Research in my group focusses on the mechanism of action of mycolactone, the lipid-like exotoxin of the Buruli ulcer infectious agent, Mycobacterium ulcerans. Mycolactone has many unusual and fascinating biological functions, and in 2014 we identified its mechanism of action - as an inhibitor of protein translocation into the endoplasmic reticulum. We are now applying this knowledge to understand more about Buruli ulcer, a neglected tropical disease, and basic cell biology. In particular, I have a Wellcome Trust Investigator Award in Science to investigate how this mechanism underpins the role of disordered blood clotting in the pathogenesis of Buruli ulcer.
After graduating from the University of Manchester with a degree in Molecular Biology, I went on to study for a PhD with David Lane at Imperial College London. My early career was in haemostasis (regulation of blood clotting), with a particular focus on the protein C anticoagulant pathway and endothelial cell biology. I had a career break and then joined the Macrophage Biology group at the Kennedy Institute of Rheumatology (Imperial College London), changing my research field to study inflammatory signalling. My first academic appointment here at the University of Surrey started when I recieved my first award from the Wellcome Trust.
As a molecular biologist, my research interests concern the molecular detail of the pathogenesis of different disease systems. My group specialises in the uncovering of “unusual” mechanisms of gene regulation in eukaryotic cells; as exemplified by our work studying mycolactone function. In order to understand this fully, we have had to (and to an extent still are) follow a trial of breadcrumbs through the fundamental concepts of molecular biology starting from gene transcription, through protein translation and on to protein fate.
University roles and responsibilities
- Director of Post-Graduate Research for the Department of Microbial Sciences
The major focus of my research is understanding the mechanism of action of the mycobacterial virulence factor mycolactone. This lipid-like molecule is synthesised by a small group of so-called mycolactone-producing mycobacteria (MPM) which each make subtly different mycolactones. Species in this group include those which are pathogenic to frogs, fish and, importantly, humans. The latter, Mycobacterium ulcerans, causes a disease called Buruli ulcer which is a serious skin infection that affects some of the poorest communities in the world. It causes massive debilitating skin ulcers, often leading to disfigurement or amputation (see http://www.who.int/buruli/en/).
Mycolactone is known to be responsible for the pathogenesis of Buruli ulcer, because it is both immunosuppressive and cytotoxic to host cells. The immunosuppression includes inhibition of the production of cytokines that drive inflammation. We first showed that production of TNF, IL-6 and Cox-2 were supressed by a post-transcriptional mechanism. Detailed investigations then identified the mechanistic target of the molecule: it prevents the translocation of proteins into the endoplasmic reticulum. Most glycosylated and secreted proteins (like cytokines) undergo a process of co-translational translocation via the Sec61 translocon which acts channel separating the two cellular compartments. Because mycolactone blocks this, the proteins get stuck in the cytosol, are recognised as being mislocated, and are destroyed by the proteasome. Therefore, many proteins that would normally pass through the ER, potentially up to 25% of total cellular protein are made as normal in the presence of mycolactone, but are immediately destroyed again. This highly unusual pathogenic molecular mechanism currently seems unique to Buruli ulcer.
We are now exploiting this knowledge in order to develop better treatments for Buruli ulcer, and other diseases where protein translocation plays an important role. We recently showed that endothelial cells are exquisitely sensitive to mycolactone; 7ng/ml mycolactone is sufficient to deplete thrombomodulin from the surface of the cells in under 24hours. Since thrombomodulin is an essential anticoagulant protein, and fibrin is commonly found in the necrotic skin tissue, this has opened up the whole area of disordered haemostasis in Buruli ulcer. I currently hold a Wellcome Trust Investigator Award in Science to fully delve into how mycolactone and changes to blood coagulation drive the pathological process.
