Genetic variation in autophagy-related genes influences the risk and phenotype of Buruli ulcer

Introduction Buruli ulcer (BU) is a severe necrotizing human skin disease caused by Mycobacterium ulcerans. Clinically, presentation is a sum of these diverse pathogenic hits subjected to critical immune-regulatory mechanisms. Among them, autophagy has been demonstrated as a cellular process of critical importance. Since microtubules and dynein are affected by mycolactone, the critical pathogenic exotoxin produced by M. ulcerans, cytoskeleton-related changes might potentially impair the autophagic process and impact the risk and progression of infection. Objective Genetic variants in the autophagy-related genes NOD2, PARK2 and ATG16L1 has been associated with susceptibility to mycobacterial diseases. Here, we investigated their association with BU risk, its severe phenotypes and its progression to an ulcerative form. Methods Genetic variants were genotyped using KASPar chemistry in 208 BU patients (70.2% with an ulcerative form and 28% in severe WHO category 3 phenotype) and 300 healthy endemic controls. Results The rs1333955 SNP in PARK2 was significantly associated with increased susceptibility to BU [odds ratio (OR), 1.43; P = 0.05]. In addition, both the rs9302752 and rs2066842 SNPs in NOD2 gee significantly increased the predisposition of patients to develop category 3 (OR, 2.23; P = 0.02; and OR 12.7; P = 0.03, respectively, whereas the rs2241880 SNP in ATG16L1 was found to significantly protect patients from presenting the ulcer phenotype (OR, 0.35; P = 0.02). Conclusion Our findings indicate that specific genetic variants in autophagy-related genes influence susceptibility to the development of BU and its progression to severe phenotypes.The research leading to these results received funding from the Health Services of the Fundação Calouste Gulbenkian under the grant Proc.N°94776 LJ; from the Fundação para a Ciência e Tecnologia (FCT), cofunded by Programa Operacional Regional do Norte (ON.2—O Novo 267 Norte); from the Quadro de Referência Estratégico Nacional (QREN) through the Fundo Europeu de Desenvolvimento Regional (FEDER) and from the Projeto Estratégico – LA 26 – 2013–2014 (PEst-C/SAU/LA0026/2013). JFM received an individual QREN fellowship (UMINHO/BPD/14/2014); CCu and AGF received an individual FCT fellowship (SFRH/BPD/96176/2013 and SFRH/BPD/68547/2010, respectively); and AC received an FCT contract (IF/00735/2014). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript

Combined Inflammatory and Metabolic Defects Reflected by Reduced Serum Protein Levels in Patients with Buruli Ulcer Disease

Buruli ulcer is a skin disease caused by Mycobacterium ulcerans that is spreading in tropical countries, with major public health and economic implications in West Africa. Multi-analyte profiling of serum proteins in patients and endemic controls revealed that Buruli ulcer disease down-regulates the circulating levels of a large array of inflammatory mediators, without impacting on the leukocyte composition of peripheral blood. Notably, several proteins contributing to acute phase reaction, lipid metabolism, coagulation and tissue remodelling were also impacted. Their down-regulation was selective and persisted after the elimination of bacteria with antibiotic therapy. It involved proteins with various functions and origins, suggesting that M. ulcerans infection causes global and chronic defects in the host’s protein metabolism. Accordingly, patients had reduced levels of total serum proteins and blood urea, in the absence of signs of malnutrition, or functional failure of liver or kidney. Interestingly, slow healers had deeper metabolic and coagulation defects at the start of antibiotic therapy. In addition to providing novel insight into Buruli ulcer pathogenesis, our study therefore identifies a unique proteomic signature for this disease

Microbiological, histological, immunological, and toxin response to antibiotic treatment in the mouse model of Mycobacterium ulcerans disease.

Mycobacterium ulcerans infection causes a neglected tropical disease known as Buruli ulcer that is now found in poor rural areas of West Africa in numbers that sometimes exceed those reported for another significant mycobacterial disease, leprosy, caused by M. leprae. Unique among mycobacterial diseases, M. ulcerans produces a plasmid-encoded toxin called mycolactone (ML), which is the principal virulence factor and destroys fat cells in subcutaneous tissue. Disease is typically first manifested by the appearance of a nodule that eventually ulcerates and the lesions may continue to spread over limbs or occasionally the trunk. The current standard treatment is 8 weeks of daily rifampin and injections of streptomycin (RS). The treatment kills bacilli and wounds gradually heal. Whether RS treatment actually stops mycolactone production before killing bacilli has been suggested by histopathological analyses of patient lesions. Using a mouse footpad model of M. ulcerans infection where the time of infection and development of lesions can be followed in a controlled manner before and after antibiotic treatment, we have evaluated the progress of infection by assessing bacterial numbers, mycolactone production, the immune response, and lesion histopathology at regular intervals after infection and after antibiotic therapy. We found that RS treatment rapidly reduced gross lesions, bacterial numbers, and ML production as assessed by cytotoxicity assays and mass spectrometric analysis. Histopathological analysis revealed that RS treatment maintained the association of the bacilli with (or within) host cells where they were destroyed whereas lack of treatment resulted in extracellular infection, destruction of host cells, and ultimately lesion ulceration. We propose that RS treatment promotes healing in the host by blocking mycolactone production, which favors the survival of host cells, and by killing M. ulcerans bacilli

