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Caroline Demangel - One of the best experts on this subject based on the ideXlab platform.
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recombinant antibodies against Mycolactone
Toxins, 2019Co-Authors: Leslie Naranjo, Caroline Demangel, Nicolas Blanchard, Fortunato Ferrara, Sara Dangelo, Frank M Erasmus, Andre A Teixera, Andrew BradburyAbstract:: In the past, it has proved challenging to generate antibodies against Mycolactone, the primary lipidic toxin A of Mycobacterium ulcerans causing Buruli ulcer, due to its immunosuppressive properties. Here we show that in vitro display, comprising both phage and yeast display, can be used to select antibodies recognizing Mycolactone from a large human naive phage antibody library. Ten different antibodies were isolated, and hundreds more identified by next generation sequencing. These results indicate the value of in vitro display methods to generate antibodies against difficult antigenic targets such as toxins, which cannot be used for immunization unless inactivated by structural modification. The possibility to easily generate anti-Mycolactone antibodies is an exciting prospect for the development of rapid and simple diagnostic/detection methods.
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Mycolactone more than just a cytotoxin
2019Co-Authors: Laure Gueninmace, Gerd Pluschke, Caroline DemangelAbstract:From their observation of necrotic areas around bacterial foci in Buruli ulcers (BUs), Connor and co-workers were the first, back in 1965, to suggest that M. ulcerans may produce a diffusible cytotoxin [1]. This hypothesis was later confirmed by injecting mycobacterial culture filtrates into the skin of guinea pigs, showing that this causes focal necrosis resembling that of naturally occurring human infections [1–3]. In 1999, George et al. succeeded in isolating a cytotoxic factor from M. ulcerans lipid extracts, and deciphered its chemical nature [4]. The M. ulcerans toxin was named Mycolactone, based on its mycobacterial origin and macrolactone structure: a 12-membered lactone ring, to which a C5-O-linked polyunsaturated acyl side chain and a C-linked upper side chain comprising C12–C20 are appended (Fig. 1). Follow-up studies showed that M. ulcerans-derived Mycolactone was in reality a mixture of two stereoisomers called A and B [6, 7] (Fig. 1). Since the initial discovery of Mycolactone A/B, eight additional Mycolactone congeners have been identified that are either produced by M. ulcerans strains of different geographical origins or genetically related fish and frog pathogens, which were initially given different species designations such as M. pseudoshottsii and M. liflandii. Comparative genome analysis later revealed that all Mycolactone-producing mycobacteria evolved from a common M. marinum-like progenitor, and are therefore ecovars of a single M. ulcerans species [8]. While the macrolide core structure and upper side chain are conserved, Mycolactone populations from different M. ulcerans sub-lineages vary in the length, number and localization of hydroxyl groups and number of double bonds of the lower side chain. These modifications of the lower polyunsaturated acyl side chain cause pronounced changes in cytotoxicity [9–11]. The origin and chemistry of natural Mycolactones, structure-activity relationships and the various approaches that were developed to generate synthetic Mycolactones have been reviewed recently [12, 13]. These aspects will therefore not be further discussed here. Instead, this chapter provides an update on our understanding of Mycolactone (A/B) biology, and discusses its proposed mechanisms of action in relation with BU pathogenesis.
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sec61 blockade by Mycolactone a central mechanism in buruli ulcer disease
Biology of the Cell, 2018Co-Authors: Caroline Demangel, Stephen HighAbstract:: Infection with Mycobacterium ulcerans results in a necrotising skin disease known as a Buruli ulcer, the pathology of which is directly linked to the bacterial production of the toxin Mycolactone. Recent studies have identified the protein translocation machinery of the endoplasmic reticulum (ER) membrane as the primary cellular target of Mycolactone, and shown that the toxin binds to the core subunit of the Sec61 complex. Mycolactone binding strongly inhibits the capacity of the Sec61 translocon to transport newly synthesised membrane and secretory proteins into and across the ER membrane. Since the ER acts as the entry point for the mammalian secretory pathway, and hence regulates initial access to the entire endomembrane system, Mycolactone-treated cells have a reduced ability to produce a range of proteins including secretory cytokines and plasma membrane receptors. The global effect of this molecular blockade of protein translocation at the ER is that the host is unable to mount an effective immune response to the underlying mycobacterial infection. Prolonged exposure to Mycolactone is normally cytotoxic, since it triggers stress responses activating the transcription factor ATF4 and ultimately inducing apoptosis.
