Search

The ecological functions of many toxins continue to remain unknown for those produced by environmental pathogens. Mycobacterium ulcerans, the causative agent of the neglected tropical disease, Buruli ulcer, produces a cytotoxic macrolide, mycolactone, whose function(s) in the environment remains elusive. Through a series of dual-choice behaviour assays, they show that mycolactone may be an interkingdom cue for the yellow fever mosquito, Aedes aegypti, seeking blood-meals as well as oviposition sites. Results provide novel insight into the evolution between bacteria and potential vectors. While further studies are needed to determine if mycolactone is an actual signal rather than simply a cue, this discovery could serve as a model for determining roles for toxins produced by other environmental pathogens and provide opportunities for developing novel strategies for disease prevention. The relationship between M. ulcerans, mycolactone, and Ae. aegypti further suggests there could be an amplification effect for the spread of pathogens responsible for other diseases, such as yellow fever and dengue.

Neglected tropical diseases (NTDs) are communicable diseases that occur under conditions of poverty and are concentrated almost exclusively in impoverished populations in the developing world. NTDs affect more than 1000 million people in tropical and subtropical countries, costing developing economies billions of dollars every year. Effective control of NTDs can be achieved with the use of large-scale delivery of single-dose preventive chemotherapy (PC) or intensified disease management (IDM) or both, as is the case for some diseases such as lymphatic filariasis, trachoma, and yaws. Several NTDs exhibit significant cutaneous manifestations that are associated with long-term disfigurement and disability, including Buruli ulcer (BU); cutaneous leishmaniasis (CL); leprosy; mycetoma; yaws; hydrocele and lymphoedema (resulting from lymphatic filariasis); and depigmentation, subcutaneous nodules, severe itching, and hanging groin (resulting from onchocerciasis). Skin examination offers an opportunity to screen people in the communities or children in schools to identify multiple conditions in a single visit. This common approach to skin diseases justifies the integrated delivery of health care interventions to both increase cost-effectiveness and expand coverage. WHO’s Department of Control of NTDs (WHO/NTD) plans to promote an integrated strategy for the skin NTDs requiring IDM. Targeting skin NTDs also provides a platform for treatment of common skin conditions and, therefore, has wider public health benefits. An informal panel of experts (writing this manuscript) was established to help develop guidance in support of the new WHO strategic direction and to develop a proposal for a change in policy for the integrated control and management of the skin NTDs. A symposium at the 2015 ASTMH meeting[1] initiated a discussion of opportunities around integration of surveillance and control of NTDs that affect the skin, but this paper moves these ideas forward and includes some initial recommendations about how these opportunities could be realised. We aim to provide specific pragmatic information and actual recommendations about potential surveillance and management approaches.

Induction of innate immunity is essential for host survival of infection. Evasion and inhibition of innate immunity constitute a strategy used by pathogens, such as the highly virulent bacterium Francisella tularensis, to ensure their replication and transmission. The mechanism and bacterial components responsible for this suppression of innate immunity by F. tularensis are not defined. In this article, we demonstrate that lipids enriched from virulent F. tularensis strain SchuS4, but not attenuated live vaccine strain, inhibit inflammatory responses in vitro and in vivo. Suppression of inflammatory responses is associated with IκBα-independent inhibition of NF-κBp65 activation and selective inhibition of activation of IFN regulatory factors. Interference with NF-κBp65 and IFN regulatory factors is also observed following infection with viable SchuS4. Together these data provide novel insight into how highly virulent bacteria selectively modulate the host to interfere with innate immune responses required for survival of infection.

Emerging infectious disease outbreaks are increasingly suspected to be a consequence of human pressures exerted on natural ecosystems. Previously, host taxonomic communities have been used as indicators of infectious disease emergence, and the loss of their diversity has been implicated as a driver of increased presence. The mechanistic details in how such pathogen-host systems function, however, may not always be explained by taxonomic variation or loss. Here we used machine learning and methods based on Gower's dissimilarity to quantify metrics of invertebrate functional diversity, in addition to functional groups and their taxonomic diversity at sites endemic and non-endemic for the model generalist pathogen Mycobacterium ulcerans, the causative agent of Buruli ulcer. Changes in these metrics allowed the rapid categorisation of the ecological niche of the mycobacterium's hosts and the ability to relate specific host traits to its presence in aquatic ecosystems. We found that taxonomic diversity of hosts and overall functional diversity loss and evenness had no bearing on the mycobacterium's presence, or whether the site was in an endemic area. These findings, however, provide strong evidence that generalist environmentally persistent bacteria such as M. ulcerans can be associated with specific functional traits rather than taxonomic groups of organisms, increasing our understanding of emerging disease ecology and origin.

