An Antigen Capture Assay for the Detection of Mycolactone, the Polyketide Toxin of Mycobacterium ulcerans.
Ontology highlight
ABSTRACT:
SUBMITTER: Warryn L
PROVIDER: S-EPMC8176938 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Mycolactone is the exotoxin produced by Mycobacterium ulcerans and is the virulence factor behind the neglected tropical disease Buruli ulcer. The toxin has a broad spectrum of biological effects within the host organism, stemming from its interaction with at least two molecular targets and the inhibition of protein uptake into the endoplasmic reticulum. Although it has been shown that the toxin can passively permeate into host cells, it is clearly lipophilic. Association with lipid carriers would have substantial implications for the toxin's distribution within a host organism, delivery to cellular targets, diagnostic susceptibility, and mechanisms of pathogenicity. Yet the toxin's interactions with, and distribution in, lipids are unknown. Herein we have used coarse-grained molecular dynamics simulations, guided by all-atom simulations, to study the interaction of mycolactone with pure and mixed lipid membranes. Using established techniques, we calculated the toxin's preferential localization, membrane translocation, and impact on membrane physical and dynamical properties. The computed water-octanol partition coefficient indicates that mycolactone prefers to be in an organic phase rather than in an aqueous environment. Our results show that in a solvated membrane environment the exotoxin mainly localizes in the water-membrane interface, with a preference for the glycerol moiety of lipids, consistent with the reported studies that found it in lipid extracts of the cell. The calculated association constant to the model membrane is similar to the reported association constant for Wiskott-Aldrich syndrome protein. Mycolactone is shown to modify the physical properties of membranes, lowering the transition temperature, compressibility modulus, and critical line tension at which pores can be stabilized. It also shows a tendency to behave as a linactant, a molecule that localizes at the boundary between different fluid lipid domains in membranes and promotes inter-mixing of domains. This property has implications for the toxin's cellular access, T-cell immunosuppression, and therapeutic potential.
| S-EPMC5814095 | biostudies-literature
Project description:BackgroundMycobacterium ulcerans, a slow-growing environmental bacterium, is the etiologic agent of Buruli ulcer, a necrotic skin disease. Skin lesions are caused by mycolactone, the main virulence factor of M. ulcerans, with dermonecrotic (destruction of the skin and soft tissues) and immunosuppressive activities. This toxin is secreted in vesicles that enhance its biological activities. Nowadays, it is well established that the main reservoir of the bacilli is localized in the aquatic environment where the bacillus may be able to colonize different niches. Here we report that plant polysaccharides stimulate M. ulcerans growth and are implicated in toxin synthesis regulation.Methodology/principal findingsIn this study, by selecting various algal components, we have identified plant-specific carbohydrates, particularly glucose polymers, capable of stimulating M. ulcerans growth in vitro. Furthermore, we underscored for the first time culture conditions under which the polyketide toxin mycolactone, the sole virulence factor of M. ulcerans identified to date, is down-regulated. Using a quantitative proteomic approach and analyzing transcript levels by RT-qPCR, we demonstrated that its regulation is not at the transcriptional or translational levels but must involve another type of regulation. M. ulcerans produces membrane vesicles, as other mycobacterial species, in which are the mycolactone is concentrated. By transmission electron microscopy, we observed that the production of vesicles is independent from the toxin production. Concomitant with this observed decrease in mycolactone production, the production of mycobacterial siderophores known as mycobactins was enhanced.Conclusions/significanceThis work is the first step in the identification of the mechanisms involved in mycolactone regulation and paves the way for the discovery of putative new drug targets in the future.
| S-EPMC3828164 | biostudies-literature
Project description:Mycolactone is a polyketide toxin produced by Mycobacterium ulcerans (Mu), the causative agent of the skin disease Buruli ulcer (BU). Surprisingly, infected tissues lack inflammatory infiltrates. Structural similarities between mycolactone and immunosuppressive agents led us to investigate the immunomodulatory properties of mycolactone on dendritic cells (DCs), the key initiators and regulators of immune responses. At noncytotoxic concentrations, phenotypic and functional maturation of both mouse and human DCs was inhibited by mycolactone. Notably, mycolactone blocked the emigration of mouse-skin DCs to draining lymph nodes, as well as their maturation in vivo. In human peripheral blood-derived DCs, mycolactone inhibited the ability to activate allogeneic T cell priming and to produce inflammatory molecules. Interestingly, production of the cytokines interleukin (IL) 12, tumor necrosis factor alpha, and IL-6 was only marginally affected, whereas production of the chemokines macrophage inflammatory protein (MIP) 1alpha, MIP-1beta, regulated on activation, normal T cell expressed and secreted, interferon gamma-inducible protein 10, and monocyte chemoattractant protein 1 was abolished at nanomolar concentrations. Importantly, mycolactone endogenously expressed by Mu mediated similar inhibitory effects on beta-chemokine production by DCs. In accordance with the histopathological features of BUs, our results suggest that bacterial production of mycolactone may limit both the initiation of primary immune responses and the recruitment of inflammatory cells to the infection site. Moreover, they highlight a potential interest in mycolactone as a novel immunosuppressive agent.
