The essential malaria protein PfCyRPA targets glycans to invade erythrocytes.

Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.

Repurposing of Tuberculosis Drug Candidates for the Treatment of Mycobacterium ulcerans Disease.

Buruli ulcer (BU) is a chronic necrotizing skin disease caused by Mycobacterium ulcerans. Historically, the disease was treated by surgical excision of the skin lesions, until an 8-week combination therapy of rifampicin and streptomycin was introduced in 2004. This treatment modality was effective and reduced recurrence rates. Rifampicin is the most efficacious antibiotic for the treatment of BU and, should rifampicin-resistant M. ulcerans strains emerge, there is currently no replacement for it. As for mycobacterial diseases in general, there is a pressing need for the development of novel, fast-acting drugs. Under market economy conditions, repurposing of new tuberculosis drug candidates is the most promising avenue for alternative BU treatments. Our drug repurposing activities have led to the identification of several actives against M. ulcerans. In particular, the cytochrome bc1 complex inhibitor telacebec (Q203) is a promising drug candidate for the treatment of BU in Africa and Australia. While an active cytochrome-bd oxidase bypass limits the potency of the cytochrome-bc1-specific inhibitor telacebec against M. tuberculosis, classical lineage M. ulcerans strains rely exclusively on cytochrome-bc1 to respire. Hence, telacebec is effective at nanomolar concentration against M. ulcerans, and a high treatment efficacy in an experimental mouse infection model indicates that treatment of BU could be substantially shortened and simplified by telacebec.

Key Contributions by the Swiss Tropical and Public Health Institute Towards New and Better Drugs for Tropical Diseases.

Thanks to its expertise in clinical research, epidemiology, infectious diseases, microbiology, parasitology, public health, translational research and tropical medicine, coupled with deeply rooted partnerships with institutions in low- and middle-income countries (LMICs), the Swiss Tropical and Public Health Institute (Swiss TPH) has been a key contributor in many drug research and development consortia involving academia, pharma and product development partnerships. Our know-how of the maintenance of parasites and their life-cycles in the laboratory, plus our strong ties to research centres and disease control programme managers in LMICs with access to field sites and laboratories, have enabled systems for drug efficacy testing in vitro and in vivo, clinical research, and modelling to support the experimental approaches. Thus, Swiss TPH has made fundamental contributions towards the development of new drugs - and the better use of old drugs - for neglected tropical diseases and infectious diseases of poverty, such as Buruli ulcer, Chagas disease, food-borne trematodiasis (e.g. clonorchiasis, fascioliasis and opisthorchiasis), human African trypanosomiasis, leishmaniasis, malaria, schistosomiasis, soil-transmitted helminthiasis and tuberculosis. In this article, we show case the success stories of molecules to which Swiss TPH has made a substantial contribution regarding their use as anti-infective compounds with the ultimate aim to improve people's health and well-being.

In vaccinated individuals serum bactericidal activity against B meningococci is abrogated by C5 inhibition but not by inhibition of the alternative complement pathway.

Introduction: Several diseases caused by the dysregulation of complement activation can be treated with inhibitors of the complement components C5 and/or C3. However, complement is required for serum bactericidal activity (SBA) against encapsulated Gram-negative bacteria. Therefore, C3 and C5 inhibition increases the risk of invasive disease, in particular by Neisseria meningitidis. As inhibitors against complement components other than C3 and C5 may carry a reduced risk of infection, we compared the effect of inhibitors targeting the terminal pathway (C5), the central complement component C3, the alternative pathway (FB and FD), and the lectin pathway (MASP-2) on SBA against serogroup B meningococci. Methods: Serum from adults was collected before and after vaccination with the meningococcal serogroup B vaccine 4CMenB and tested for meningococcal killing. Since the B capsular polysaccharide is structurally similar to certain human polysaccharides, 4CMenB was designed to elicit antibodies against meningococcal outer membrane proteins. Results: While only a few pre-vaccination sera showed SBA against the tested B meningococcal isolates, 4CMenB vaccination induced potent complement-activating IgG titers against isolates expressing a matching allele of the bacterial cell surface-exposed factor H-binding protein (fHbp). SBA triggered by these cell surface protein-specific antibodies was blocked by C5 and reduced by C3 inhibition, whereas alternative (factor B and D) and lectin (MASP-2) pathway inhibitors had no effect on the SBA of post-4CMenB vaccination sera. Discussion: Compared to the SBA triggered by A,C,W,Y capsule polysaccharide conjugate vaccination, SBA against B meningococci expressing a matching fHbp allele was remarkably resilient against the alternative pathway inhibition.