Lifecourse socioeconomic status and type 2 diabetes: the role of chronic inflammation in the English Longitudinal Study of Ageing

We examined the association between lifecourse socioeconomic status (SES) and the risk of type 2 diabetes at older ages, ascertaining the extent to which adult lifestyle factors and systemic inflammation explain this relationship. Data were drawn from the English Longitudinal Study of Ageing (ELSA) which, established in 2002, is a representative cohort study of ?50-year olds individuals living in England. SES indicators were paternal social class, participants? education, participants? wealth, and a lifecourse socioeconomic index. Inflammatory markers (C-reactive protein and fibrinogen) and lifestyle factors were measured repeatedly; diabetes incidence (new cases) was monitored over 7.5 years of follow-up. Of the 6218 individuals free from diabetes at baseline (44% women, mean aged 66 years), 423 developed diabetes during follow-up. Relative to the most advantaged people, those in the lowest lifecourse SES group experienced more than double the risk of diabetes (hazard ratio 2.59; 95% Confidence Interval (CI)?=?1.81?3.71). Lifestyle factors explained 52% (95%CI:30?85) and inflammatory markers 22% (95%CI:13?37) of this gradient. Similar results were apparent with the separate SES indicators. In a general population sample, socioeconomic inequalities in the risk of type 2 diabetes extend to older ages and appear to partially originate from socioeconomic variations in modifiable factors which include lifestyle and inflammation

Phage therapy is effective against infection by Mycobacterium ulcerans in a murine footpad model

Author Summary: Buruli Ulcer (BU), caused by Mycobacterium ulcerans, is a necrotizing disease of the skin, subcutaneous tissue and bone. Standard treatment of BU patients consists of a combination of the antibiotics rifampicin and streptomycin for 8 weeks. However, in advanced stages of the disease, surgical resection of the destroyed skin is still required. The use of bacterial viruses (bacteriophages) for the control of bacterial infections has been considered as an alternative or a supplement to antibiotic chemotherapy. By using a mouse model of M. ulcerans footpad infection, we show that mice treated with a single subcutaneous injection of the mycobacteriophage D29 present decreased footpad pathology associated with a reduction of the bacterial burden. In addition, D29 treatment induced increased levels of IFN-γ and TNF in M. ulcerans -infected footpads, correlating with a predominance of a mononuclear infiltrate. These findings suggest the potential use of phage therapy in BU, as a novel therapeutic approach against this disease, particularly in advanced stages where bacteria are found primarily in an extracellular location in the subcutaneous tissue, and thus immediately accessible by lytic phages.This work was supported by a grant from the Health Services of Fundacao Calouste Gulbenkian, and the Portuguese Science and Technology Foundation (FCT) fellowships SFRH/BPD/64032/2009, SFRH/BD/41598/2007, and SFRH/BPD/68547/2010 to GT, TGM, and AGF, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Photodegradation of the Mycobacterium ulcerans Toxin, Mycolactones: Considerations for Handling and Storage

Background: Mycolactones are toxins secreted by M. ulcerans, the etiological agent of Buruli ulcer. These toxins, which are the main virulence factors of the bacilli, are responsible for skin lesions. Considering their specificity for M. ulcerans and their presence in skin lesions even at early stages, mycolactones are promising candidates for the development of a diagnostic tool for M. ulcerans infection. Stability of purified mycolactones towards light and heat has not yet been investigated, despite the importance of such parameters in the selection of strategies for a diagnosis tool development. In this context, the effects of UV, light and temperature on mycolactone stability and biological activity were studied. Methodology/Principal Findings: To investigate the effect of these physical parameters, mycolactones were exposed to different wavelengths in several solvents and temperatures. Structural changes and biological activity were monitored. Whilst high temperature had no effect on mycolactones, UV irradiation (UV-A, UV-B and UV-C) and sunlight exposure caused a considerable degradation, as revealed by LC-MS and NMR analysis, correlated with a loss of biological activity. Moreover, effect of UVs on mycolactone caused a photodegradation rather than a phototransformation due to the identification of degradation product. Conclusion/Significance: This study demonstrates the high sensitivity of mycolactones to UVs as such it defines instruction

