

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Low-Temperature Storage Improves the Over-Time Stability of Implantable Glucose and Lactate Biosensors

Sensors ◽

10.3390/s19020422 ◽

2019 ◽

Vol 19 (2) ◽

pp. 422 ◽

Cited By ~ 7

Author(s):

Giulia Puggioni ◽

Giammario Calia ◽

Paola Arrigo ◽

Andrea Bacciu ◽

Gianfranco Bazzu ◽

...

Keyword(s):

Storage Conditions ◽

Time Stability ◽

Low Temperature Storage ◽

Specificity And Sensitivity ◽

Short Period ◽

Analytical Parameter ◽

The Impact ◽

And Storage ◽

Over Time ◽

Temperature Storage

Molecular biomarkers are very important in biology, biotechnology and even in medicine, but it is quite hard to convert biology-related signals into measurable data. For this purpose, amperometric biosensors have proven to be particularly suitable because of their specificity and sensitivity. The operation and shelf stability of the biosensor are quite important features, and storage procedures therefore play an important role in preserving the performance of the biosensors. In the present study two different designs for both glucose and lactate biosensor, differing only in regards to the containment net, represented by polyurethane or glutharaldehyde, were studied under different storage conditions (+4, −20 and −80 °C) and monitored over a period of 120 days, in order to evaluate the variations of kinetic parameters, as VMAX and KM, and LRS as the analytical parameter. Surprisingly, the storage at −80 °C yielded the best results because of an unexpected and, most of all, long-lasting increase of VMAX and LRS, denoting an interesting improvement in enzyme performances and stability over time. The present study aimed to also evaluate the impact of a short-period storage in dry ice on biosensor performances, in order to simulate a hypothetical preparation-conservation-shipment condition.

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Bactericidal Activity of Rifampin-Amikacin against Mycobacterium ulcerans in Mice

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.10.3193-3196.2002 ◽

2002 ◽

Vol 46 (10) ◽

pp. 3193-3196 ◽

Cited By ~ 65

Author(s):

Herve Dega ◽

Abdelhalim Bentoucha ◽

Jerome Robert ◽

Vincent Jarlier ◽

Jacques Grosset

Keyword(s):

Clinical Trial ◽

Bactericidal Activity ◽

Buruli Ulcer ◽

Acid Fast Bacillus ◽

Drug Dosage ◽

Mycobacterium Ulcerans ◽

World Health ◽

Health Organization ◽

The Impact ◽

Lesion Index

ABSTRACT To identify the most active curative treatment of Buruli ulcer, two regimens incorporating the use of rifampin (RIF) were compared with the use of RIF alone in a mouse footpad model of Mycobacterium ulcerans infection. Treatments began after footpad swelling from infection and continued for 12 weeks with five doses weekly of one of the following regimens: (i) 10 mg of RIF/kg alone; (ii) 10 mg of RIF/kg and 100 mg of amikacin (AMK)/kg; and (iii) 10 mg of RIF/kg, 100 mg of clarithromycin (CLR)/kg, and 50 mg of sparfloxacin (SPX)/kg. The activity of each regimen was assessed in terms of the reduction of the average lesion index and acid-fast bacillus (AFB) and CFU counts. All three regimens displayed various degrees of bactericidal activity against M. ulcerans. The ranking of bactericidal activity was found to be as follows: RIF-AMK > RIF-CLR-SPX > RIF. RIF-AMK was able to cure M. ulcerans-infected mice and prevent relapse 26 weeks after completion of treatment. To determine the impact of different rhythms of administration of RIF-AMK on the suppression of M.ulcerans growth, mice were given the RIF-AMK combination for 4 weeks but doses were administered either 5 days a week or twice or once weekly. After completion of treatment, the mice were kept under supervision for 30 additional weeks. M. ulcerans was considered to have grown in the footpad if swelling was visually observed and harvests contained more than 5 × 105 AFB per footpad. The proportion of mice in which growth of M. ulcerans occurred, irrespective of drug dosage, was compared with the control mice to determine the proportion of M. ulcerans killed. Each dosage of RIF-AMK was bactericidal for M. ulcerans (P < 0.001), but the effect was significantly stronger in mice treated 5 days per week. The promising results of RIF-AMK treatment in M. ulcerans-infected mice support the clinical trial that is currently in progress under World Health Organization auspices in Ghana.

