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Buruli ulcer (BU) is a skin infection caused by Mycobacterium ulcerans and a neglected tropical disease of the skin (skin NTD). Antibiotic treatments are available but, to be effective in the absence of surgery, BU must be detected at its earliest stages (an innocuous-looking lump under the skin) and adherence to prescribed drugs must be high. This study aimed to develop multisensory medical illustrations of BU to support communication with at-risk communities. We used a Think Aloud method to explore community health workers' (n = 6) experiences of BU with a focus on the role of their five senses, since these non-medical disease experts are familiar with the day-to-day challenges presented by BU. Thematic analysis of the transcripts identified three key themes relating to 'Detection,' 'Help Seeking,' and 'Adherence' with a transcending theme 'Senses as key facilitators of health care'. New medical illustrations, for which we coin the phrase "5D illustrations" (signifying the contribution of the five senses) were then developed to reflect these themes. The senses therefore facilitated an enriched narrative enabling the production of relevant and useful visuals for health communication. The medical artist community could utilise sensory experiences to create dynamic medical illustrations for use in practice.
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Úlcera de Buruli , Ilustración Médica , Humanos , Úlcera de Buruli/tratamiento farmacológico , Conducta de Búsqueda de Ayuda , Femenino , Masculino , Adulto , Agentes Comunitarios de SaludRESUMEN
The drivers of tissue necrosis in Mycobacterium ulcerans infection (Buruli ulcer disease) have historically been ascribed solely to the directly cytotoxic action of the diffusible exotoxin, mycolactone. However, its role in the clinically-evident vascular component of disease aetiology remains poorly explained. We have now dissected mycolactone's effects on primary vascular endothelial cells in vitro and in vivo. We show that mycolactone-induced changes in endothelial morphology, adhesion, migration, and permeability are dependent on its action at the Sec61 translocon. Unbiased quantitative proteomics identified a profound effect on proteoglycans, driven by rapid loss of type II transmembrane proteins of the Golgi, including enzymes required for glycosaminoglycan (GAG) synthesis, combined with a reduction in the core proteins themselves. Loss of the glycocalyx is likely to be of particular mechanistic importance, since knockdown of galactosyltransferase II (beta-1,3-galactotransferase 6; B3Galt6), the GAG linker-building enzyme, phenocopied the permeability and phenotypic changes induced by mycolactone. Additionally, mycolactone depleted many secreted basement membrane components and microvascular basement membranes were disrupted in vivo. Remarkably, exogenous addition of laminin-511 reduced endothelial cell rounding, restored cell attachment and reversed the defective migration caused by mycolactone. Hence supplementing mycolactone-depleted extracellular matrix may be a future therapeutic avenue, to improve wound healing rates.
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Equine viral arteritis is an infectious disease of equids caused by equine arteritis virus (EAV), an RNA virus of the family Arteriviridae. Dendritic cells (DC) are important modulators of the immune response with the ability to present antigen to naïve T cells and can be generated in vitro from monocytes (MoDC). DC are important targets for many viruses and this interaction is crucial for the establishment-or rather not-of an anti-viral immunity. Little is known of the effect EAV has on host immune cells, particularly DC. To study the interaction of eqDC with EAV in vitro, an optimized eqMoDC system was used, which was established in a previous study. MoDC were infected with strains of different genotypes and pathogenicity. Virus replication was determined through titration and qPCR. The effect of the virus on morphology, phenotype and function of cells was assessed using light microscopy, flow cytometry and in vitro assays. This study confirms that EAV replicates in monocytes and MoDC. The replication was most efficient in mature MoDC, but variable between strains. Only the virulent strain caused a significant down-regulation of certain proteins such as CD14 and CD163 on monocytes and of CD83 on mature MoDC. Functional studies conducted after infection showed that EAV inhibited the endocytic and phagocytic capacity of Mo and mature MoDC with minimal effect on immature MoDC. Infected MoDC showed a reduced ability to stimulate T cells. Ultimately, EAV replication resulted in an apoptosis-mediated cell death. Thus, EAV evades the host anti-viral immunity both by inhibition of antigen presentation early after infection and through killing infected DC during replication.