High-Frequency Changes in Pilin Glycosylation Patterns during Neisseria meningitidis Serogroup a Meningitis Outbreaks in the African Meningitis Belt.

In the meningitis belt of sub-Saharan Africa, there are cyclic meningococcal epidemics that coincide with clonal waves of Neisseria meningitidis carriage and invasive disease. In the framework of longitudinal colonization and disease studies in Ghana and Burkina Faso, meningococcal isolates belonging to the closely related hypervirulent A:ST-5, A:ST-7, and A:ST-2859 clones have been collected from 1998 to 2011 during meningococcal outbreaks. A comparative whole-genome sequencing study with 100 of these isolates identified the pilin glycosylation (pgl) locus as one hot spot of recombination. Frequent exchange of pgl genes in N. meningitidis by lateral gene transfer results in differences in the glycosylation patterns of pilin and other cell surface glycoproteins. In this study, we looked at both recombination and phase variation of the pgl genes of these clinical isolates and analyzed the glycan structures resulting from different pgl alleles and their variable expression. Our results indicate that the basal O-linked sugar of the glycans expressed by these isolates is masked by various additional mono- or disaccharide structures whose expression is highly variable due to the phase-variable expression of pgl genes. We also observed a distinct glycoform in two isolates with pgl loci that were modified by recombination. These data suggest that variation in N. meningitidis protein glycosylation could be crucial for bacterial adaptation to evade herd immunity in semi-immune populations. Investigating pilin glycosylation in N. meningitidis can shed light on the mechanisms by which this pathogen evades the host immune response, and may help identify potential targets for novel therapies and vaccines.

The malaria blood stage antigen PfCyRPA formulated with the TLR-4 agonist adjuvant GLA-SE elicits parasite growth inhibitory antibodies in experimental animals.

BACKGROUND: Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. In contrast to several previously clinically tested merozoite vaccine candidate antigens, PfCyRPA is not polymorphic, making it a promising candidate antigen for blood stage vaccine development. METHODS: Mice and rabbits were immunized with vaccine formulations of recombinantly expressed PfCyRPA adjuvanted either with the glucopyranosyl lipid A (GLA) containing adjuvants GLA-LSQ, GLA-SE, GLA-Alum or with Nanoalum. ELISA and indirect immunofluorescence assays (IFA) were used to analyse elicited IgG titers and the P. falciparum growth inhibitory activity was determined with a standardized in vitro [3H]-hypoxanthine incorporation assay. RESULTS: In the mouse experiments, the GLA adjuvanted formulations were superior to the Nanoalum formulation with respect to antibody titer development, IFA sero-conversion rates and in vitro parasite growth-inhibitory activity. In rabbits, the highest titers of parasite growth inhibitory antibodies were obtained with the GLA-SE formulation. Comparable mean ELISA IgG endpoint titers were reached in rabbits after three immunizations with GLA-SE adjuvanted PfCyRPA doses of 5, 25 and 100 µg, but with 100 µg of antigen, only two immunizations were required to reach this titer. CONCLUSION: PfCyRPA formulated with the human-compatible adjuvant GLA-SE represents an attractive vaccine candidate for early clinical testing in a controlled P. falciparum blood stage challenge trial.