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proteomics reveals scope of Mycolactone mediated sec61 blockade and distinctive stress signature
Molecular & Cellular Proteomics, 2018Co-Authors: Jeandavid Morel, Laure Gueninmace, Anja O Paatero, Francis Impens, Jonathan W Yewdell, Delphi Van Haver, Natalia Pietrosemoli, Ville O Paavilainen, Caroline DemangelAbstract:Mycolactone is a bacteria-derived macrolide that blocks the biogenesis of a large array of secretory and integral transmembrane proteins (TMP) through potent inhibition of the Sec61 translocon. Here, we used quantitative proteomics to delineate the direct and indirect effects of Mycolactone-mediated Sec61 blockade in living cells. In T lymphocytes, dendritic cells and sensory neurons, Sec61 substrates downregulated by Mycolactone were in order of incidence: secretory proteins (with a signal peptide but no transmembrane domain), TMPs with a signal peptide (Type I) and TMPs without signal peptide and a cytosolic N terminus (Type II). TMPs without a signal peptide and the opposite N terminus topology (Type III) were refractory to Mycolactone inhibition. This rule applied comparably to single- and multi-pass TMPs, and extended to exogenous viral proteins. Parallel to its broad-spectrum inhibition of Sec61-mediated protein translocation, Mycolactone rapidly induced cytosolic chaperones Hsp70/Hsp90. Moreover, it activated an atypical endoplasmic reticulum stress response, differing from conventional unfolded protein response by the down-regulation of Bip. In addition to refining our mechanistic understanding of Sec61 inhibition by Mycolactone, our findings thus reveal that Sec61 blockade induces proteostatic stress in the cytosol and the endoplasmic reticulum.
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Mycolactone displays anti inflammatory effects on the nervous system
PLOS Neglected Tropical Diseases, 2017Co-Authors: Caroline Demangel, Caroline Isaac, A Mauborgne, Alfonso Grimaldi, M Pohl, Cristina Limatola, Yves BoucherAbstract:Background Mycolactone is a macrolide produced by the skin pathogen Mycobacterium ulcerans, with cytotoxic, analgesic and immunomodulatory properties. The latter were recently shown to result from Mycolactone blocking the Sec61-dependent production of pro-inflammatory mediators by immune cells. Here we investigated whether Mycolactone similarly affects the inflammatory responses of the nervous cell subsets involved in pain perception, transmission and maintenance. We also investigated the effects of Mycolactone on the neuroinflammation that is associated with chronic pain in vivo. Methodology/ Principle findings Sensory neurons, Schwann cells and microglia were isolated from mice for ex vivo assessment of Mycolactone cytotoxicity and immunomodulatory activity by measuring the production of proalgesic cytokines and chemokines. In all cell types studied, prolonged (>48h) exposure to Mycolactone induced significant cell death at concentrations >10 ng/ml. Within the first 24h treatment, nanomolar concentrations of Mycolactone efficiently suppressed the cell production of pro-inflammatory mediators, without affecting their viability. Notably, Mycolactone also prevented the pro-inflammatory polarization of cortical microglia. Since these cells critically contribute to neuroinflammation, we next tested if Mycolactone impacts this pathogenic process in vivo. We used a rat model of neuropathic pain induced by chronic constriction of the sciatic nerve. Here, Mycolactone was injected daily for 3 days in the spinal canal, to ensure its proper delivery to spinal cord. While this treatment failed to prevent injury-induced neuroinflammation, it decreased significantly the local production of inflammatory cytokines without inducing detectable cytotoxicity. Conclusion/ Significance The present study provides in vitro and in vivo evidence that Mycolactone suppresses the inflammatory responses of sensory neurons, Schwann cells and microglia, without affecting the cell viability. Together with previous studies using peripheral blood leukocytes, our work implies that Mycolactone-mediated analgesia may, at least partially, be explained by its anti-inflammatory properties.
Pamela L C Small - One of the best experts on this subject based on the ideXlab platform.
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mycobacterium ulcerans toxin Mycolactone may enhance host seeking and oviposition behaviour by aedes aegypti l diptera culicidae
Environmental Microbiology, 2017Co-Authors: M L Sanders, Pamela L C Small, Heather R Jordan, C Serewispond, Longyu Zheng, Mark Eric Benbow, Jeffery K TomberlinAbstract:Summary The ecological functions of many toxins continue to remain unknown for those produced by environmental pathogens. Mycobacterium ulcerans, the causative agent of the neglected tropical disease, Buruli ulcer, produces a cytotoxic macrolide, Mycolactone, whose function(s) in the environment remains elusive. Through a series of dual-choice behaviour assays, they show that Mycolactone may be an interkingdom cue for the yellow fever mosquito, Aedes aegypti, seeking blood-meals as well as oviposition sites. Results provide novel insight into the evolution between bacteria and potential vectors. While further studies are needed to determine if Mycolactone is an actual signal rather than simply a cue, this discovery could serve as a model for determining roles for toxins produced by other environmental pathogens and provide opportunities for developing novel strategies for disease prevention. The relationship between M. ulcerans, Mycolactone, and Ae. aegypti further suggests there could be an amplification effect for the spread of pathogens responsible for other diseases, such as yellow fever and dengue.