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Equartevirus , Animales , Caballos , Equartevirus/genética , Monocitos , Virulencia , Células Dendríticas , Diferenciación CelularRESUMEN
The plant-derived macrocyclic resin glycoside ipomoeassin F (Ipom-F) binds to Sec61α and significantly disrupts multiple aspects of Sec61-mediated protein biogenesis at the endoplasmic reticulum, ultimately leading to cell death. However, extensive assessment of Ipom-F as a molecular tool and a therapeutic lead is hampered by its limited production scale, largely caused by intramolecular assembly of the macrocyclic ring. Here, using in vitro and/or in cellula biological assays to explore the first series of ring-opened analogues for the ipomoeassins, and indeed all resin glycosides, we provide clear evidence that macrocyclic integrity is not required for the cytotoxic inhibition of Sec61-dependent protein translocation by Ipom-F. Furthermore, our modeling suggests that open-chain analogues of Ipom-F can interact with multiple sites on the Sec61α subunit, most likely located at a previously identified binding site for mycolactone and/or the so-called lateral gate. Subsequent in silico-aided design led to the discovery of the stereochemically simplified analogue 3 as a potent, alternative lead compound that could be synthesized much more efficiently than Ipom-F and will accelerate future ipomoeassin research in chemical biology and drug discovery. Our work may also inspire further exploration of ring-opened analogues of other resin glycosides.
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Antineoplásicos , Glicoconjugados , Antineoplásicos/química , Glicoconjugados/química , Glicósidos/farmacología , Canales de Translocación SEC/metabolismoRESUMEN
Buruli ulcer (BU) is a neglected tropical disease caused by subcutaneous infection with Mycobacterium ulcerans and its exotoxin mycolactone. BU displays coagulative necrosis and widespread fibrin deposition in affected skin tissues. Despite this, the role of the vasculature in BU pathogenesis remains almost completely unexplored. We hypothesise that fibrin-driven ischemia can be an 'indirect' route to mycolactone-dependent tissue necrosis by a mechanism involving vascular dysfunction. Here, we tracked >900 vessels within contiguous tissue sections from eight BU patient biopsies. Our aim was to evaluate their vascular and coagulation biomarker phenotype and explore potential links to fibrin deposition. We also integrated this with our understanding of mycolactone's mechanism of action at Sec61 and its impact on proteins involved in maintaining normal vascular function. Our findings showed that endothelial cell dysfunction is common in skin tissue adjacent to necrotic regions. There was little evidence of primary haemostasis, perhaps due to mycolactone-dependent depletion of endothelial von Willebrand factor. Instead, fibrin staining appeared to be linked to the extrinsic pathway activator, tissue factor (TF). There was significantly greater than expected fibrin staining around vessels that had TF staining within the stroma, and this correlated with the distance it extended from the vessel basement membrane. TF-induced fibrin deposition in these locations would require plasma proteins outside of vessels, therefore we investigated whether mycolactone could increase vascular permeability in vitro. This was indeed the case, and leakage was further exacerbated by IL-1ß. Mycolactone caused the loss of endothelial adherens and tight junctions by the depletion of VE-cadherin, TIE-1, TIE-2 and JAM-C; all Sec61-dependent proteins. Taken together, our findings suggest that both vascular and lymphatic vessels in BU lesions become "leaky" during infection, due to the unique action of mycolactone, allowing TF-containing structures and plasma proteins into skin tissue, ultimately leading to local coagulopathy and tissue ischemia.