Inhibition of the different complement pathways has varying impacts on the serum bactericidal activity and opsonophagocytosis against Haemophilus influenzae type b.

Defense against Haemophilus influenzae type b (Hib) is dependent on antibodies and complement, which mediate both serum bactericidal activity (SBA) and opsonophagocytosis. Here we evaluated the influence of capsule-specific antibodies and complement inhibitors targeting the central component C3, the alternative pathway (AP; fB, fD), the lectin pathway (LP; MASP-2) and the terminal pathway (C5) on both effector functions. Findings may be relevant for the treatment of certain diseases caused by dysregulation of the complement system, where inhibitors of complement factors C3 or C5 are used. Inhibitors against other complement components are being evaluated as potential alternative treatment options that may carry a reduced risk of infection by encapsulated bacteria. Serum and reconstituted blood of healthy adults were tested for bactericidal activity before and after vaccination with the Hib capsule-conjugate vaccine ActHIB. Most sera had bactericidal activity prior to vaccination, but vaccination significantly enhanced SBA titers. Independently of the vaccination status, both C3 and C5 inhibition abrogated SBA, whereas inhibition of the LP had no effect. AP inhibition had a major inhibitory effect on SBA of pre- vaccination serum, but vaccination mitigated this inhibition for all disease isolates tested. Despite this, SBA-mediated killing of some Hib isolates remained retarded. Even for the most serum-resistant isolate, SBA was the dominating defense mechanism in reconstituted whole blood, as addition of blood cells to the serum did not enhance bacterial killing. Limited Fc receptor-mediated opsonophagocytosis was unmasked when bacterial killing by the membrane attack complex was blocked. In the presence of C3 or C5 inhibitors, addition of post-vaccination, but not of pre-vaccination serum to the blood cells triggered opsonophagocytosis, leading to suppression of bacterial multiplication. Taken together, our data indicate that for host defense against Hib, killing by SBA is more efficient than by blood cell opsonophagocytosis. However, additional defense mechanisms, such as bacterial clearance by spleen and liver, may play an important role in preventing Hib-mediated sepsis, in particular for Hib isolates with increased serum-resistance. Results indicate potentially improved safety profile of AP inhibitors over C3 and C5 inhibitors as alternative therapeutic agents in patients with increased susceptibility to Hib infection.

Perceived water-related risk factors of Buruli ulcer in two villages of south-central Côte d'Ivoire.

BACKGROUND: Buruli ulcer, caused by Mycobacterium ulcerans, is a neglected tropical skin disease that is primarily endemic in West and Central Africa, including Côte d'Ivoire. Studies indicate that M. ulcerans infections are caused by contact with an environmental reservoir of the bacteria, governed by specific human biological conditions. Yet, the nature of this reservoir and the exact mode of transmission remain unknown. METHODOLOGY: To identify ecologic risk factors of Buruli ulcer in south-central Côte d'Ivoire, we pursued a qualitative study matched with geo-referencing inquiry. Embedded in a broader integrated wound management research project, we (i) mapped households and water sources of laboratory confirmed Buruli ulcer cases and (ii) interviewed 12 patients and four health care workers to assess exposure to surface water and to deepen the understanding of perceived transmission pathways. PRINCIPAL FINDINGS: Water availability, accessibility, and affordability were reported as key determinants for choosing water resources. Furthermore, perceived risks were related to environmental, structural, and individual factors. Despite the presence of improved water sources (e.g., drilled wells), communities heavily relied on unprotected surface water for a multitude of activities. The nearby Bandama River and seasonal waterbodies were frequently used for washing, bathing, and collection of water for drinking and cooking. Many residents also reported to cross the river on a daily basis for agricultural chores, and hence, are exposed to stagnant water during farming activities. CONCLUSIONS/SIGNIFICANCE: Our study in two Buruli ulcer endemic villages in south-central Côte d'Ivoire revealed a wide range of water-related domestic activities that might expose people to an increased risk of contracting the disease. Environmental, biological, social, and cultural risk factors are closely interlinked and should be considered in future investigations of Buruli ulcer transmission. Active participation of the communities is key to better understand their circumstances to advance research and fight against Buruli ulcer and other neglected tropical diseases.