Buruli ulcer, caused by Mycobacterium ulcerans, is highly endemic in West Africa. While the mode of transmission is unknown, many studies associate Buruli ulcer with different types of water exposure. We present results from the largest study to date to test for M. ulcerans in aquatic sites and identify environmental attributes associated with its presence. Environmental samples from 98 aquatic sites in the Greater Accra, Ashanti, and Volta regions of Ghana were tested for the presence of M. ulcerans DNA by polymerase chain reaction. The proportion of aquatic sites positive for M. ulcerans varied by region: Ashanti 66% (N = 39), Greater Accra 34% (N = 29), and Volta 0% (N = 30). We explored the spatial distribution of M. ulcerans positive and negative water bodies and found no significant clusters. We also determined both highly localized water attributes and broad scale remotely sensed land cover and terrain environmental characteristics associated with M. ulcerans presence through logistic regression. Our results concur with published results regarding conditions suitable for M. ulcerans growth and associations with Buruli ulcer disease burden with regards to water characteristics and disturbed environments, but differ from others with regards to spatial associations and topographic effects such as elevation and wetness. While our results suggest M. ulcerans is an environmental organism existing in a specific ecological niche, they also reveal variation in the elements defining this niche across the sites considered. In addition, despite the causal association between Buruli ulcer and M. ulcerans, we observed no significant statistical association between case reports of Buruli ulcer and presence of M. ulcerans in nearby waterbodies.

The pathogenicity of Mycobacterium ulcerans (Buruli ulcer) depends on cytotoxic effect of its exotoxin mycolactone. Since epidermis represents a barrier against infectious agents and balanced apoptosis is essential in epidermal homeostasis, we explored if mycolactone A/B induces apoptosis on two human keratinocyte populations, stem cells (KSC) and transit amplifying cells (TAC), and on human keratinocyte line, HaCaT. Treatment of TAC with 1 and 10 ng/ml mycolactone-induced 60 and 90% apoptosis. KSC were more resistant than TAC: 50 and 75% of cells underwent apoptosis after 10 and 100 ng/ml toxin-treatment. Higher doses (1000 ng/ml) induced about 30% apoptosis on HaCaT. In contrast, mycolactone A/B was devoid of toxicity neither on human hepatoma HuH7 nor on human embryonic kidney HEK 293 T cell lines. In conclusion, mycolactone induces apoptosis in human keratinocytes, thus contributing to Buruli ulcer lesions development.

Mycolactone is an immunosuppressive cytotoxin responsible for the clinical manifestation of Buruli ulcer in humans. It was believed to be confined to its etiologic agent, Mycobacterium ulcerans . However, the identification of other mycolactone-producing mycobacteria (MPMs) in other species, including Mycobacterium marinum , indicated a more complex taxonomic relationship. This highlighted the need for research on the biology, evolution, and distribution of such emerging and potentially infectious strains. The reliable genetic fingerprinting analyses presented here aim at both the unraveling of phylogenetic relatedness and of dispersal between environmental and pathogenic mycolactone producers and the identification of genetic prerequisites that enable lateral gene transfer of such plasmids. This will allow for the identification of environmental reservoirs of virulence plasmids that encode enzymes required for the synthesis of mycolactone. Based on dynamic chromosomal loci identified earlier in M. ulcerans , we characterized large sequence polymorphisms for the phylogenetic analysis of MPMs. Here, we identify new insertional-deletional events and single-nucleotide polymorphisms that confirm and redefine earlier strain differentiation markers. These results support other data showing that all MPMs share a common ancestry. In addition, we found unique genetic features specific for M. marinum strain M, the genome sequence strain which is used widely in research.

