SNP-Typisierung des Buruli-Ulkus-Erregers

Zusammenfassung: Mithilfe vergleichender Genomanalysen konnten wir eine Feintypisierungsmethode für Mycobacterium ulcerans, dem genetisch monomorphen Erreger des Buruli-Ulkus, entwickel

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

Use of recombinant virus replicon particles for vaccination against Mycobacterium ulcerans disease

Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a necrotizing disease of the skin and subcutaneous tissue, which is most prevalent in rural regions of West African countries. The majority of clinical presentations seen in patients are ulcers on limbs that can be treated by eight weeks of antibiotic therapy. Nevertheless, scarring and permanent disabilities occur frequently and Buruli ulcer still causes high morbidity. A vaccine against the disease is so far not available but would be of great benefit if used for prophylaxis as well as therapy. In the present study, vesicular stomatitis virus-based RNA replicon particles encoding the M. ulcerans proteins MUL2232 and MUL3720 were generated and the expression of the recombinant antigens characterized in vitro. Immunisation of mice with the recombinant replicon particles elicited antibodies that reacted with the endogenous antigens of M. ulcerans cells. A prime-boost immunization regimen with MUL2232-recombinant replicon particles and recombinant MUL2232 protein induced a strong immune response but only slightly reduced bacterial multiplication in a mouse model of M. ulcerans infection. We conclude that a monovalent vaccine based on the MUL2232 antigen will probably not sufficiently control M. ulcerans infection in humans

Mycobacterium ulcerans mouse model refinement for pre-clinical profiling of vaccine candidates

Buruli Ulcer is a neglected tropical disease leading to extensive disabilities and morbidity in West Africa. In this paper we sought to characterize various strains of Mycobacterium ulcerans (M.ulcerans) with different origins and laboratory passage records while refining a mouse model for Buruli ulcer. We described, compared and followed the kinetics of the histo-pathological outcome of infection of a collection of strains at various anatomical sites of infection in order to find a suitable model for further immunization studies. Moreover we compared the outcome of infection in C57Bl/6 and Balbc/J mice. Specifically we described thoroughly one M. ulcerans strain characterized by slow growth rate and limited tissue necrosis, which presents close ressemblance with the infection kinetics in humans. This strain caused macrophages as well as T and B cells infiltration, correlating with mycobacterial proliferation at the site of infection as well as in the draining lymph nodes, making it a suitable strain to screen vaccine candidates efficacy

Experimental infection of the pig with Mycobacterium ulcerans : a novel model for studying the pathogenesis of Buruli ulcer disease

Buruli ulcer (BU) is a slowly progressing, necrotising disease of the skin caused by infection with Mycobacterium ulcerans. Non-ulcerative manifestations are nodules, plaques and oedema, which may progress to ulceration of large parts of the skin. Histopathologically, BU is characterized by coagulative necrosis, fat cell ghosts, epidermal hyperplasia, clusters of extracellular acid fast bacilli (AFB) in the subcutaneous tissue and lack of major inflammatory infiltration. The mode of transmission of BU is not clear and there is only limited information on the early pathogenesis of the disease available.; For evaluating the potential of the pig as experimental infection model for BU, we infected pigs subcutaneously with different doses of M. ulcerans. The infected skin sites were excised 2.5 or 6.5 weeks after infection and processed for histopathological analysis. With doses of 2×107 and 2×106 colony forming units (CFU) we observed the development of nodular lesions that subsequently progressed to ulcerative or plaque-like lesions. At lower inoculation doses signs of infection found after 2.5 weeks had spontaneously resolved at 6.5 weeks. The observed macroscopic and histopathological changes closely resembled those found in M. ulcerans disease in humans.; Our results demonstrate that the pig can be infected with M. ulcerans. Productive infection leads to the development of lesions that closely resemble human BU lesions. The pig infection model therefore has great potential for studying the early pathogenesis of BU and for the development of new therapeutic and prophylactic interventions

An efficient system to generate monoclonal antibodies against membrane-associated proteins by immunisation with antigen-expressing mammalian cells

ABSTRACT: BACKGROUND: The generation of monoclonal antibodies specific for protein antigens usually depends on purified recombinant protein for both immunisation and hybridoma screening. Purification of recombinant protein in sufficient yield and purity is a tedious undertaking and can be demanding especially in the case of membrane proteins. Furthermore, antibodies generated against a purified recombinant protein are frequently incapable of binding to the endogenous protein in its native context. RESULTS: We describe a strategy to generate monoclonal antibodies against membrane or membrane-associated proteins that completely bypasses any need for purified recombinant antigen. This approach utilises stably transfected mammalian cells expressing recombinant antigens on their cell surface for immunisation of mice. The transfected cells are also used for measuring seroconversion, hybridoma selection and antibody characterisation. By presenting the antigen in its native conformation for immunisation and hybridoma selection, this procedure promotes the generation of antibodies capable of binding to the endogenous protein. In the present study, we applied this approach successfully for three predicted GPI-anchored proteins of the malaria parasite Plasmodium falciparum. CONCLUSIONS: The described entirely cell-based technology is a fast and efficient approach for obtaining antibodies reactive with endogenous cell-surface proteins in their native conformatio

Improved Protective Efficacy of a Species-Specific DNA Vaccine Encoding Mycolyl-Transferase Ag85A from Mycobacterium ulcerans by Homologous Protein Boosting