Skin wounds in a rural setting of Côte d'Ivoire: Population-based assessment of the burden and clinical epidemiology.

BACKGROUND: Data on the burden and clinical epidemiology of skin wounds in rural sub-Saharan Africa is scant. The scale of the problem including preventable progression to chronic wounds, disability and systemic complications is largely unaddressed. METHODS: We conducted a cross-sectional study combining active (household-based survey) and passive case finding (health services-based survey) to determine the burden and clinical epidemiology of wounds within the Taabo Health and Demographic Surveillance System (HDSS) in rural Côte d'Ivoire. Patients identified with wounds received free care and were invited to participate in the wound management study simultaneously carried out in the survey area. The data were analysed for wound prevalence, stratified by wound and patient characteristics. RESULTS: 3842 HDSS-registered persons were surveyed. Overall wound prevalence derived from combined active and passive case finding was 13.0%. 74.1% (403/544) of patients were below the age of 15 years. Most frequent aetiologies were mechanical trauma (85.3%), furuncles (5.1%), burns (2.9%) and Buruli ulcer (2.2%). Most wounds were acute and smaller than 5 cm2 in size. 22.0% (176/799) of wounds showed evidence of secondary bacterial infection. 35.5% (22/62) of chronic wounds had persisted entirely neglected for years. Buruli ulcer prevalence was 2.3 per 1000 individuals and considerably higher than expected from an annual incidence of 0.01 per 1000 individuals as reported by WHO for Côte d'Ivoire at the time of the study. CONCLUSIONS: Skin wounds are highly prevalent in rural West Africa, where they represent a widely neglected problem. The HDSS-based survey with combined active and passive case finding adopted in this study provides a better estimate than school- and health institution-based surveys which underestimate the frequency of skin wounds and, particularly, of neglected tropical diseases of the skin, such as Buruli ulcer and yaws. A comparison with country-specific WHO data suggests underreporting of Buruli ulcer cases. TRIAL REGISTRATION: Registration at ClinicalTrials.gov NCT03957447.

Community-based wound management in a rural setting of Côte d'Ivoire.

BACKGROUND: Wounds are a neglected health problem in rural communities of low-income countries, mostly caused by trauma and ulcerative skin diseases including Neglected Tropical Diseases (NTDs) and associated with systemic complications and disability. Rural communities have limited access to high quality health services-based wound care. METHODS: We conducted a prospective observational study on wound management at three levels-community (C), health centre (HC), district hospital (DH)-in a rural community of Côte d'Ivoire. Patients with skin wounds actively identified in a house-to-house survey and passively in the health services in a defined area of the Taabo Health and Demographic Surveillance System were asked to participate and followed-up longitudinally. Endpoints were proportion of wounds closed, time to wound closure, wound size over time, frequency of secondary bacterial infection, need for recapturing after follow-up interruption, and duration of treatment stratified by health service level and wound aetiology. RESULTS: We enrolled 561 patients with 923 wounds between May 2019 and March 2020. The observation period ended in March 2021. Median age was 10 years (IQR 7-15), 63.0% of patients were male. Almost all (99.5%, 870/874) wounds closed within the observation period, 5.3% (49/923) were lost to follow-up. Wounds primarily treated in C, HC and DH closed within a median time of 10, 16 and 170 days, respectively. Median time to acute wound and chronic wound closure was 13 and 72 days, respectively. Wounds treated in C, HC and DH presented with secondary bacterial infections in 10.3% (36/350), 31.0% (133/429) and 100% (5/5) of cases, respectively. Recapturing was required in 68.3% (630/923) of wounds with participants reporting wound closure as the main reason for not attending follow-up. CONCLUSIONS: We describe a wound management model based on national and WHO recommendations focusing on early identification and treatment in the community with potential for broad implementation in low-income countries. TRIAL REGISTRATION: Registration at ClinicalTrials.gov (NCT03957447).