A number of studies have suggested that Mycobacterium ulcerans , the etiological agent of Buruli ulcer, may be transmitted to humans by insect bites. M. ulcerans has been isolated from a predaceous aquatic insect, and PCR detection of M. ulcerans DNA in aquatic environments suggests that the organism is widely distributed within many invertebrate taxa and functional feeding groups. Thus, M. ulcerans may be concentrated through different trophic links. However, the specific environmental niche of M. ulcerans and route of transmission to humans remain a mystery. In this study, a biologically relevant infection model in which M. ulcerans -infected mosquito larvae were fed to a species of predaceous hemiptera (African Belostomatidae) was used to demonstrate the persistent colonization of M. ulcerans and subsequent transmission of bacteria to naïve prey. The association of M. ulcerans with specific anatomical compartments showed that M. ulcerans accumulates preferentially on the exoskeleton. In contrast, few organisms were found in dissected guts or salivary glands. No difference was found between the ability of wild-type M. ulcerans and an M. ulcerans isogenic mycolactone-negative mutant to colonize belostomatids. These data show that African belostomatids can successfully be colonized by M. ulcerans and support the trophic transfer of M. ulcerans within the environment.

Biting water bugs were not correlated with pathogen occurrence., Buruli ulcer is a necrotizing skin disease caused by Mycobacterium ulcerans and associated with exposure to aquatic habitats. To assess possible transmission of M. ulcerans by aquatic biting insects, we conducted a field examination of biting water bugs (Hemiptera: Naucoridae, Belostomatidae, Nepidae) in 15 disease-endemic and 12 non–disease-endemic areas of Ghana, Africa. From collections of 22,832 invertebrates, we compared composition, abundance, and associated M. ulcerans positivity among sites. Biting hemipterans were rare and represented a small percentage (usually

Mycobacterium marinum, a ubiquitous pathogen of fish and amphibia, is a near relative of Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans. The genome of the M strain of M. marinum comprises a 6,636,827-bp circular chromosome with 5424 CDS, 10 prophages, and a 23-kb mercury-resistance plasmid. Prominent features are the very large number of genes (57) encoding polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and the most extensive repertoire yet reported of the mycobacteria-restricted PE and PPE proteins, and related-ESX secretion systems. Some of the NRPS genes comprise a novel family and seem to have been acquired horizontally. M. marinum is used widely as a model organism to study M. tuberculosis pathogenesis, and genome comparisons confirmed the close genetic relationship between these two species, as they share 3000 orthologs with an average amino acid identity of 85%. Comparisons with the more distantly related Mycobacterium avium subspecies paratuberculosis and Mycobacterium smegmatis reveal how an ancestral generalist mycobacterium evolved into M. tuberculosis and M. marinum. M. tuberculosis has undergone genome downsizing and extensive lateral gene transfer to become a specialized pathogen of humans and other primates without retaining an environmental niche. M. marinum has maintained a large genome so as to retain the capacity for environmental survival while becoming a broad host range pathogen that produces disease strikingly similar to M. tuberculosis. The work described herein provides a foundation for using M. marinum to better understand the determinants of pathogenesis of tuberculosis.

We have used RNASeq and qRT-PCR to study mRNA levels for all σ-factors in different Mycobacterium marinum strains under various growth and stress conditions. We also studied their levels in M. marinum from infected fish and mosquito larvae. The annotated σ-factors were expressed and transcripts varied in relation to growth and stress conditions. Some were highly abundant such as sigA, sigB, sigC, sigD, sigE and sigH while others were not. The σ-factor mRNA profiles were similar after heat stress, during infection of fish and mosquito larvae. The similarity also applies to some of the known heat shock genes such as the α-crystallin gene. Therefore, it seems probable that the physiological state of M. marinum is similar when exposed to these different conditions. Moreover, the mosquito larvae data suggest that this is the state that the fish encounter when infected, at least with respect to σ-factor mRNA levels. Comparative genomic analysis of σ-factor gene localizations in three M. marinum strains and Mycobacterium tuberculosis H37Rv revealed chromosomal rearrangements that changed the localization of especially sigA, sigB, sigD, sigE, sigF and sigJ after the divergence of these two species. This may explain the variation in species-specific expression upon exposure to different growth conditions.