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a mycobacterium ulcerans toxin Mycolactone induces apoptosis in primary human keratinocytes and in hacat cells
Microbes and Infection, 2010Co-Authors: Chiarella Bozzo, Pamela L C Small, Francesca Graziola, Rossana Tiberio, Ginevra Pertusi, Guido Valente, Enrico Colombo, Giorgio LeighebAbstract:The pathogenicity of Mycobacterium ulcerans (Buruli ulcer) depends on cytotoxic effect of its exotoxin Mycolactone. Since epidermis represents a barrier against infectious agents and balanced apoptosis is essential in epidermal homeostasis, we explored if Mycolactone A/B induces apoptosis on two human keratinocyte populations, stem cells (KSC) and transit amplifying cells (TAC), and on human keratinocyte line, HaCaT. Treatment of TAC with 1 and 10 ng/ml Mycolactone-induced 60 and 90% apoptosis. KSC were more resistant than TAC: 50 and 75% of cells underwent apoptosis after 10 and 100 ng/ml toxin-treatment. Higher doses (1000 ng/ml) induced about 30% apoptosis on HaCaT. In contrast, Mycolactone A/B was devoid of toxicity neither on human hepatoma HuH7 nor on human embryonic kidney HEK 293 T cell lines. In conclusion, Mycolactone induces apoptosis in human keratinocytes, thus contributing to Buruli ulcer lesions development.
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antioxidants protect keratinocytes against m ulcerans Mycolactone cytotoxicity
PLOS ONE, 2010Co-Authors: Alvar Gronberg, Pamela L C Small, Louise Zettergren, Kerstin Bergh, Mona Stahle, Johan Heilborn, Kristian Angeby, Hannah AkuffoAbstract:BACKGROUND: Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, Mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of Mycolactone on keratinocytes has not been reported previously and the mechanism of Mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS). We have studied the effect of Mycolactone in vitro on human keratinocytes--key cells in wound healing--and tested the hypothesis that the cytotoxic effect of Mycolactone is mediated by ROS. METHODOLOGY/PRINCIPAL FINDINGS: The effect of Mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to Mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe(2+), completely prevented Mycolactone mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that Mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in Mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease.
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heterogeneity in the stereochemistry of Mycolactones isolated from m marinum toxins produced by fresh vs saltwater fish pathogens
Chemical Communications, 2009Co-Authors: Katrina L Jackson, Yoshito Kishi, Heather Williamson, Lydia Mosi, Pamela L C SmallAbstract:A novel Mycolactone has been identified from Mycobacterium marinum infecting freshwater fish.
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Mycolactone inhibits monocyte cytokine production by a posttranscriptional mechanism
Journal of Immunology, 2009Co-Authors: Rachel E Simmonds, Pamela L C Small, Ferdinand V Lali, Tim Smallie, Brian M J FoxwellAbstract:The virulence and immunosuppressive activity of Mycobacterium ulcerans is attributed to Mycolactone, a macrolide toxin synthesized by the bacteria. We have explored the consequence and mechanism of Mycolactone pretreatment of primary human monocytes activated by a wide range of TLR ligands. The production of cytokines (TNF, IL-1β, IL-6, IL-10, and IFN-γ-inducible protein-10), chemokines (IL-8), and intracellular effector molecules (exemplified by cyclooxygenase-2) was found to be powerfully and dose dependently inhibited by Mycolactone, irrespective of the stimulating ligand. However, Mycolactone had no effect on the activation of signaling pathways that are known to be important in inducing these genes, including the MAPK and NF-κB pathways. Unexpectedly, LPS-dependent transcription of TNF, IL-6, and cyclooxygenase-2 mRNA was found not to be inhibited, implying that Mycolactone has a novel mechanism of action and must function posttranscriptionally. We propose that Mycolactone mediates its effects by inhibiting the translation of a specific subset of proteins in primary human monocytes. This mechanism is distinct from rapamycin, another naturally occurring immunosuppressive lactone. The current findings also suggest that monocyte-derived cytokine transcript and protein levels may not correlate in Buruli ulcer lesions, and urge caution in the interpretation of RT-PCR data obtained from patient biopsy samples.