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Buruli Ulcer, a Prototype for Ecosystem-Related Infection, Caused byMycobacterium ulcerans

Clinical Microbiology Reviews ◽

10.1128/cmr.00045-17 ◽

2017 ◽

Vol 31 (1) ◽

Cited By ~ 12

Author(s):

Dezemon Zingue ◽

Amar Bouam ◽

Roger B. D. Tian ◽

Michel Drancourt

Keyword(s):

Point Of Care ◽

Buruli Ulcer ◽

Mycobacterium Ulcerans ◽

Content Type ◽

Laboratory Confirmation ◽

Route Of Transmission ◽

Skin Contamination ◽

Insect Bites ◽

The Impact ◽

Specific Sequences

SUMMARYBuruli ulcer is a noncontagious disabling cutaneous and subcutaneous mycobacteriosis reported by 33 countries in Africa, Asia, Oceania, and South America. The causative agent,Mycobacterium ulcerans, derives fromMycobacterium marinumby genomic reduction and acquisition of a plasmid-borne, nonribosomal cytotoxin mycolactone, the major virulence factor.M. ulcerans-specific sequences have been readily detected in aquatic environments in food chains involving small mammals. Skin contamination combined with any type of puncture, including insect bites, is the most plausible route of transmission, and skin temperature of <30°C significantly correlates with the topography of lesions. After 30 years of emergence and increasing prevalence between 1970 and 2010, mainly in Africa, factors related to ongoing decreasing prevalence in the same countries remain unexplained. Rapid diagnosis, including laboratory confirmation at the point of care, is mandatory in order to reduce delays in effective treatment. Parenteral and potentially toxic streptomycin-rifampin is to be replaced by oral clarithromycin or fluoroquinolone combined with rifampin. In the absence of proven effective primary prevention, avoiding skin contamination by means of clothing can be implemented in areas of endemicity. Buruli ulcer is a prototype of ecosystem pathology, illustrating the impact of human activities on the environment as a source for emerging tropical infectious diseases.

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Ketogenic diet impairs Mycobacterium ulcerans growth and toxin production, enhancing hosts’ response to the infection in an experimental mouse model

The Journal of Infectious Diseases ◽

10.1093/infdis/jiab236 ◽

2021 ◽

Author(s):

Mélanie Foulon ◽

Marie Robbe-Saule ◽

Lucille Esnault ◽

Marine Malloci ◽

Anthony Mery ◽

...

Keyword(s):

Mouse Model ◽

Ketogenic Diet ◽

Buruli Ulcer ◽

Mycobacterium Ulcerans ◽

World Health ◽

Ketogenic Diets ◽

Experimental Mouse Model ◽

Experimental Mouse ◽

The Impact

Abstract Ketogenic diets have been used to treat diverse conditions, and there is growing evidence of their benefits for tissue repair and in inflammatory disease treatment. However, their role in infectious diseases has been little studied. Buruli ulcer (Mycobacterium ulcerans infection) is a chronic infectious disease characterized by large skin ulcerations caused by mycolactone, the major virulence factor of the bacillus. Here, we investigated the impact of ketogenic diet on this cutaneous disease in an experimental mouse model. This diet prevented ulceration, by modulating bacterial growth and host inflammatory response. β-hydroxybutyrate, the major ketone body produced during ketogenic diet and diffusing in tissues, impeded M. ulcerans growth and mycolactone production in vitro underlying its potential key role in infection. These results pave the way for the development of new patient management strategies involving shorter courses of treatment and improving wound healing, in line with the major objectives of the World Health Organization.

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Pharmacognostic studies of Morinda brevipes S. Y. Hu

Bangladesh Journal of Botany ◽

10.3329/bjb.v48i3.47718 ◽

2019 ◽

Vol 48 (3) ◽

pp. 401-407

Author(s):

Rong Li ◽

Xiaojing Lin ◽

Genqiu Tang ◽

Junni Li ◽

Dong Wang ◽

...

Keyword(s):

Calcium Oxalate ◽

Transverse Section ◽

Volatile Oils ◽

Needle Crystal ◽

Cork Cell ◽

Registration Number ◽

Phytochemical Studies ◽

First Time ◽

Layer Chromatography ◽

Inequality Type

DNA barcoding of ITS and psbA-trnH regions, histochemistry as well as thin layer chromatography (TLC) of Morinda brevipes S.Y. Hu were analyzed. Transverse section of root revealed the presence of cortex, xylem, cork cell, stone cells, and calcium oxalate sandy crystal. The lower epidermis cells showed many stoma in paracytic or inequality type. Spiral vessel and tiny calcium oxalate needle crystal usually appeared in the powder. TLC showed the presence of emodin in M. brevipes. Phytochemical studies revealed the existence of carbohydrates, saponins, tannins, flavones, anthraquinones, alkaloids and volatile oils. The ITS and psbAtrnH sequences were found for the first time which were submitted to NCBI to obtain the GenBank registration number. This study might play an important role in the identification, and utilization of M. brevipes for various purposes.

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