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Úlcera de Buruli/metabolismo , Fibrina/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Interleucina-1beta/metabolismo , Macrólidos/metabolismo , Mycobacterium ulcerans/metabolismo , Piel , Tromboplastina/metabolismo , Adolescente , Adulto , Anciano , Úlcera de Buruli/microbiología , Úlcera de Buruli/patología , Niño , Femenino , Células Endoteliales de la Vena Umbilical Humana/microbiología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Masculino , Persona de Mediana Edad , Piel/irrigación sanguínea , Piel/metabolismo , Piel/microbiologíaRESUMEN
The Mycobacterium ulcerans exotoxin, mycolactone, is responsible for the immunosuppression and tissue necrosis that characterizes Buruli ulcer. Mycolactone inhibits SEC61-dependent co-translational translocation of proteins into the endoplasmic reticulum and the resultant cytosolic translation triggers degradation of mislocalized proteins by the ubiquitin-proteasome system. Inhibition of SEC61 by mycolactone also activates multiple EIF2S1/eIF2α kinases in the integrated stress response (ISR). Here we show mycolactone increased canonical markers of selective macroautophagy/autophagy LC3B-II, ubiquitin and SQSTM1/p62 in diverse disease-relevant primary cells and cell lines. Increased formation of puncta positive for the early autophagy markers WIPI2, RB1CC1/FIP200 and ATG16L1 indicates increased initiation of autophagy. The mycolactone response was SEC61A1-dependent and involved a pathway that required RB1CC1 but not ULK. Deletion of Sqstm1 reduced cell survival in the presence of mycolactone, suggesting this response protects against the increased cytosolic protein burden caused by the toxin. However, reconstitution of baseline SQSTM1 expression in cells lacking all autophagy receptor proteins could not rescue viability. Translational regulation by EIF2S1 in the ISR plays a key role in the autophagic response to mycolactone. Mycolactone-dependent induction of SQSTM1 was reduced in eif2ak3-/-/perk-/- cells while the p-EIF2S1 antagonist ISRIB reversed the upregulation of SQSTM1 and reduced RB1CC1, WIPI2 and LC3B puncta formation. Increased SQSTM1 staining could be seen in Buruli ulcer patient skin biopsy samples, reinforcing genetic data that suggests autophagy is relevant to disease pathology. Since selective autophagy and the ISR are both implicated in neurodegeneration, cancer and inflammation, the pathway uncovered here may have a broad relevance to human disease.Abbreviations: ATF4: activating transcription factor 4; ATG: autophagy related; BAF: bafilomycin A1; ATG16L1: autophagy related 16 like 1; BU: Buruli ulcer; CQ: chloroquine; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; CALCOCO2: calcium binding and coiled-coil domain 2; DMSO: dimethyl sulfoxide; EIF2S1: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; GFP: green fluorescent protein; HDMEC: human dermal microvascular endothelial cells; HFFF: human fetal foreskin fibroblasts; ISR: integrated stress response; ISRIB: integrated stress response inhibitor; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; Myco: mycolactone; NBR1: NBR1 autophagy cargo receptor; NFE2L2: nuclear factor, erythroid 2 like 2; OPTN: optineurin; PFA: paraformaldehyde; PtdIns3P: phosphatidylinositol-3-phosphate; RB1CC1: RB1-inducible coiled coil 1; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; ULK: unc-51 like autophagy activating kinase; UPS: ubiquitin-proteasome system; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type.
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Autofagia , Úlcera de Buruli , Factor 2 Eucariótico de Iniciación/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/fisiología , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrólidos , Ratones , Factor 2 Procariótico de Iniciación/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Canales de Translocación SEC/metabolismo , Proteína Sequestosoma-1/metabolismo , Ubiquitina/metabolismoRESUMEN
The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61α because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61α in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon 'breathing' during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactone's stabilisation of the latter underpins the phenotype observed.
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Calcio/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Macrólidos/farmacología , Canales de Translocación SEC/metabolismo , Animales , Células HCT116 , Células HEK293 , Humanos , Ratones , Células RAW 264.7RESUMEN
Buruli ulcer (BU), caused by Mycobacterium ulcerans, is a devastating necrotizing skin disease. Key to its pathogenesis is mycolactone, the exotoxin virulence factor that is both immunosuppressive and cytotoxic. The discovery that the essential Sec61 translocon is the major cellular target of mycolactone explains much of the disease pathology, including the immune blockade. Sec61 inhibition leads to a loss in production of nearly all cytokines from monocytes, macrophages, dendritic cells and T cells, as well as antigen presentation pathway proteins and costimulatory molecules. However, there has long been evidence that the immune system is not completely incapable of responding to M. ulcerans infection. In particular, IL-1ß was recently shown to be present in BU lesions, and to be induced from M. ulcerans-exposed macrophages in a mycolactone-dependent manner. This has important implications for our understanding of BU, showing that mycolactone can act as the "second signal" for IL-1ß production without inhibiting the pathways of unconventional secretion it uses for cellular release. In this Perspective article, we validate and discuss this recent advance, which is entirely in-line with our understanding of mycolactone's inhibition of the Sec61 translocon. However, we also show that the IL-1 receptor, which uses the conventional secretory pathway, is sensitive to mycolactone blockade at Sec61. Hence, a more complete understanding of the mechanisms regulating IL-1ß function in skin tissue, including the transient intra-macrophage stage of M. ulcerans infection, is urgently needed to uncover the double-edged sword of IL-1ß in BU pathogenesis, treatment and wound healing.
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Úlcera de Buruli/inmunología , Interleucina-1beta/inmunología , Macrólidos/metabolismo , Macrófagos/inmunología , Canales de Translocación SEC/metabolismo , Humanos , Mycobacterium ulcerans/patogenicidadRESUMEN
Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.