Richard O. Phillips - One of the best experts on this subject based on the ideXlab platform.
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recent advances role of Mycolactone in the pathogenesis and monitoring of mycobacterium ulcerans infection buruli ulcer disease
Cellular Microbiology, 2016Co-Authors: Fred Stephen Sarfo, Richard O. Phillips, Mark Wansbroughjones, Rachel E SimmondsAbstract:Infection of subcutaneous tissue with Mycobacterium ulcerans can lead to chronic skin ulceration known as Buruli ulcer. The pathogenesis of this neglected tropical disease is dependent on a lipid-like toxin, Mycolactone, which diffuses through tissue away from the infecting organisms. Since its identification in 1999, this molecule has been intensely studied to elucidate its cytotoxic and immunosuppressive properties. Two recent major advances identifying the underlying molecular targets for Mycolactone have been described. First, it can target scaffolding proteins (such as Wiskott Aldrich Syndrome Protein), which control actin dynamics in adherent cells and therefore lead to detachment and cell death by anoikis. Second, it prevents the co-translational translocation (and therefore production) of many proteins that pass through the endoplasmic reticulum for secretion or placement in cell membranes. These pleiotropic effects underpin the range of cell-specific functional defects in immune and other cells that contact Mycolactone during infection. The dose and duration of Mycolactone exposure for these different cells explains tissue necrosis and the paucity of immune cells in the ulcers. This review discusses recent advances in the field, revisits older findings in this context and highlights current developments in structure-function studies as well as methodology that make Mycolactone a promising diagnostic biomarker.
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Recent advances: role of Mycolactone in the pathogenesis and monitoring of Mycobacterium ulcerans infection/Buruli ulcer disease
Cellular Microbiology, 2015Co-Authors: Fred Stephen Sarfo, Richard O. Phillips, Mark Wansbrough-jones, Rachel E SimmondsAbstract:Infection of subcutaneous tissue with Mycobacterium ulcerans can lead to chronic skin ulceration known as Buruli ulcer. The pathogenesis of this neglected tropical disease is dependent on a lipid-like toxin, Mycolactone, which diffuses through tissue away from the infecting organisms. Since its identification in 1999, this molecule has been intensely studied to elucidate its cytotoxic and immunosuppressive properties. Two recent major advances identifying the underlying molecular targets for Mycolactone have been described. First, it can target scaffolding proteins (such as Wiskott Aldrich Syndrome Protein), which control actin dynamics in adherent cells and therefore lead to detachment and cell death by anoikis. Second, it prevents the co-translational translocation (and therefore production) of many proteins that pass through the endoplasmic reticulum for secretion or placement in cell membranes. These pleiotropic effects underpin the range of cell-specific functional defects in immune and other cells that contact Mycolactone during infection. The dose and duration of Mycolactone exposure for these different cells explains tissue necrosis and the paucity of immune cells in the ulcers. This review discusses recent advances in the field, revisits older findings in this context and highlights current developments in structure-function studies as well as methodology that make Mycolactone a promising diagnostic biomarker.
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metabolomic profiles delineate Mycolactone signature in buruli ulcer disease
Scientific Reports, 2015Co-Authors: Richard O. Phillips, Timothy P Stinear, Laure Gueninmace, Mark Wansbroughjones, Fatoumata Niang, Fred S Sarfo, Michael FrimpongAbstract:Infection of human skin with Mycobacterium ulcerans, the causative agent of Buruli ulcer, is associated with the systemic diffusion of a bacterial macrolide named Mycolactone. Patients with progressive disease show alterations in their serum proteome, likely reflecting the inhibition of secreted protein production by Mycolactone at the cellular level. Here, we used semi-quantitative metabolomics to characterize metabolic perturbations in serum samples of infected individuals, and human cells exposed to Mycolactone. Among the 430 metabolites profiled across 20 patients and 20 healthy endemic controls, there were significant differences in the serum levels of hexoses, steroid hormones, acylcarnitines, purine, heme, bile acids, riboflavin and lysolipids. In parallel, analysis of 292 metabolites in human T cells treated or not with Mycolactone showed alterations in hexoses, lysolipids and purine catabolites. Together, these data demonstrate that M. ulcerans infection causes systemic perturbations in the serum metabolome that can be ascribed to Mycolactone. Of particular importance to Buruli ulcer pathogenesis is that changes in blood sugar homeostasis in infected patients are mirrored by alterations in hexose metabolism in Mycolactone-exposed cells.