Vaccination with plasmid DNA encoding Ag85A from M. bovis BCG can partially protect C57BL/6 mice against a subsequent footpad challenge with M. ulcerans. Unfortunately, this cross-reactive protection is insufficient to completely control the infection. Although genes encoding Ag85A from M. bovis BCG (identical to genes from M. tuberculosis) and from M. ulcerans are highly conserved, minor sequence differences exist, and use of the specific gene of M. ulcerans could possibly result in a more potent vaccine. Here we report on a comparison of immunogenicity and protective efficacy in C57BL/6 mice of Ag85A from M. tuberculosis and M. ulcerans, administered as a plasmid DNA vaccine, as a recombinant protein vaccine in adjuvant or as a combined DNA prime-protein boost vaccine. All three vaccination formulations induced cross-reactive humoral and cell-mediated immune responses, although species-specific Th1 type T cell epitopes could be identified in both the NH2-terminal region and the COOH-terminal region of the antigens. This partial species-specificity was reflected in a higher—albeit not sustained—protective efficacy of the M. ulcerans than of the M. tuberculosis vaccine, particularly when administered using the DNA prime-protein boost protocol

Structural and functional characterisation of the Toll like receptor 9 of Aotus nancymaae , a non-human primate model for malaria vaccine development

In the absence of suitable rodent animal models for Plasmodium falciparum malaria, the efficacy testing of asexual blood-stage vaccine candidates in Aotus nancymaae represents a tool to select between different formulations before conducting expensive human clinical trials. CpG oligonucleotides (ODN) specifically promote the production of pro-inflammatory and Th1-type cytokines and they enhance the immunogenicity of co-administered antigens. Toll like receptor 9 (TLR-9) binds directly and sequence-specifically to single-stranded un-methylated CpG-DNA mediating the biological effects of CpG ODN. We cloned and functionally characterised the TLR-9 cDNA of A. nancymaae. The cDNA encompassed 3,099bp predicted to code for 1,032 amino acid residues. Results of homology searches to human TLR-9 suggested that the receptor is 93 and 94% identical at the nucleotide and amino acid sequence levels, respectively. Stimulation of splenocytes of A. nancymaae with CpG ODN resulted in proliferative responses in all animals analysed. FACS analysis of cultures incubated with CpG ODN 2006 indicated that the B cell marker CD20 was up-regulated consistent with B cell activation. The high level of sequence conservation of Aona-TLR-9 reinforces the suitability of A. nancymaae as animal model for malaria subunit vaccine developmen

Evaluating decontamination protocols for the isolation of Mycobacterium ulcerans from swabs

Mycobacterium ulcerans (M. ulcerans) is the causative agent of Buruli Ulcer (BU) disease. In order to inhibit the growth of the microbial contaminants during culture of M. ulcerans, it is necessary to decontaminate BU samples with effective chemical agents. This study aimed at investigating some selected chemicals as potential decontamination agents for the isolation of M. ulcerans from swabs.; Povidone iodine at 0.5 and 1% exhibited the lowest contamination and recovery rate for microbial contaminants and M. ulcerans. The most effective decontamination method was the protocol using 2% cetylpyridinium chloride/4% sodium chloride (recovery rate = 53%, contamination rate = 14%). The observed difference between the recovery rate of 2% CPC/4% NaC and the other protocols was however not statistically significant (p = 0.76).; Two percent (2%) cetylpyridinium chloride/4% sodium chloride can be conveniently used as an alternative decontamination method for the isolation of M. ulcerans from swabs

Evolution of two distinct phylogenetic lineages of the emerging human pathogen Mycobacterium ulcerans

<p>Abstract</p> <p>Background</p> <p>Comparative genomics has greatly improved our understanding of the evolution of pathogenic mycobacteria such as <it>Mycobacterium tuberculosis</it>. Here we have used data from a genome microarray analysis to explore insertion-deletion (InDel) polymorphism among a diverse strain collection of <it>Mycobacterium ulcerans</it>, the causative agent of the devastating skin disease, Buruli ulcer. Detailed analysis of large sequence polymorphisms in twelve regions of difference (RDs), comprising irreversible genetic markers, enabled us to refine the phylogenetic succession within <it>M. ulcerans</it>, to define features of a hypothetical <it>M. ulcerans </it>most recent common ancestor and to confirm its origin from <it>Mycobacterium marinum</it>.</p> <p>Results</p> <p><it> M. ulcerans </it>has evolved into five InDel haplotypes that separate into two distinct lineages: (i) the "classical" lineage including the most pathogenic genotypes – those that come from Africa, Australia and South East Asia; and (ii) an "ancestral" <it>M. ulcerans </it>lineage comprising strains from Asia (China/Japan), South America and Mexico. The ancestral lineage is genetically closer to the progenitor <it>M. marinum </it>in both RD composition and DNA sequence identity, whereas the classical lineage has undergone major genomic rearrangements.</p> <p>Conclusion</p> <p>Results of the InDel analysis are in complete accord with recent multi-locus sequence analysis and indicate that <it>M. ulcerans </it>has passed through at least two major evolutionary bottlenecks since divergence from <it>M. marinum</it>. The classical lineage shows more pronounced reductive evolution than the ancestral lineage, suggesting that there may be differences in the ecology between the two lineages. These findings improve the understanding of the adaptive evolution and virulence of <it>M. ulcerans </it>and pathogenic mycobacteria in general and will facilitate the development of new tools for improved diagnostics and molecular epidemiology.</p