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Macrólidos/farmacología , Microsomas/química , Ribosomas/química , Canales de Translocación SEC/química , Animales , Sitios de Unión , Sistema Libre de Células/metabolismo , Perros , Expresión Génica , Células HCT116 , Células HEK293 , Humanos , Macrólidos/química , Macrólidos/aislamiento & purificación , Microsomas/metabolismo , Simulación de Dinámica Molecular , Mutación , Mycobacterium ulcerans/química , Mycobacterium ulcerans/patogenicidad , Páncreas/química , Páncreas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Ribosomas/metabolismo , Canales de Translocación SEC/antagonistas & inhibidores , Canales de Translocación SEC/genética , Canales de Translocación SEC/metabolismo , Homología Estructural de Proteína , Especificidad por SustratoRESUMEN
Background: The innate immune response is a tightly regulated process that reacts rapidly in response to pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS). Evidence is accumulating that microRNAs contribute to this, although few studies have examined the early events that constitute the "primary" response. Methods: LPS-dependent changes to miRNA expression were studied in primary human monocyte-derived macrophages (1°MDMs). An unbiased screen by microarray was validated by qPCR and a method for the absolute quantitation of miRNAs was also developed, utilising 5' phosphorylated RNA oligonucleotide templates. RNA immunoprecipitation was performed to explore incorporation of miRNAs into the RNA-induced silencing complex (RISC). The effect of miRNA functional inhibition on TNF expression (mRNA and secretion) was investigated. Results: Of the 197 miRNAs expressed in 1°MDMs, only five were induced >1.5-fold. The most strongly induced was miR-155-3p, the partner strand to miR-155-5p, which are both derived from the BIC gene (B cell integration cluster, MIR155HG). The abundance of miR-155-3p was induced transiently ~250-fold at 2-4hrs and then returned towards baseline, mirroring the BIC mRNA. Other PAMPs, IL-1ß, and TNF caused similar responses. IL-10, NF-κB, and JNK inhibition suppressed these responses, unlike cytokine-suppressing mycolactone. Absolute quantitation showed that miRNA abundance varies widely from donor-to-donor, and showed that miR-155-3p abundance is substantially less than miR-155-5p in unstimulated cells. However, at its peak there were 446-1,113 copies/cell, and miR-155-3p was incorporated into the RISC with an efficiency similar to miR-16-5p and miR-155-5p. Inhibition of neither miRNA affected TNF expression in 1°MDMs, but technical challenges here are noted. Conclusions: Dynamic regulation of miRNAs during the primary response is rare, with the exception of miR-155-3p, which transiently achieves levels that might have a biological effect. Further work on this candidate would need to overcome the technical challenges of the broad-ranging effects of liposomes on 1°MDMs.
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Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used chemical proteomics to identify Sec61α (protein transport protein Sec61 subunit alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions, enabling a biotin analogue of ipomoeassin F to pull down Sec61α from live cells, yet it is also reversible, as judged by several experiments including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of TNF biogenesis after washout. Sec61α forms the central subunit of the ER protein translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile demonstrated by specific amino acid single-point mutations in Sec61α provides compelling evidence that Sec61α is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to Sec61α is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61α function and to further investigate its potential as a therapeutic target for drug discovery.
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Glicoconjugados/farmacología , Canales de Translocación SEC/antagonistas & inhibidores , Sitios de Unión/efectos de los fármacos , Glicoconjugados/química , Humanos , Estructura Molecular , Transporte de Proteínas/efectos de los fármacos , Canales de Translocación SEC/metabolismoRESUMEN
PURPOSE OF THE REVIEW: Buruli ulcer (BU) is a necrotizing and disabling cutaneous disease caused by Mycobacterium ulcerans, one of the skin-related neglected tropical diseases (skin NTDs). This article aims to review the current knowledge of this disease and challenges ahead. RECENT FINDINGS: Around 60,000 cases of BU have been reported from over 33 countries between 2002 and 2017. Encouraging findings for development of point-of-care tests for BU are being made, and its treatment is currently in the transition period from rifampicin plus streptomycin (injection) to all-oral regimen. A major recent advance in our understanding of its pathogenesis has been agreement on the mechanism of action of the major virulence toxin mycolactone in host cells, targeting the Sec61 translocon during a major step in protein biogenesis. SUMMARY: BU is distributed mainly in West Africa, but cases are also found in other parts of the world. We may be underestimating its true disease burden, due to the limited awareness of this disease. More awareness and more understanding of BU will surely contribute in enhancing our fight against this skin NTD.