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simple rapid mycobacterium ulcerans disease diagnosis from clinical samples by fluorescence of Mycolactone on thin layer chromatography
PLOS Neglected Tropical Diseases, 2015Co-Authors: Anita Wadagni, Estelle Marion, Vaddela Sudheer Babu, Yalan Xing, Michael Frimpong, Delphin Mavinga Phanzu, Anthony Ablordey, Emmanuel Kacou, Mirabelle Gbedevi, Richard O. PhillipsAbstract:Introduction Mycobacterium ulcerans infection, known as Buruli ulcer, is a disease of the skin and subcutaneous tissues which is an important but neglected tropical disease with its major impact in rural parts of West and Central Africa where facilities for diagnosis and management are poorly developed. We evaluated fluorescent thin layer chromatography (f-TLC) for detection of Mycolactone in the laboratory using samples from patients with Buruli ulcer and patients with similar lesions that gave a negative result on PCR for the IS2404 repeat sequence of M. ulcerans Methodology/Principal findings Mycolactone and DNA extracts from fine needle aspiration (FNA), swabs and biopsy specimen were used to determine the sensitivity and specificity of f-TLC when compared with PCR for the IS2404. For 71 IS2404 PCR positive and 28 PCR negative samples the sensitivity was 73.2% and specificity of 85.7% for f-TLC. The sensitivity was similar for swabs (73%), FNAs (75%) and biopsies (70%). Conclusions We have shown that Mycolactone can be detected from M. ulcerans infected skin tissue by f-TLC technique. The technique is simple, easy to perform and read with minimal costs. In this study it was undertaken by a member of the group from each endemic country. It is a potentially implementable tool at the district level after evaluation in larger field studies.
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Kinetics of Mycolactone in human subcutaneous tissue during antibiotic therapy for Mycobacterium ulcerans disease
BMC Infectious Diseases, 2014Co-Authors: Fred S Sarfo, Richard O. Phillips, Jihui Zhang, Mohammed K Abass, Justice Abotsi, Yaw A Amoako, Yaw Adu-sarkodie, Clive Robinson, Mark H Wansbrough-jonesAbstract:Background Mycobacterium ulcerans ( M. ulcerans ) causes a devastating necrotising infection of skin tissue leading to progressive ulceration. M. ulcerans is the only human pathogen that secretes Mycolactone, a polyketide molecule with potent cytotoxic and immunomodulatory properties. These unique features make Mycolactone an attractive biomarker for M. ulcerans disease. We sought to measure the concentration of Mycolactone within lesions of patients with Buruli ulcer before, during and after antibiotic treatment to evaluate its association with the clinical and bacteriological response to therapy. Methods Biopsies of M. ulcerans infected skin lesions were obtained from patients before, during and after antibiotic therapy. Lipids were extracted from the biopsies and concentration of Mycolactone was assayed by mass spectrometry and a cytotoxicity assay and correlated with clinical and bacteriological response to therapy. Results Baseline concentration of Mycolactone measured by mass spectrometry predicted time to complete healing of small nodules and ulcers. Even though intra-lesional concentrations of Mycolactone declined with antibiotic treatment, the toxin was still present after antibiotic treatment for 6 weeks and also 4 weeks after the end of treatment for 8 weeks in a subgroup of patients with slowly healing lesions. Additionally viable bacilli were detected in a proportion of these slowly healing lesions during and after treatment. Conclusions Our findings indicate that baseline intra-lesional Mycolactone concentration and its kinetics with antibiotic therapy are important prognostic determinants of clinical and bacteriological response to antibiotic treatment for Mycobacterium ulcerans disease. Mycolactone may be a useful biomarker with potential utility in optimising antibiotic therapy.
Timothy P Stinear - One of the best experts on this subject based on the ideXlab platform.
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The pMUM Megaplasmid of Mycobacterium ulcerans and Closely Related Mycobacteria: A Blueprint for the Synthesis of Mycolactones
Microbial Megaplasmids, 2020Co-Authors: Sacha J Pidot, Nicholas J Tobias, Timothy P StinearAbstract:Mycobacterium ulcerans is the third most common mycobacterial infection of humans and causes a destructive disease of subcutaneous tissue known as Buruli ulcer. A cytotoxic lipid known as Mycolactone mediates the characteristic necrosis seen in Buruli ulcers. A family of highly related Mycolactone structural variants have been discovered, all of which are produced by large polyketide synthases (PKSs), encoded by genes on large plasmids harbored by M. ulcerans strains. The prototype Mycolactone plasmid is pMUM001, a 174-kb circular molecule from a West African epidemic strain of M. ulcerans. A striking feature of pMUM001 is that it has one-third of its DNA devoted to three very large genes that encode the mycol-actone biosynthetic machinery. The plasmid (pMUM002) from M. ulcerans subsp. liflandii has recently been completely sequenced, and partial sequence has been obtained for a further pMUM plasmid, pMUM003, from M. ulcerans DL240490. Interstrain comparisons of pMUM sequences has revealed the highly mutable nature of the Mycolactone PKS genes and given us greater insight into the origins of these large replicons.