Infection with Mycobacterium ulcerans causes Buruli Ulcer, a neglected tropical disease. Mosquito vectors are suspected to participate in the transmission and environmental maintenance of the bacterium. However, mechanisms and consequences of mosquito contamination by M. ulcerans are not well understood. We evaluated the metabolome of the Anopheles gambiae mosquito to profile the metabolic changes associated with bacterial colonization. Contamination of mosquitoes with live M. ulcerans bacilli results in disruptions to lipid metabolic pathways of the mosquito, specifically the utilization of glycerolipid molecules, an affect that was not observed in mosquitoes exposed to dead M. ulcerans. These results are consistent with aberrations of lipid metabolism described in other mycobacterial infections, implying global host-pathogen interactions shared across diverse saprophytic and pathogenic mycobacterial species. This study implicates features of the bacterium, such as the putative M. ulcerans encoded phospholipase enzyme, which promote virulence, survival and active adaptation in concert with mosquito development and provides significant groundwork for enhanced studies of the vector-pathogen interactions using metabolomics profiling. Lastly, metabolic and survival data suggest an interaction which is unlikely to contribute to transmission of M. ulcerans by A. gambiae and more likely to contribute to persistence of M. ulcerans in waters cohabitated by both organisms.

Mycobacterium ulcerans and Mycobacterium marinum are closely related pathogens which share an aquatic environment. The pathogenesis of these organisms in humans is limited by their inability to grow above 35°C. M. marinum causes systemic disease in fish but produces localized skin infections in humans. M. ulcerans causes Buruli ulcer, a severe human skin lesion. At the molecular level, M. ulcerans is distinguished from M. marinum by the presence of a virulence plasmid which encodes a macrolide toxin, mycolactone, as well as by hundreds of insertion sequences, particularly IS 2404 . There has been a global increase in reports of fish mycobacteriosis. An unusual clade of M. marinum has been reported from fish in the Red and Mediterranean Seas and a new mycobacterial species, Mycobacterium pseudoshottsii , has been cultured from fish in the Chesapeake Bay, United States. We have discovered that both groups of fish pathogens produce a unique mycolactone toxin, mycolactone F. Mycolactone F is the smallest mycolactone (molecular weight, 700) yet identified. The core lactone structure of mycolactone F is identical to that of M. ulcerans mycolactones, but a unique side chain structure is present. Mycolactone F produces apoptosis and necrosis on cultured cells but is less potent than M. ulcerans mycolactones. Both groups of fish pathogens contain IS 2404 . In contrast to M. ulcerans and conventional M. marinum , mycolactone F-producing mycobacteria are incapable of growth at above 30°C. This fact is likely to limit their virulence for humans. However, such isolates may provide a reservoir for horizontal transfer of the mycolactone plasmid in aquatic environments.

Buruli ulcer (Mycobacterium ulcerans infection) is an emerging disease of developing tropical and subtropical countries. This environmental mycobacterium causes severe morbidity in communities associated with rivers and standing water bodies (particularly those created as a result of human environmental disturbance such as deforestation and agriculture) and subsequent water quality changes. Neither the reservoir nor the mode of transmission is known, but data from laboratory studies suggest that biting aquatic insects may be involved. M ulcerans has been shown to colonize and grow within specific water bugs (Naucoridae), which then transmit M ulcerans to mice through their bites. PCR evidence suggests that the mycobacterium is present in water, biofilms of aquatic plants, detritus, invertebrates, and fish; however, systematic ecological studies that would provide a more comprehensive understanding of M ulcerans distribution in the environment have been lacking. Several hypotheses are explored in relation ...