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Mycolactone is the exotoxin virulence factor of Mycobacterium ulcerans that causes the neglected tropical disease Buruli ulcer. We recently showed it to be a broad spectrum inhibitor of Sec61-dependent co-translational translocation of proteins into the endoplasmic reticulum (ER). An outstanding question is the molecular pathway linking this to its known cytotoxicity. We have now used translational profiling to better understand the reprogramming that occurs in cells exposed to mycolactone. Gene ontology identified enrichment in genes involved in cellular response to stress, and apoptosis signalling among those showing enhanced translation. Validation of these results supports a mechanism by which mycolactone activates an integrated stress response meditated by phosphorylation of eIF2α via multiple kinases (PERK, GCN, PKR) without activation of the ER stress sensors IRE1 or ATF6. The response therefore uncouples the integrated stress response from ER stress, and features translational and transcriptional modes of genes expression that feature the key regulatory transcription factor ATF4. Emphasising the importance of this uncoupled response in cytotoxicity, downstream activation of this pathway is abolished in cells expressing mycolactone-resistant Sec61α variants. Using multiple genetic and biochemical approaches, we demonstrate that eIF2α phosphorylation is responsible for mycolactone-dependent translation attenuation, which initially protects cells from cell death. However, chronic activation without stress remediation enhances autophagy and apoptosis of cells by a pathway facilitated by ATF4 and CHOP. Our findings demonstrate that priming events at the ER can result in the sensing of stress within different cellular compartments.
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Factor de Transcripción Activador 4/metabolismo , Toxinas Bacterianas/toxicidad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Factor 2 Eucariótico de Iniciación/metabolismo , Macrólidos/toxicidad , Canales de Translocación SEC/metabolismo , Factor de Transcripción Activador 4/genética , Animales , Línea Celular , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Humanos , Ratones , Transporte de Proteínas/efectos de los fármacos , Canales de Translocación SEC/genéticaRESUMEN
Mycolactone is the exotoxin virulence factor produced by Mycobacterium ulcerans, the pathogen responsible for Buruli ulcer. The skin lesions and immunosuppression that are characteristic of this disease result from the action of mycolactone, which targets the Sec61 complex and inhibits the co-translational translocation of secretory proteins into the endoplasmic reticulum. In this study, we investigate the effect of mycolactone on the Sec61-dependent biogenesis of different classes of transmembrane protein (TMP). Our data suggest that the effect of mycolactone on TMP biogenesis depends on how the nascent chain initially engages the Sec61 complex. For example, the translocation of TMP lumenal domains driven by an N-terminal cleavable signal sequence is efficiently inhibited by mycolactone. In contrast, the effect of mycolactone on protein translocation that is driven solely by a non-cleavable signal anchor/transmembrane domain depends on which flanking region is translocated. For example, while translocation of the region N-terminal to a signal anchor/transmembrane domain is refractive to mycolactone, C-terminal translocation is efficiently inhibited. Our findings highlight the diversity of Sec61-dependent translocation and provide a molecular basis for understanding the effect of mycolactone on the biogenesis of different TMPs.
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Macrólidos/farmacología , Proteínas de la Membrana/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Canales de Translocación SEC/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Humanos , Proteínas de la Membrana/química , Dominios Proteicos , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The virulence factor mycolactone is responsible for the immunosuppression and tissue necrosis that characterise Buruli ulcer, a disease caused by infection with Mycobacterium ulcerans In this study, we confirm that Sec61, the protein-conducting channel that coordinates entry of secretory proteins into the endoplasmic reticulum, is a primary target of mycolactone, and characterise the nature of its inhibitory effect. We conclude that mycolactone constrains the ribosome-nascent-chain-Sec61 complex, consistent with its broad-ranging perturbation of the co-translational translocation of classical secretory proteins. In contrast, the effect of mycolactone on the post-translational ribosome-independent translocation of short secretory proteins through the Sec61 complex is dependent on both signal sequence hydrophobicity and the translocation competence of the mature domain. Changes to protease sensitivity strongly suggest that mycolactone acts by inducing a conformational change in the pore-forming Sec61α subunit. These findings establish that mycolactone inhibits Sec61-mediated protein translocation and highlight differences between the co- and post-translational routes that the Sec61 complex mediates. We propose that mycolactone also provides a useful tool for further delineating the molecular mechanisms of Sec61-dependent protein translocation.