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metabolomic profiles delineate Mycolactone signature in buruli ulcer disease
Scientific Reports, 2015Co-Authors: Richard O. Phillips, Timothy P Stinear, Laure Gueninmace, Mark Wansbroughjones, Fatoumata Niang, Fred S Sarfo, Michael FrimpongAbstract:Infection of human skin with Mycobacterium ulcerans, the causative agent of Buruli ulcer, is associated with the systemic diffusion of a bacterial macrolide named Mycolactone. Patients with progressive disease show alterations in their serum proteome, likely reflecting the inhibition of secreted protein production by Mycolactone at the cellular level. Here, we used semi-quantitative metabolomics to characterize metabolic perturbations in serum samples of infected individuals, and human cells exposed to Mycolactone. Among the 430 metabolites profiled across 20 patients and 20 healthy endemic controls, there were significant differences in the serum levels of hexoses, steroid hormones, acylcarnitines, purine, heme, bile acids, riboflavin and lysolipids. In parallel, analysis of 292 metabolites in human T cells treated or not with Mycolactone showed alterations in hexoses, lysolipids and purine catabolites. Together, these data demonstrate that M. ulcerans infection causes systemic perturbations in the serum metabolome that can be ascribed to Mycolactone. Of particular importance to Buruli ulcer pathogenesis is that changes in blood sugar homeostasis in infected patients are mirrored by alterations in hexose metabolism in Mycolactone-exposed cells.
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transfer stable maintenance and expression of the Mycolactone polyketide megasynthase mls genes in a recombination impaired mycobacterium marinum
Microbiology, 2009Co-Authors: Jessica L Porter, Hui Hong, Nicholas J Tobias, Kellie L Tuck, Grant A Jenkin, Timothy P StinearAbstract:The human pathogen Mycobacterium ulcerans produces a polyketide metabolite called Mycolactone with potent immunomodulatory activity. M. ulcerans strain Agy99 has a 174 kb plasmid called pMUM001 with three large genes (mlsA1, 51 kb; mlsA2, 7.2 kb; mlsB, 43 kb) that encode type I polyketide synthases (PKS) required for the biosynthesis of Mycolactone, as demonstrated by transposon mutagenesis. However, there have been no reports of transfer of the mls locus to another mycobacterium to demonstrate that these genes are sufficient for Mycolactone production because in addition to their large size, the mls genes contain a high level of internal sequence repetition, such that the entire 102 kb locus is composed of only 9.5 kb of unique DNA. The combination of their large size and lack of stability during laboratory passage makes them a challenging prospect for transfer to a more rapidly growing and genetically tractable host. Here we describe the construction of two bacterial artificial chromosome Escherichia coli/Mycobacterium shuttle vectors, one based on the pMUM001 origin of replication bearing mlsB, and the other based on the mycobacteriophage L5 integrase, bearing mlsA1 and mlsA2. The combination of these two constructs permitted the two-step transfer of the entire 174 kb pMUM001 plasmid to Mycobacterium marinum, a rapidly growing non-Mycolactone-producing mycobacterium that is a close genetic relative of M. ulcerans. To improve the stability of the mls locus in M. marinum, recA was inactivated by insertion of a hygromycin-resistance gene using double-crossover allelic exchange. As expected, the ΔrecA mutant displayed increased susceptibility to UV killing and a decreased frequency of homologous recombination. Southern hybridization and RT-PCR confirmed the stable transfer and expression of the mls genes in both wild-type M. marinum and the recA mutant. However, neither Mycolactone nor its predicted precursor metabolites were detected in either strain. These experiments show that it is possible to successfully manipulate and stably transfer the large mls genes, but that other bacterial host factors appear to be required to facilitate Mycolactone production.
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Transfer, stable maintenance and expression of the Mycolactone polyketide megasynthase mls genes in a recombination-impaired Mycobacterium marinum.