Scalable Process for High-Yield Production of PfCyRPA Using Insect Cells for Inclusion in a Malaria Virosome-Based Vaccine Candidate.

Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) has been identified as a promising blood-stage candidate antigen to include in a broadly cross-reactive malaria vaccine. In the last couple of decades, substantial effort has been committed to the development of scalable cost-effective, robust, and high-yield PfCyRPA production processes. Despite insect cells being a suitable expression system due to their track record for protein production (including vaccine antigens), these are yet to be explored to produce this antigen. In this study, different insect cell lines, culture conditions (baculovirus infection strategy, supplementation schemes, culture temperature modulation), and purification strategies (affinity tags) were explored aiming to develop a scalable, high-yield, and high-quality PfCyRPA for inclusion in a virosome-based malaria vaccine candidate. Supplements with antioxidants improved PfCyRPA volumetric titers by 50% when added at the time of infection. In addition, from three different affinity tags (6x-His, 4x-His, and C-tag) evaluated, the 4x-His affinity tag was the one leading to the highest PfCyRPA purification recovery yields (61%) and production yield (26 mg/L vs. 21 mg/L and 13 mg/L for 6x-His and C-tag, respectively). Noteworthy, PfCyRPA expressed using High Five cells did not show differences in protein quality or stability when compared to its human HEK293 cell counterpart. When formulated in a lipid-based virosome nanoparticle, immunized rabbits developed functional anti-PfCyRPA antibodies that impeded the multiplication of P. falciparum in vitro. This work demonstrates the potential of using IC-BEVS as a qualified platform to produce functional recombinant PfCyRPA protein with the added benefit of being a non-human expression system with short bioprocessing times and high expression levels.

Aberrant stromal tissue factor localisation and mycolactone-driven vascular dysfunction, exacerbated by IL-1ß, are linked to fibrin formation in Buruli ulcer lesions.

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.

Efficacy of an Acid-Oxidizing Solution against Mycobacterium ulcerans.

For the treatment of chronic wounds, acid-oxidizing solutions (AOSs) with broad-spectrum microbicidal activity without disturbing granulation tissue formation have been developed. We found AOSs to efficiently kill Mycobacterium ulcerans, the causative agent of Buruli ulcer, which is able to survive harsh decontamination treatments. Topical AOS treatment of Buruli ulcer lesions may support the recommended antibiotic therapy (oral rifampin and clarithromycin), prevent contamination of the environment by the mycobacteria, and control secondary infections, which are a prevalent wound management problem in resource-poor settings where Buruli ulcer is endemic.

Inhibition of the SEC61 translocon by mycolactone induces a protective autophagic response controlled by EIF2S1-dependent translation that does not require ULK1 activity.

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.

Overview: Mycobacterium ulcerans Disease (Buruli Ulcer).

Enhanced international research efforts since the establishment of the Global BU Initiative in 1998 by the WHO have helped to advance our understanding of the epidemiology, and pathogenesis of Mycobacterium ulcerans infections. Improved methods to cultivate the extremely slow-growing pathogen from BU lesions have laid the groundwork for a variety of studies using M. ulcerans isolates, including the analysis of the genome and proteome of the pathogen, as well as drug susceptibility testing and analyses of host-pathogen interactions in vitro and in animal models. The identification of specific, high-copy number target sequences in the genome of M. ulcerans has enabled the development of diagnostic tests and assays to detect the pathogen in the environment. Important research questions remain about the reservoir(s) of M. ulcerans in aquatic environments, factors leading to or promoting transmission to hosts, and host-pathogen interactions resulting in chronic infection versus spontaneous healing.