Summary Mycobacterium ulcerans produces an extracellular cutaneous infection (Buruli ulcer) characterized by immunosuppression. This is in stark contrast to all other pathogenic Mycobacteria species that cause intracellular, granulomatous infections. The unique mycobacterial pathology of M. ulcerans infection is attributed to a plasmid-encoded immunomodulatory macrolide toxin, mycolactone. In this article we explore the role of mycolactone in the virulence of M. ulcerans using mycolactone and genetically defined mycolactone negative mutants. In a guinea pig infection model wild-type (WT) M. ulcerans produces an extracellular infection whereas mycolactone negative mutants produce an intracellular inflammatory infection similar to that of Mycobacterium marinum. Although mycolactone negative mutants are avirulent, they persist for at least 6 weeks. Chemical complementation of M. ulcerans mutants with mycolactone restores WT M. ulcerans pathology. Mycolactone negative mutants are capable of growth within macrophages in vitro whereas macrophages are killed by WT M. ulcerans. The ability of mycolactone to caused delayed cell death via apoptosis has been reported. However, mycolactone also causes cell death via necrosis. In vitro mycolactone has antiphagocytic properties. Neither WT M. ulcerans nor mycolactone negative strains are strong neutrophil attractants. These results suggest that mycolactone is largely responsible for the unique pathology produced by M. ulcerans.

Mycobacterium ulcerans , the causative agent of Buruli ulcer, produces a macrolide toxin, mycolactone A/B, which is thought to play a major role in virulence. A disease similar to Buruli ulcer recently appeared in United States frog colonies following importation of the West African frog, Xenopus tropicalis . The taxonomic position of the frog pathogen has not been fully elucidated, but this organism, tentatively designated Mycobacterium liflandii , is closely related to M. ulcerans and Mycobacterium marinum , and as further evidence is gathered, it will most likely be considered a subspecies of one of these species. In this paper we show that M. liflandii produces a novel plasmid-encoded mycolactone, mycolactone E. M. liflandii contains all of the genes in the mycolactone cluster with the exception of that encoding CYP140A2, a putative p450 monooxygenase. Although the core lactone structure is conserved in mycolactone E, the fatty acid side chain differs from that of mycolactone A/B in the number of hydroxyl groups and double bonds. The cytopathic phenotype of mycolactone E is identical to that of mycolactone A/B, although it is less potent. To further characterize the relationship between M. liflandii and M. ulcerans , strains were analyzed for the presence of the RD1 region genes, esxA (ESAT-6) and esxB (CFP-10). The M. ulcerans genome strain has a deletion in RD1 and lacks these genes. The results of these studies show that M. liflandii contains both esxA and esxB .

Mycobacterium ulcerans was first identified as the causative agent of Buruli ulcer; this cutaneous tissue-destructive process represents the third most important mycobacterial disease in humans after tuberculosis and leprosy. More recently other life traits were documented. M. ulcerans is mainly detected in humid tropical zones as part of a complex ecosystem comprising algae, aquatic insect predators of the genus Naucoris, and very likely their vegetarian preys. Coelomic plasmatocytes could be the first cells of Naucoris cimicoides to be involved in the infection process, acting as shuttle cells that deliver M. ulcerans to the salivary glands as suggested by both in vitro and in vivo approaches. Furthermore, a key element for the early and long-term establishment of M. ulcerans in Naucoridae is demonstrated by the fact that only mycolactone toxin-producing M. ulcerans isolates are able to invade the salivary glands, a site where they proliferate. Later, the raptorial legs of Naucoris are covered by M. ulcerans-containing material that displays features of biofilms.

Mycobacterium ulcerans is an environmental organism which is responsible for the disease Buruli ulcer, a necrotizing skin disease emerging in west Africa. M. ulcerans produces the polyketide-derived macrolide mycolactone, which is required for the immunosuppression and tissue damage which characterizes Buruli ulcer. We have extracted lipids from the cell envelope and culture filtrate from 52 isolates of Mycobacterium species, analyzed them with thin-layer chromatography, and tested them in a murine fibroblast cell line (L929) cytotoxicity assay to investigate whether these mycobacterial species produce mycolactone. For these studies chloroform-methanol (2:1, vol/vol) extracts were prepared from representative fast- and slow-growing mycobacterial species. Isolates tested included 16 uncharacterized, slow-growing, environmental mycobacterial species isolated from areas in which M. ulcerans infection is endemic. Although several strains of mycobacteria studied produced cytopathic lipids, none of these produced a phenotype on cultured cells consistent with that produced by mycolactone. Two mycobacterial species, M. scrofulaceum and M. kansasii , and eight of the environmental mycobacterial isolates contained cell-associated lipids cytopathic to fibroblasts at concentrations of 33 to 1,000 μg/ml. In contrast, mycolactone produces cytotoxicity at less than 2 ng/ml. Analysis of 16S rRNA sequences from the eight environmental isolates suggests that these are novel mycobacterial species. Results from these studies suggest that, although production of cytopathic lipids is relatively common among mycobacterial species, the production of mycolactone as a cell-associated or secreted molecule appears so far to be restricted to M. ulcerans .