Microbiology (Reading England), 2009Co-Authors: Jessica L Porter, Hui Hong, Nicholas J Tobias, Kellie L Tuck, Grant A Jenkin, Timothy P StinearAbstract:The human pathogen Mycobacterium ulcerans produces a polyketide metabolite called Mycolactone with potent immunomodulatory activity. M. ulcerans strain Agy99 has a 174 kb plasmid called pMUM001 with three large genes (mlsA1, 51 kb; mlsA2, 7.2 kb; mlsB, 43 kb) that encode type I polyketide synthases (PKS) required for the biosynthesis of Mycolactone, as demonstrated by transposon mutagenesis. However, there have been no reports of transfer of the mls locus to another mycobacterium to demonstrate that these genes are sufficient for Mycolactone production because in addition to their large size, the mls genes contain a high level of internal sequence repetition, such that the entire 102 kb locus is composed of only 9.5 kb of unique DNA. The combination of their large size and lack of stability during laboratory passage makes them a challenging prospect for transfer to a more rapidly growing and genetically tractable host. Here we describe the construction of two bacterial artificial chromosome Escherichia coli/Mycobacterium shuttle vectors, one based on the pMUM001 origin of replication bearing mlsB, and the other based on the mycobacteriophage L5 integrase, bearing mlsA1 and mlsA2. The combination of these two constructs permitted the two-step transfer of the entire 174 kb pMUM001 plasmid to Mycobacterium marinum, a rapidly growing non-Mycolactone-producing mycobacterium that is a close genetic relative of M. ulcerans. To improve the stability of the mls locus in M. marinum, recA was inactivated by insertion of a hygromycin-resistance gene using double-crossover allelic exchange. As expected, the DeltarecA mutant displayed increased susceptibility to UV killing and a decreased frequency of homologous recombination. Southern hybridization and RT-PCR confirmed the stable transfer and expression of the mls genes in both wild-type M. marinum and the recA mutant. However, neither Mycolactone nor its predicted precursor metabolites were detected in either strain. These experiments show that it is possible to successfully manipulate and stably transfer the large mls genes, but that other bacterial host factors appear to be required to facilitate Mycolactone production.
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buruli ulcer reductive evolution enhances pathogenicity of mycobacterium ulcerans
Nature Reviews Microbiology, 2009Co-Authors: Caroline Demangel, Timothy P Stinear, Stewart T ColeAbstract:Buruli ulcer is an emerging human disease caused by infection with a slow-growing pathogen, Mycobacterium ulcerans, that produces Mycolactone, a cytotoxin with immunomodulatory properties. The disease is associated with wetlands in certain tropical countries, and evidence for a role of insects in transmission of this pathogen is growing. Comparative genomic analysis has revealed that M. ulcerans arose from Mycobacterium marinum, a ubiquitous fast-growing aquatic species, by horizontal transfer of a virulence plasmid that carries a cluster of genes for Mycolactone production, followed by reductive evolution. Here, the ecology, microbiology, evolutionary genomics and immunopathology of Buruli ulcer are reviewed.
Yoshito Kishi - One of the best experts on this subject based on the ideXlab platform.
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Mycolactone cytotoxicity in schwann cells could explain nerve damage in buruli ulcer
PLOS Neglected Tropical Diseases, 2017Co-Authors: Junichiro En, Sho Kitamoto, Suguru Yonezawa, Norihisa Ishii, Yoshito Kishi, Akira Kawashima, Masamichi GotoAbstract:Buruli ulcer is a chronic painless skin disease caused by Mycobacterium ulcerans. The local nerve damage induced by M. ulcerans invasion is similar to the nerve damage evoked by the injection of Mycolactone in a Buruli ulcer mouse model. In order to elucidate the mechanism of this nerve damage, we tested and compared the cytotoxic effect of synthetic Mycolactone A/B on cultured Schwann cells, fibroblasts and macrophages. Mycolactone induced much higher cell death and apoptosis in Schwann cell line SW10 than in fibroblast line L929. These results suggest that Mycolactone is a key substance in the production of nerve damage of Buruli ulcer.
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design synthesis and cytotoxicity of stabilized Mycolactone analogs
Bioorganic & Medicinal Chemistry Letters, 2017Co-Authors: Vaddela Sudheer Babu, Ya Zhou, Yoshito KishiAbstract:Abstract On exposure to visible light, Mycolactone A/B, the causative toxin of Buruli ulcer, rearranges to a mixture of four photo-Mycolactones apparently via a rare photochemically-induced [ 4 π s + 2 π a ] cycloaddition. In order to prevent the rearrangement, two C6′-C7′ dihydroMycolactone analogs 6′α- 15 and 6′β- 15 were designed and synthesized. 6′α- 15 and 6′β- 15 were shown to be stable under not only photochemical, but also acidic and basic conditions. Cytotoxicity was tested against arbitrarily chosen four cell lines (human Hek-293, human lung carcinoma A-549, human melanoma LOX-IMVI, and mouse L-929), thereby revealing that: (1) both analogs maintain potent cytotoxicity; (2) 6′β- 15 exhibits significantly higher potency against human cell lines than 6′α- 15 ; (3) in comparison with parent Mycolactone A/B, 6′β- 15 exhibits equal potency against human Hek-293, whereas significantly lower potency against human lung carcinoma A-549 and human melanoma LOX-IMVI.