Investigation of Mycobacterium ulcerans Glycan Interactions Using Glycan Array and Surface Plasmon Resonance.

Many pathogenic bacteria utilize glycan-based interactions to bind to host cells. Glycan array analysis and surface plasmon resonance are glycobioanalytical techniques that have been used to investigate the glycointeractions of a range of pathogens. The analysis of the glycointeractome, particularly the binding of host glycans by Mycobacteria, has been limited. In this chapter, we outline methodologies that have been successfully implemented for studying Mycobacterium ulcerans glycointeractions.

Overview: Mycolactone , the Macrolide Toxin of Mycobacterium ulcerans.

The acquisition by a Mycobacterium marinum-like progenitor of a plasmid encoding enzymes for the biosynthesis of the highly potent macrolide toxin mycolactone has set off the evolution of M. ulcerans toward a new mycobacterial species. While the selective advantage of producing mycolactone for survival in environmental niche(s) of the pathogen is unclear, there is no doubt that the cytotoxic, immunomodulatory, and analgesic properties of mycolactone are key for the establishment and progression of M. ulcerans infections in the host. Improved procedures for the isolation, handling, and detection of the amphiphilic and light-sensitive toxin have facilitated studies to unravel molecular mechanisms of mycolactone action on host cells in vitro and on cellular and immune responses in animal models. The pivotal role of mycolactone in the pathology of Buruli ulcer and the fact that the toxin has not been associated with other pathogens make it an ideal target for therapeutics/vaccines aiming at mycolactone neutralization and for the development of assays for the diagnosis of the disease.

Overview: Development of Drugs Against Mycobacterium ulcerans.

For many years, wide margin surgical excision of Buruli ulcer lesions has been the main approach for the treatment of Mycobacterium ulcerans disease. The WHO now recommends an eight-week course of oral antibiotics with a combination of rifampicin and clarithromycin in Africa. However, disease management is complicated by social stigma, lack of awareness, and limited access to healthcare facilities, resulting in underreporting and frequently late initiation of medical treatment. Inadequate initial treatment can drive permanent disabilities and also limited compliance to the eight-week therapy is a limitation. Therefore, search for a faster and more simple treatment modality is ongoing, focusing primarily on the testing of new tuberculosis drug candidates for the treatment of M. ulcerans disease.

Alternative Complement Pathway Inhibition Does Not Abrogate Meningococcal Killing by Serum of Vaccinated Individuals.

Dysregulation of complement activation causes a number of diseases, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. These conditions can be treated with monoclonal antibodies (mAbs) that bind to the complement component C5 and prevent formation of the membrane attack complex (MAC). While MAC is involved in uncontrolled lysis of erythrocytes in these patients, it is also required for serum bactericidal activity (SBA), i.e. clearance of encapsulated bacteria. Therefore, terminal complement blockage in these patients increases the risk of invasive disease by Neisseria meningitidis more than 1000-fold compared to the general population, despite obligatory vaccination. It is assumed that alternative instead of terminal pathway inhibition reduces the risk of meningococcal disease in vaccinated individuals. To address this, we investigated the SBA with alternative pathway inhibitors. Serum was collected from adults before and after vaccination with a meningococcal serogroup A, C, W, Y capsule conjugate vaccine and tested for meningococcal killing in the presence of factor B and D, C3, C5 and MASP-2 inhibitors. B meningococci were not included in this study since the immune response against protein-based vaccines is more complex. Unsurprisingly, inhibition of C5 abrogated killing of meningococci by all sera. In contrast, both factor B and D inhibitors affected meningococcal killing in sera from individuals with low, but not with high bactericidal anti-capsular titers. While the anti-MASP-2 mAb did not impair SBA, inhibition of C3 impeded meningococcal killing in most, but not in all sera. These data provide evidence that vaccination can provide protection against invasive meningococcal disease in patients treated with alternative pathway inhibitors.