Current group members
Belinda Hall (Post-Doc)
Louise Hsieh (Post-Doc)
Jane Newcombe (Post-Doc)
Katherine Corfield (PhD Student)
Jo Butler (Interdisciplinary PhD Student in Medical Illustration)
Aloysius Loglo (Collaborative PhD Student at KCCR, Kumasi, Ghana)
Lucy Eke (Collaborative PhD Student in Virology)
Efi Mavrogiannaki (Collaborative PhD Student in Chemistry)
Elizabeth Gyamfi (Visiting PhD Student from WACCBIP, Accra, Ghana)
Past group members
Joy Ogbechi (PhD Student)
Scott Dos Santos (Euromasters Student)
Sweta Jain (MSc Student)
Phil Biggin (Oxford): MD Simulations of mycolactone interactions
Adolfo Cavalie (Saarland University Faculty of Medicine): Calcium leak via the Sec61 channel
Mark Field (Dundee): Evolution of the Sec61 translocon in eukaryotes
Matt Higgins (Oxford): Structural Biology
Stephen High (University of Manchester): Protein translocation
Rebecca Hoyle (Southampton: Mathematical modelling of Buruli ulcer lesions
Nick Ktistakis (Babraham): Early stage induction of autophagy by mycolactone
Lydia Mosi (WACCBIP, Ghana): Ultra-long read sequencing of the M. ulcerans genome
Richard Phillips (KCCR, Ghana): Buruli ulcer clinical research
Gerd Pluschke (Swiss Tropical and Public Health Institute): Buruli ulcer pathogenesis
Anne Willis (MRC Toxicology Unit): Translational control
Indicators of esteem
Member of the Wellcome Trust Expert Review Group on Pathogen Biology
Editorial board of Tuberculosis
Member of the Biochemical Society's Awards Committee
Visiting Research Fellow at University of Southampton
Visiting Lecturer at the University of Ghana
Postgraduate research supervision
Aloysius Loglo (2018-current) Collaborative supervisor KCCR, Ghana
The role of diet and coagulation in the pathogenesis of Buruli ulcer
Katherine Corfield (2017-current) Principal supervisor University of Surrey
The evolution of mycolactone-dependent inhibition of the Sec61 translocon
Efi Mavrogiannaki (2017-current) Co-supervisor University of Surrey
New approaches to chemical synthesis of mycolactone
Fatumah Atuhaire (2017-current) Collaborative supervisor University of Southampton
Mathematical modelling of Buruli ulcer lesion formation
Jo Culley (2017-current) Principal supervisor University of Surrey
Utilising medical illustration to improve understanding & communication of disfiguring skin NTDs
Lucy Eke (2017-current) Co-supervisor University of Surrey
Realising the therapeutic potential of exotoxins
Elizabeth Gyamfi (2016-current) Collaborative supervisor University of Ghana
Probing the diversity of Mycolactone-producing mycobacteria using MinION Sequencing
Joy Ogbechi (2013-2016) Principal supervisor University of Surrey
Investigating the mechanisms behind the tissue necrosis in Mycobacterium ulcerans infection
BMS2045: Introduction to Immunology
BMS2036: Molecular Biology and Genetics, from Genes to Biological Function
BMS3054: Clinical Immunology and Immunohaematology
MSc in Medical Microbiology
MMIM024: Pathogenesis of Infectious Diseases
MMIM027: Research Methods 2 (Module Coordinator)
Courses I teach on
Toll-like receptors (TLRs) are central to innate immunity and yet their expression is widespread and not restricted to professional inflammatory cells. TLRs have been reported on adipocytes and have been implicated in obesity-associated pathologies such as diabetes. Why TLRs are found on adipocytes is not clear although one hypothesis is that they may coordinate energy utilization for the energy intensive process of an immune response. We have explored TLR signalling in primary human in vitro differentiated adipocytes and investigated the specific adapter molecules that are involved. Only lipopolysaccharide (LPS), poly(I:C), Pam3CSK4 and MALP-2 could induce the production of IL-6, IL-8 and MCP-1 by adipocytes. Poly(I:C) alone caused a strong induction of type I interferons, as assessed by IP-10 production. Using siRNA, it was confirmed that LPS-dependent signalling in adipocytes occurs via TLR4 utilizing the adapter molecules MyD88, Mal and TRIF and caused rapid degradation of IκBα. Pam3CSK4 signalling utilized TLR2, MyD88 and Mal (but not TRIF). However, the response to poly(I:C) observed in these cells appeared not to require TRIF, but MyD88 was required for induction of NFκB-dependent cytokines by Poly(I:C). Despite this, IκBα degradation could not be detected in poly(I:C) stimulated adipocytes at any time-point up to 4 h. Indeed, IL-6 transcription was not induced until 8-16 h after exposure. These data suggest that Pam3CSK4 and LPS signal via the expected routes in human adipocytes, whereas poly(I:C)/TLR3 signalling may act via a TRIF-independent, MyD88-dependent route.
Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation chan-nel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compoundsinhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, makingthis a promising target for therapeutic intervention. To understand the mode of inhibition and provide insightinto the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61inhibited by theMycobacterium ulceransexotoxin mycolactone via electron cryo-microscopy. Unexpect-edly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedgingopen the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively trans-port substrate proteins implies that signal peptides and transmembrane domains pass through the site occu-pied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhib-itors and reveals novel features of translocon function and dynamics.
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.
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.
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 amongst 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 transcription modes of genes expression that feature the key regulatory transcription factor ATF4. Emphasizing 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.