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Stereoselective total synthesis and stereochemistry confirmation of photo-Mycolactones
Tetrahedron Letters, 2015Co-Authors: Xiaoyong Li, Vaddela Sudheer Babu, Yoshito KishiAbstract:Abstract With use of the LiTMP-induced Hodgson cyclopropanation of an epoxide-olefin to a bicyclo[3.1.0]hexanol as the key step, a stereoselective total synthesis of photo-Mycolactones was achieved. Each of the four diastereomeric epoxide-olefins, selectively prepared from (R)- and (S)-glycidols, yielded the corresponding unique bicyclo[3.1.0]hexanol. Four bicyclo[3.1.0]hexanols 16, dia-16, 19, and dia-19 were converted into four primary alcohols 17, dia-17, 20, and dia-20, the intermediates used in the previous work on photo-Mycolactones. This synthesis confirmed the stereochemistry previously proposed for photo-Mycolactones.
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accelerated detection of Mycolactone production and response to antibiotic treatment in a mouse model of mycobacterium ulcerans disease
PLOS Neglected Tropical Diseases, 2014Co-Authors: Paul J Converse, Yalan Xing, Eric L Nuermberger, Sandeep Tyagi, Siyang Li, Deepak V Almeida, J Grosset, Yoshito KishiAbstract:Diagnosis of the neglected tropical disease, Buruli ulcer, can be made by acid-fast smear microscopy, specimen culture on mycobacterial growth media, polymerase chain reaction (PCR), and/or histopathology. All have drawbacks, including non-specificity and requirements for prolonged culture at 32°C, relatively sophisticated laboratory facilities, and expertise, respectively. The causative organism, Mycobacterium ulcerans, produces a unique toxin, Mycolactone A/B (ML) that can be detected by thin layer chromatography (TLC) or mass spectrometric analysis. Detection by the latter technique requires sophisticated facilities. TLC is relatively simple but can be complicated by the presence of other lipids in the specimen. A method using a boronate-assisted fluorogenic chemosensor in TLC can overcome this challenge by selectively detecting ML when visualized with UV light. This report describes modifications in the fluorescent TLC (F-TLC) procedure and its application to the mouse footpad model of M. ulcerans disease to determine the kinetics of Mycolactone production and its correlation with footpad swelling and the number of colony forming units in the footpad. The response of all three parameters to treatment with the current standard regimen of rifampin (RIF) and streptomycin (STR) or a proposed oral regimen of RIF and clarithromycin (CLR) was also assessed. ML was detectable before the onset of footpad swelling when there were 105 CFU per footpad. Mycolactone concentrations increased as swelling increased whereas CFU levels reached a plateau. Treatment with either RIF+STR or RIF+CLR resulted in comparable reductions of Mycolactone, footpad swelling, and CFU burden. Storage in absolute ethanol appears critical to successful detection of ML in footpads and would be practical for storage of clinical samples. F-TLC may offer a new tool for confirmation of suspected clinical lesions and be more specific than smear microscopy, much faster than culture, and simpler than PCR.
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photochemistry of Mycolactone a b the causative toxin of buruli ulcer
Journal of the American Chemical Society, 2012Co-Authors: Yalan Xing, Sudhir M. Hande, Yoshito KishiAbstract:Photochemistry of Mycolactone A/B and related unsaturated fatty acid esters is reported. On exposure to visible light, Mycolactone A/B gave a mixture of four photoMycolactones. Pentaenoates and tetraenoates, representing the unsaturated fatty acid portion of Mycolactone A/B, were found to show the reactivity profile parallel with that of Mycolactone A/B. The structure of the four photoMycolactones was elucidated via (1) structure determination of the four photoproducts in the tetraenoate series; (2) their transformation to the photoproducts in the pentaenoate and then Mycolactone series. Triplet quenchers did not affect the photochemical transformation, thereby indicating an event at the singlet state. A concerted, photochemically allowed [4πs + 2πa] cycloaddition was suggested to account for the observed result. This study provided the structurally defined and homogeneous material, which allowed demonstration that photoMycolactones exhibit significantly reduced cytotoxicity, compared with Mycolactone A/B.