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe necrotizing skin disease endemic in tropical countries. Clinical evidence suggests that M. ulcerans isolates from Asia, Mexico, and Australia may be less virulent than isolates from Africa. In vivo studies suggest that mycolactone, a polyketide-derived macrolide toxin, plays a major role in the tissue destruction and immune suppression which occur in cases of Buruli ulcer. Mycolactones were extracted from 34 isolates of M. ulcerans representing strains from Africa, Malaysia, Asia, Australia, and Mexico. Thin-layer chromatography, mass spectroscopic analysis, and cytopathic assays of partially purified mycolactones from these isolates revealed that M. ulcerans produces a heterogeneous mixture of mycolactone variants. Mycolactone A/B, the most biologically active mycolactone species, was identified by mass spectroscopy as [M + Na] + at m/z 765.5 in all cytotoxic isolates except for those from Mexico. Mycolactone C [M+Na] + at m/z 726.3 was the dominant mycolactone species in eight Australian isolates, and mycolactone D [M+Na] + m/z 781.2 was characteristic of two Asian strains. Mycolactone species are conserved within specific geographic areas, suggesting that there may be a correlation between mycolactone profile and virulence. In addition, the core lactone, [M+Na] + m/z 447.4, was identified as a minor species, supporting the hypothesis that mycolactones are synthesized by two polyketide synthases. A cytopathic assay of the core lactone showed that this molecule is sufficient for cytotoxicity, although it is much less potent than the complete mycolactone.

Diarrheal disease is one of the most common diseases of humans. The causative agents of infectious diarrhea and dysentery can be viral, bacterial, or protozoal. Ideally, subjects in a volunteer study should be representative of the population in which diarrheal disease occurs. However, some enteric pathogens, such as rotavirus and enteropathogenic Escherichia coli (EPEC), cause diarrhea primarily in infants and small children. Pathogens which are transmitted almost exclusively through the ingestion of common food are believed to require a higher infective dose to produce disease than organisms capable of spreading directly from person to person. This is because food-borne pathogens have often had the opportunity to replicate to high concentrations in foods before consumption. Both volunteer studies and epidemiological data concur in assigning a low infective dose to Shigella and Campylobacter jejuni diarrhea. Evidence is particularly strong in the case of Shigella species, in which numerous volunteer studies with three different Shigella species have shown that the infective dose for shigellosis is between 10 and 500 organisms. Most of the enteric pathogens which require a very large inoculum to cause disease, such as V. cholerae and enterotoxigenic E. coli (ETEC), are extracellular pathogens whose mode of virulence involves adherence and toxin secretion. The abilities of organisms to survive passage through the normally acidic stomach is probably an important factor for determining infective dose, but other, more specific determinants such as the ability to survive and replicate within cells are likely to play significant roles as well.

Mycolactones are a family of lipophilic small molecules and virulence factors produced as secondary metabolites by Mycobacterium ulcerans and some highly related aquatic mycobacteria. This chapter describes what is known about mycolactones and their unique role in the pathogenesis of Buruli ulcer, explains their unusual biosynthetic locus, and highlights the key questions that remain to be answered. A recent report describes an abundant extracellular matrix produced by M. ulcerans that harbors vesicles that are rich in mycolactones. Mycolactones are related to antibacterial macrolides such as erythromycin and immunosuppressants such as FK506 because they all share a polyketide-derived macrolactone core. The apoptotic ability of mycolactone F is significantly less than that of the other mycolactones; a feature that may be associated with its shorter acyl side chain. All of these mycolactone-producing mycobacteria, including M. ulcerans, form a distinct lineage within a genetically more diverse assemblage of Mycobacterium marinum that have clearly evolved from a common M. marinum-like progenitor. All mycolactone-producing mycobacteria found so far have been discovered through their ability to cause disease in vertebrates, but they may be acting as “indicator” species and represent only a proportion of the mycolactones that exist in nature.