| S-EPMC2118602 | biostudies-literature
Project description:Buruli ulcer is a neglected tropical disease caused by the environmental pathogen, Mycobacterium ulcerans whose major virulence factor is mycolactone, a lipid cytotoxic molecule. Buruli ulcer has high morbidity, particularly in rural West Africa where the disease is endemic. Data have shown that infected lesions of Buruli ulcer patients can be colonized by quorum sensing bacteria such as Staphylococcus aureus, S. epidermidis, and Pseudomonas aeruginosa, but without typical pathology associated with those pathogens' colonization. M. ulcerans pathogenesis may not only be an individual act but may also be dependent on synergistic or antagonistic mechanisms within a polymicrobial network. Furthermore, co-colonization by these pathogens may promote delayed wound healing, especially after the initiation of antibiotic therapy. Hence, it is important to understand the interaction of M. ulcerans with other bacteria encountered during skin infection. We added mycolactone to S. aureus and incubated for 3, 6 and 24 h. At each timepoint, S. aureus growth and hemolytic activity was measured, and RNA was isolated to measure virulence gene expression through qPCR and RNASeq analyses. Results showed that mycolactone reduced S. aureus hemolytic activity, suppressed hla promoter activity, and attenuated virulence genes, but did not affect S. aureus growth. RNASeq data showed mycolactone greatly impacted S. aureus metabolism. These data are relevant and significant as mycolactone and S. aureus sensing and response at the transcriptional, translational and regulation levels will provide insight into biological mechanisms of interspecific interactions that may play a role in regulation of responses such as effects between M. ulcerans, mycolactone, and S. aureus virulence that will be useful for treatment and prevention.
| S-EPMC8175560 | biostudies-literature
Project description:Buruli ulcer, a neglected tropical infectious disease, is caused by Mycobacterium ulcerans. Without treatment, its lesions can progress to chronic skin ulcers, but spontaneous healing is observed in 5% of cases, suggesting the possible establishment of a host strategy counteracting the effects of M. ulcerans. We reveal here a skin-specific local humoral signature of the spontaneous healing process, associated with a rise in antibody-producing cells and specific recognition of mycolactone by the mouse IgG2a immunoglobulin subclass. We demonstrate the production of skin-specific antibodies neutralizing the immunomodulatory activity of the mycolactone toxin, and confirm the role of human host machinery in triggering effective local immune responses by the detection of anti-mycolactone antibodies in patients with Buruli ulcer. Our findings pave the way for substantial advances in both the diagnosis and treatment of Buruli ulcer in accordance with the most recent challenges issued by the World Health Organization.
| S-EPMC7043917 | biostudies-literature
Project description:Mycobacterium ulcerans (MU), an emerging human pathogen harbored by aquatic insects, is the causative agent of Buruli ulcer, a devastating skin disease rife throughout Central and West Africa. Mycolactone, an unusual macrolide with cytotoxic and immunosuppressive properties, is responsible for the massive s.c. tissue destruction seen in Buruli ulcer. Here, we show that MU contains a 174-kb plasmid, pMUM001, bearing a cluster of genes encoding giant polyketide synthases (PKSs), and polyketide-modifying enzymes, and demonstrate that these are necessary and sufficient for mycolactone synthesis. This is a previously uncharacterized example of plasmid-mediated virulence in a Mycobacterium, and the emergence of MU as a pathogen most likely reflects the acquisition of pMUM001 by horizontal transfer. The 12-membered core of mycolactone is produced by two giant, modular PKSs, MLSA1 (1.8 MDa) and MLSA2 (0.26 MDa), whereas its side chain is synthesized by MLSB (1.2 MDa), a third modular PKS highly related to MLSA1. There is an extreme level of sequence identity within the different domains of the MLS cluster (>97% amino acid identity), so much so that the 16 ketosynthase domains seem functionally identical. This is a finding of significant consequence for our understanding of polyketide biochemistry. Such detailed knowledge of mycolactone will further the investigation of its mode of action and the development of urgently needed therapeutic strategies to combat Buruli ulcer.
| S-EPMC337055 | biostudies-literature
Project description:Mycolactone is a diffusible lipid toxin produced by Mycobacterium ulcerans, the causative agent of Buruli ulcer disease. Altough bacterially derived mycolactone has been shown to traffic from cutaneous foci of infection to the bloodstream, the mechanisms underpinning its access to systemic circulation and import by host cells remain largely unknown. Using biophysical and cell-based approaches, we demonstrate that mycolactone specific association to serum albumin and lipoproteins is necessary for its solubilization and is a major mechanism to regulate its bioavailability. We also demonstrate that Scavenger Receptor (SR)-B1 contributes to the cellular uptake of mycolactone. Overall, we suggest a new mechanism of transport and cell entry, challenging the dogma that the toxin enters host cells via passive diffusion.
