Professor Rachel Simmonds FRSB

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-1β 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-1β 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-1β 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-1β in BU pathogenesis, treatment and wound healing.

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.

This book reviews successes and (remaining) challenges in vaccine development for the selected Neglected Tropical Diseases (NTD) of Leprosy, Leishmaniasis, Meliodoisis and Tuberculosis, which are a continuous burden for millions of people in affected areas worldwide. Written by frontline researchers, the volume deep-dives into different vaccine strategies, provides biotechnological background information and also tackles animal models in NTD therapeutics research. By bringing together state-of-the-art expert knowledge, the book contributes to the aim of ultimately ending the epidemics of neglected tropical diseases, complying with UN Sustainable Development Goal 3, Health and Well-Being.The volume highlights the activities of the research network VALIDATE (VAccine deveLopment for complex Intracellular neglecteD pAThogEns), funded by the Medical Research Council in the UK. The four NTDs discussed in the book were selected as these are in the focus of VALIDATE’s research. The book targets scientists and clinicians working on NTDs, as well as all readers with a background in biomedicine and interest in vaccine development. This is an open access book.

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 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.

A medical illustration of Buruli ulcer (BU) at the plaque stage with a raised portion of skin. BU is part of a set of skin related neglected topical diseases (skin NTDs), which are chronic infectious diseases found in tropical and sub-tropical regions of the world. The purpose to illustrate how BU plaque with raised skin presents, provided for use within visual communications and for educational purposes regarding skin NTDs. The original purpose for use by health care workers in Ghana, Africa.

A medical illustration of Buruli ulcer (BU) at the plaque stage with oedema which is additional swelling. BU is part of a set of skin related neglected topical diseases (skin NTDs), which are chronic infectious diseases found in tropical and sub-tropical regions of the world. The purpose to illustrate how BU plaque with oedema presents on the skin, provided for use within visual communication and for educational purposes regarding skin NTDs. The original purpose for use by health care workers in Ghana, Africa.

A medical illustration of Buruli ulcer (BU) at the small lesion stage. BU is part of a set of skin related neglected topical diseases (skin NTDs), which are chronic infectious diseases found in tropical and sub-tropical regions of the world. The purpose to illustrate how BU at the small lesion stage presents on the skin, provided for use within visual communications and for educational purposes regarding skin NTDs. The original purpose for use by health care workers in Ghana, Africa.

A medical illustration of Buruli ulcer (BU) at the large lesion stage. BU is part of a set of skin related neglected topical diseases (skin NTDs), which are chronic infectious diseases found in tropical and sub-tropical regions of the world. The purpose to illustrate how BU with a large lesion presents on the skin, provided for use within visual communications and for educational purposes regarding skin NTDs. The original purpose for use by health care workers in Ghana, Africa.

The plant-derived macrocyclic resin glycoside ipomoeassin F (Ipom-F) binds to Sec61 alpha 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 Sec61 alpha 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.

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.

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.

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.

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.

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.

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.

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.

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 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.