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Úlcera de Buruli/patología , Macrólidos/metabolismo , Mycobacterium ulcerans/patogenicidad , Canales de Translocación SEC/antagonistas & inhibidores , Canales de Translocación SEC/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Humanos , Transporte de Proteínas/fisiología , Ribosomas/metabolismoRESUMEN
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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Úlcera de Buruli/microbiología , Úlcera de Buruli/patología , Macrólidos/toxicidad , Mycobacterium ulcerans/metabolismo , Mycobacterium ulcerans/patogenicidad , Animales , Citotoxinas/toxicidad , Humanos , Inmunosupresores/toxicidadRESUMEN
A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2 ng/ml and as early as 8 hrs after exposure. TM activates protein C by altering thrombin's substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells' ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone's effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.
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Antibacterianos/uso terapéutico , Úlcera de Buruli/tratamiento farmacológico , Fibrina/metabolismo , Macrólidos/uso terapéutico , Mycobacterium ulcerans/fisiología , Trombomodulina/metabolismo , Úlcera de Buruli/diagnóstico , Úlcera de Buruli/metabolismo , Úlcera de Buruli/microbiología , Células Endoteliales/metabolismo , Humanos , Macrólidos/metabolismo , Necrosis/microbiología , Piel/microbiología , Piel/patologíaRESUMEN
Infection with Mycobacterium ulcerans is characterised by tissue necrosis and immunosuppression due to mycolactone, the necessary and sufficient virulence factor for Buruli ulcer disease pathology. Many of its effects are known to involve down-regulation of specific proteins implicated in important cellular processes, such as immune responses and cell adhesion. We have previously shown mycolactone completely blocks the production of LPS-dependent proinflammatory mediators post-transcriptionally. Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages. Instead, it inhibits the production of these, along with nearly all other (induced and constitutive) proteins that transit through the ER. This is due to a blockade of protein translocation and subsequent degradation of aberrantly located protein. Several lines of evidence support this transformative explanation of mycolactone function. First, cellular TNF and Cox-2 can be once more detected if the action of the 26S proteasome is inhibited concurrently. Second, restored protein is found in the cytosol, indicating an inability to translocate. Third, in vitro translation assays show mycolactone prevents the translocation of TNF and other proteins into the ER. This is specific as the insertion of tail-anchored proteins into the ER is unaffected showing that the ER remains structurally intact. Fourth, metabolic labelling reveals a near-complete loss of glycosylated and secreted proteins from treated cells, whereas cytosolic proteins are unaffected. Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types. These studies provide a new mechanism underlying mycolactone's observed pathological activities both in vitro and in vivo. Mycolactone-dependent inhibition of protein translocation into the ER not only explains the deficit of innate cytokines, but also the loss of membrane receptors, adhesion molecules and T-cell cytokines that drive the aetiology of Buruli ulcer.
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Retículo Endoplásmico/metabolismo , Mediadores de Inflamación/metabolismo , Macrólidos/metabolismo , Mycobacterium ulcerans/patogenicidad , Animales , Úlcera de Buruli/metabolismo , Úlcera de Buruli/microbiología , Úlcera de Buruli/patología , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Ciclooxigenasa 2/metabolismo , Retículo Endoplásmico/patología , Interleucina-6/metabolismo , Lipopolisacáridos/toxicidad , Ratones , Mycobacterium ulcerans/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Mycolactone is a polyketide macrolide lipid-like secondary metabolite synthesized by Mycobacterium ulcerans, the causative agent of BU (Buruli ulcer), and is the only virulence factor for this pathogen identified to date. Prolonged exposure to high concentrations of mycolactone is cytotoxic to diverse mammalian cells (albeit with varying efficiency), whereas at lower doses it has a spectrum of immunosuppressive activities. Combined, these pleiotropic properties have a powerful influence on local and systemic cellular function that should explain the pathophysiology of BU disease. The last decade has seen significant advances in our understanding of the molecular mechanisms underlying these effects in a range of different cell types. The present review focuses on the current state of our knowledge of mycolactone function, and its molecular and cellular targets, and seeks to identify commonalities between the different functional and cellular systems. Since mycolactone influences fundamental cellular processes (cell division, cell death and inflammation), getting to the root of how mycolactone achieves this could have a profound impact on our understanding of eukaryotic cell biology.