Corticosteroid-Induced Immunosuppression ultimately does not compromise the efficacy of antibiotherapy in murine mycobacterium ulcerans Infection

Buruli ulcer (BU) is a necrotizing disease of the skin, subcutaneous tissue and bone caused by Mycobacterium ulcerans. It has been suggested that the immune response developed during the recommended rifampicin/streptomycin (RS) antibiotherapy is protective, contributing to bacterial clearance. On the other hand, paradoxical reactions have been described during or after antibiotherapy, characterized by pathological inflammatory responses. This exacerbated inflammation could be circumvented by immunosuppressive drugs. Therefore, it is important to clarify if the immune system contributes to bacterial clearance during RS antibiotherapy and if immunosuppression hampers the efficacy of the antibiotic regimen. METHODOLOGY/PRINCIPAL FINDINGS: We used the M. ulcerans infection footpad mouse model. Corticosteroid-induced immunosuppression was achieved before experimental infection and maintained during combined RS antibiotherapy by the administration of dexamethasone (DEX). Time-lapsed analyses of macroscopic lesions, bacterial burdens, histology and immunohistochemistry were performed in M. ulcerans-infected footpads. We show here that corticosteroid-immunosuppressed mice are more susceptible to M. ulcerans, with higher bacterial burdens and earlier ulceration. Despite this, macroscopic lesions remised during combined antibiotic/DEX treatment and no viable bacteria were detected in the footpads after RS administration. This was observed despite a delayed kinetics in bacterial clearance, associated with a local reduction of T cell and neutrophil numbers, when compared with immunocompetent RS-treated mice. In addition, no relapse was observed following an additional 3 month period of DEX administration. CONCLUSIONS/SIGNIFICANCE: These findings reveal a major role of the RS bactericidal activity for the resolution of M. ulcerans experimental infections even during immunosuppression, and support clinical investigation on the potential use of corticosteroids or other immunosuppressive/anti-inflammatory drugs for the management of BU patients undergoing paradoxical reactions.This work was supported by a grant from the Health Services of Fundação Calouste Gulbenkian, and the Portuguese Science and Technology Foundation (FCT) fellowships SFRH/BD/41598/2007, SFRH/BPD/64032/2009, SFRH/BPD/68547/2010 and SFRH/BD/33573/2009 to TGM, GT, AGF, and JBG, respectively. MS is a Ciência 2007 fello

Cellular Immunity Confers Transient Protection in Experimental Buruli Ulcer following BCG or Mycolactone-Negative Mycobacterium ulcerans Vaccination

BACKGROUND: Buruli ulcer (BU) is an emerging infectious disease caused by Mycobacterium ulcerans that can result in extensive necrotizing cutaneous lesions due to the cytotoxic exotoxin mycolactone. There is no specific vaccine against BU but reports show some degree of cross-reactive protection conferred by M. bovis BCG immunization. Alternatively, an M. ulcerans-specific immunization could be a better preventive strategy. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used the mouse model to characterize the histological and cytokine profiles triggered by vaccination with either BCG or mycolactone-negative M. ulcerans, followed by footpad infection with virulent M. ulcerans. We observed that BCG vaccination significantly delayed the onset of M. ulcerans growth and footpad swelling through the induction of an earlier and sustained IFN-gamma T cell response in the draining lymph node (DLN). BCG vaccination also resulted in cell-mediated immunity (CMI) in M. ulcerans-infected footpads, given the predominance of a chronic mononuclear infiltrate positive for iNOS, as well as increased and sustained levels of IFN-gamma and TNF. No significant IL-4, IL-17 or IL-10 responses were detected in the footpad or the DLN, in either infected or vaccinated mice. Despite this protective Th1 response, BCG vaccination did not avoid the later progression of M. ulcerans infection, regardless of challenge dose. Immunization with mycolactone-deficient M. ulcerans also significantly delayed the progression of footpad infection, swelling and ulceration, but ultimately M. ulcerans pathogenic mechanisms prevailed. CONCLUSIONS/SIGNIFICANCE: The delay in the emergence of pathology observed in vaccinated mice emphasizes the relevance of protective Th1 recall responses against M. ulcerans. In future studies it will be important to determine how the transient CMI induced by vaccination is compromised