Transmission of M. ulcerans, the etiological agent of Buruli ulcer, from the environment to humans remains an enigma despite decades of research. Major transmission hypotheses propose 1) that M. ulcerans is acquired through an insect bite or 2) that bacteria enter an existing wound through exposure to a contaminated environment. In studies reported here, a guinea pig infection model was developed to determine whether Buruli ulcer could be produced through passive inoculation of M. ulcerans onto a superficial abrasion. The choice of an abrasion model was based on the fact that most bacterial pathogens infecting the skin are able to infect an open lesion, and that abrasions are extremely common in children. Our studies show that after a 90d infection period, an ulcer was present at intra-dermal injection sites of all seven animals infected, whereas topical application of M. ulcerans failed to establish an infection. Mycobacterium ulcerans was cultured from all injection sites whereas infected abrasion sites healed and were culture negative. A 14d experiment was conducted to determine how long organisms persisted after inoculation. Mycobacterium ulcerans was isolated from abrasions at one hour and 24 hours post infection, but cultures from later time points were negative. Abrasion sites were qPCR positive up to seven days post infection, but negative at later timepoints. In contrast, M. ulcerans DNA was detected at intra-dermal injection sites throughout the study. M. ulcerans was cultured from injection sites at each time point. These results suggest that injection of M. ulcerans into the skin greatly facilitates infection and lends support for the role of an invertebrate vector or other route of entry such as a puncture wound or deep laceration where bacteria would be contained within the lesion. Infection through passive inoculation into an existing abrasion appears a less likely route of entry., Author Summary Buruli ulcer, a severe skin disease in West and Central Africa results in significant disability. The causative bacterium, M. ulcerans has been detected in many aquatic sources, but how bacteria enter the skin is an enigma. Two major hypotheses for infection are 1) that bacteria are injected into the skin through the bite of an aquatic insect, or 2) that bacteria enter open wounds on a person's body. In this study, we use a guinea pig infection model to evaluate whether application of M. ulcerans to an open abrasion produces Buruli ulcer. Our results show that despite topical application of very large numbers of M. ulcerans, we are unable to produce infection in open abrasions. These results are extremely surprising because most bacteria such as Staphylococcus or Streptococcus can readily infect abrasions. In contrast, intra-dermal injection of M. ulcerans into the skin of guinea pigs consistently produced an ulcer. Our studies are the first to explore the route of infection of M. ulcerans in an experimental model. These results suggest that Buruli ulcer is not likely to be due to passive entry of bacteria into pre-existing abrasions and supports the role of biting invertebrates, puncture wounds, or lacerations as a requirement for infection.

Pathogens that use multiple host species are an increasing public health issue due to their complex transmission, which makes them difficult to mitigate. Here, we explore the possibility of using networks of ecological interactions among potential host species to identify the particular disease-source species to target to break down transmission of such pathogens. We fit a mathematical model on prevalence data of Mycobacterium ulcerans in western Africa and we show that removing the most abundant taxa for this category of pathogen is not an optimal strategy to decrease the transmission of the mycobacterium within aquatic ecosystems. On the contrary, we reveal that the removal of some taxa, especially Oligochaeta worms, can clearly reduce rates of pathogen transmission and should be considered as a keystone organism for its transmission because it leads to a substantial reduction in pathogen prevalence regardless of the network topology. Besides its potential application for the understanding of M. ulcerans ecology, we discuss about how networks of species interactions can modulate transmission of multi-host pathogens.