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 thatmicroRNAs contribute to this, although few studies have examined the earlyevents that constitute the “primary” response. LPS-dependent changes to miRNA expression were studied inMethods:primary human monocyte-derived macrophages (1°MDMs). An unbiasedscreen by microarray was validated by qPCR and a method for the absolutequantitation of miRNAs was also developed, utilising 5’ phosphorylatedRNA oligonucleotide templates. RNA immunoprecipitation was performedto explore incorporation of miRNAs into the RNA-induced silencingcomplex (RISC). The effect of miRNA functional inhibition on TNFexpression (mRNA and secretion) was investigated. Of the 197 miRNAs expressed in 1°MDMs, only five were inducedResults:>1.5-fold. The most strongly induced was miR-155-3p, the partner strand tomiR-155-5p, which are both derived from the MIR155HG/BIC gene(pri-miR-155). The abundance of miR-155-3p was induced transiently~250-fold at 2-4hrs and then returned towards baseline, mirroringpri-miR-155. Other PAMPs, IL-1β, and TNF caused similar responses.IL-10, NF-κB, and JNK inhibition reduced these responses,unlike cytokine-suppressing mycolactone. Absolute quantitation revealedthat miRNA abundance varies widely from donor-to-donor, and showed thatmiR-155-3p abundance is substantially less than miR-155-5p inunstimulated 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 tomiR-16-5p and miR-155-5p. Inhibition of neither miRNA affected TNFsecretion after 2hrs in 1°MDMs, but technical challenges here are noted. Dynamic regulation of miRNAs during the primary responseConclusions:is rare, with the exception of miR-155-3p. Further work is required toestablish whether its low abundance, even at the transient peak, issufficient for biological activity and to determine whether there are specificmechanisms determining its biogenesis from miR-155 precursor.
In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
Mycolactone is the exotoxin virulence factor produced by Mycobacterium ulcerans, the pathogen responsible for Buruli ulcer. The skin lesions and immunosuppression 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, 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 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.
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 2ng/ml and as early as 8hrs 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.
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.
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.
Buruli Ulcer (BU) is the third most common mycobacterium disease following only tuberculosis and leprosy. Though BU is thought to be associated with large-and small-scale disturbances to the landscape and bodies of water frequented by human populations, primary prevention of BU is difficult because the mode of transmission is not known. This chapter reviews the most common environmental risk factors for BU and recent research into understanding its transmission. It is predicted that the proteins affected by mycolactone may share an underlying mechanism of production that could explain their co-regulation. Early work on the mechanism of suppression by mycolac-tone was carried out in Jurkat T cells using ASLs and focused on the suppression of IL-2 production. A multidisciplinary approach to treatment and patient care is essential for optimizing treatment outcomes. Physiotherapy is paramount minimizing and/or preventing disabilities.
Additional key publications
Simmonds RE and Foxwell BM. NF-kB and its relevance to arthritis and inflammation (Review). Rheumatology (Oxford) 2008: 47; 584-90
Rance J, Follows GA, Cockerill PN, Bonifer C, Lane DA, Simmonds RE. Regulation of the human endothelial cell protein C receptor gene promoter by multiple Sp1 binding sites. Blood 2003:101; 4393-4401
Rezende SM, Lane DA, Mille-Baker B, Samama M, Conard J and Simmonds RE. Protein S Gla-domain mutations causing impaired Ca2+-induced phospholipid binding and severe functional protein S deficiency. Blood 2002: 100; 2812-9
Simmonds RE and Lane DA. The Endothelial Cell Protein C Receptor: A Candidate Genetic Risk Factor for Thrombosis (Invited Commentary). Thromb Haemost 2001: 86; 939-41
Simmonds RE and Lane DA. Structural and functional implications of the intron/exon organisation of the human endothelial cell protein C/activated protein C receptor (EPCR) gene. Comparison with the structure of CD1/Major Histocompatibility Complex alpha1 and alpha2 domains. Blood 1999: 94; 632-41
Simmonds RE, Ireland H, Lane DA, Zöller B, García de Frutos P and Dahlbäck B . Clarification of the risk of venous thrombosis associated with hereditary protein S deficiency by investigation of a large kindred with a characterised gene defect. Ann Int Med 1998; 128; 8-14
Simmonds RE, Zöller B, Ireland H, Thompson E, García de Frutos P, Dahlbäck B and Lane DA. Genetic and phenotypic analysis of a large (122 member) protein S-deficient kindred provides an explanation for the familial coexistence of type I and type III plasma phenotypes. Blood 1997; 89; 4364-70