| S-EPMC8481864 | biostudies-literature
Project description:Background:Buruli ulcer is a disease of the skin and soft tissues caused by infection with a slow growing pathogen, Mycobacterium ulcerans. A vaccine for this disease is not available but M. ulcerans possesses a giant plasmid pMUM001 that harbours the polyketide synthase (PKS) genes encoding a multi-enzyme complex needed for the production of its unique lipid toxin called mycolactone, which is central to the pathogenesis of Buruli ulcer. We have studied the immunogenicity of enzymatic domains in humans with M. ulcerans disease, their contacts, as well as non-endemic areas controls. Methods:Between March 2013 and August 2015, heparinized whole blood was obtained from patients confirmed with Buruli ulcer. The blood samples were diluted 1 in 10 in Roswell Park Memorial Institute (RPMI) medium and incubated for 5 days with recombinant mycolactone PKS domains and mycolyltransferase antigen 85A (Ag85A). Blood samples were obtained before and at completion of antibiotic treatment for 8 weeks and again 8 weeks after completion of treatment. Supernatants were assayed for interferon-? (IFN-?) and interleukin-5 (IL-5) by enzyme-linked immunosorbent assay. Responses were compared with those of contacts and non-endemic controls. Results:More than 80% of patients and contacts from endemic areas produced IFN-? in response to all the antigens except acyl carrier protein type 3 (ACP3) to which only 47% of active Buruli ulcer cases and 71% of contacts responded. The highest proportion of responders in cases and contacts was to load module ketosynthase domain (Ksalt) (100%) and enoylreductase (100%). Lower IL-5 responses were induced in a smaller proportion of patients ranging from 54% after ketoreductase type B stimulation to only 21% with ketosynthase type C (KS C). Among endemic area contacts, the, highest proportion was 73% responding to KS C and the lowest was 40% responding to acyltransferase with acetate specificity type 2. Contacts of Buruli ulcer patients produced significantly higher IFN-? and IL-5 responses compared with those of patients to PKS domain antigens and to mycolyltransferase Ag85A of M. ulcerans. There was low or no response to all the antigens in non-endemic areas controls. IFN-? and IL-5 responses of patients improved after treatment when compared to baseline results. Discussion:The major response to PKS antigen stimulation was IFN-? and the strongest responses were observed in healthy contacts of patients living in areas endemic for Buruli ulcer. Patients elicited lower responses than healthy contacts, possibly due to the immunosuppressive effect of mycolactone, but the responses were enhanced after antibiotic treatment. A vaccine made up of the most immunogenic PKS domains combined with the mycolyltransferase Ag85A warrants further investigation.
| S-EPMC6078848 | biostudies-literature
Project description:There is no effective vaccine against Buruli ulcer. In experimental footpad infection of C57BL/6 mice with M. ulcerans, a prime-boost vaccination protocol using plasmid DNA encoding mycolyltransferase Ag85A of M. ulcerans and a homologous protein boost has shown significant, albeit transient protection, comparable to the one induced by M. bovis BCG. The mycolactone toxin is an obvious candidate for a vaccine, but by virtue of its chemical structure, this toxin is not immunogenic in itself. However, antibodies against some of the polyketide synthase domains involved in mycolactone synthesis, were found in Buruli ulcer patients and healthy controls from the same endemic region, suggesting that these domains are indeed immunogenic. Here we have analyzed the vaccine potential of nine polyketide synthase domains using a DNA prime/protein boost strategy. C57BL/6 mice were vaccinated against the following domains: acyl carrier protein 1, 2, and 3, acyltransferase (acetate) 1 and 2, acyltransferase (propionate), enoylreductase, ketoreductase A, and ketosynthase load module. As positive controls, mice were vaccinated with DNA encoding Ag85A or with M. bovis BCG. Strongest antigen specific antibodies could be detected in response to acyltransferase (propionate) and enoylreductase. Antigen-specific Th1 type cytokine responses (IL-2 or IFN-γ) were induced by vaccination against all antigens, and were strongest against acyltransferase (propionate). Finally, vaccination against acyltransferase (propionate) and enoylreductase conferred some protection against challenge with virulent M. ulcerans 1615. However, protection was weaker than the one conferred by vaccination with Ag85A or M. bovis BCG. Combinations of these polyketide synthase domains with the vaccine targeting Ag85A, of which the latter is involved in the integrity of the cell wall of the pathogen, and/or with live attenuated M. bovis BCG or mycolactone negative M. ulcerans may eventually lead to the development of an efficacious BU vaccine.
| S-EPMC3879250 | biostudies-literature
Project description: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.
| S-EPMC4125307 | biostudies-literature