Buruli ulcer is a chronic and progressive necrotizing ulcer for which there is no medical treatment. Historically, a soluble toxin (factor) derived from the causative Mycobacterium ulcerans was found to induce the massive necrosis of skin and s.c. tissue seen in this condition. However, the persistence of the disease is thought to be caused by a lack of any immune response. We therefore investigated whether the factor was related to immunosuppression. A protocol to partially purify the factor was developed, and its effects on immune competent cells were tested. The factor produced >95% inhibition of LPS-induced release of TNF and IL-10 from human monocytes and caused a loss of adherence of these cells without cell death. The factor also blocked the production of IL-2 from activated T lymphocytes. The factor had no effect on TNF-induced cytotoxicity, but abrogated TNF-induced NF-κB activation. Surprisingly, a synergy was observed between the factor and phorbol ester-directed NF-κB activation. The factor had no effect on IL-1- or LPS-induced NF-κB activity, indicating selective activity of the factor. The factor did not inhibit the degradation of IκBα induced by TNF, indicating that the target for its activity lies within an undefined part of the TNF signaling mechanism. The data indicate that the localized immunosuppression associated with Buruli ulcer relates to the activity of the released factor, and this may provide a target for future therapeutic strategies for this intractable disease.

Gastric acidity is recognized as the first line of defense against food-borne pathogens, and the ability of pathogens to resist this pH corresponds to their oral infective dose (ID). Naturally occurring and genetically engineered acid-sensitive enteric pathogens were examined for their ability to survive under acidic conditions of pH 2.5 for 2 h at 37°C when inoculated onto ground beef. Each of the strains displayed significantly high survival rates under these normally lethal conditions. The acid-sensitive pathogens Campylobacter jejuni and Vibrio cholerae , which were protected at lower levels from acid-induced killing by ground beef under these conditions, were sensitive to killing in acidified media at pH 5.0 but survived at pH 6.0. Salmonella inoculated onto the surface of preacidified ground beef could not survive if the pH on the surface of the beef was 2.61 or lower but was viable if the surface pH was 3.27. This implies that the pH of the microenvironment occupied by the bacteria on the surface of the food source is critical for their survival. Salmonella was also shown to be protected from killing when inoculated onto boiled egg white, a food source high in protein and low in fat. These results may explain why Salmonella species have a higher oral ID of approximately 10 5 cells when administered under defined conditions but have been observed to cause disease at doses as low as 50 to 100 organisms when consumed as part of a contaminated food source. They may also help explain why some pathogens are associated primarily with food-borne modes of transmission rather than fecal-oral transmission.

Mycobacterium marinum, like Mycobacterium tuberculosis, is a slow-growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter-capture vector pFPV27, we have constructed a library of 200-1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence-activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2-20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis. These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.

Buruli ulcer (BU) is an emerging, but neglected tropical disease, where there has been a reported association with disturbed aquatic habitats and proposed aquatic macroinvertebrate vectors such as biting Hemiptera. An initial step in understanding the potential role of macroinvertebrates in the ecology of BU is to better understand the entire community, not just one or two taxa, in relation to the pathogen, Mycobacterium ulcerans, at a large spatial scale. For the first time at a country-wide scale this research documents that M. ulcerans was frequently detected from environmental samples taken from BU endemic regions, but was not present in 30 waterbodies of a non-endemic region. There were significant differences in macroinvertebrate community structure and identified potential indicator taxa in relation to pathogen presence. These results suggest that specific macroinvertebrate taxa or functional metrics may potentially be used as aquatic biological indicators of M. ulcerans. Developing ecological indicators of this pathogen is a first step for understanding the disease ecology of BU and should assist future studies of transmission.

Numerous studies have associated Buruli ulcer (BU) disease with disturbed aquatic habitats; however, the natural reservoir, distribution, and transmission of the pathogen, Mycobacterium ulcerans, remain unknown. To better understand the role of aquatic plants in the ecology of this disease, a large-scale survey was conducted in waterbodies of variable flow throughout three regions of Ghana, Africa. Our objectives were to characterize plant communities and identify potential relationships with M. ulcerans and other mycolactone-producing mycobacteria (MPM). Waterbodies with M. ulcerans had significantly different aquatic plant communities, with submerged terrestrial plants identified as indicators of M. ulcerans presence. Mycobacterium ulcerans and MPM were detected on 14 plant taxa in emergent zones from both lotic and lentic waterbodies in endemic regions; however, M. ulcerans was not detected in the non-endemic Volta region. These findings support the hypothesis that plants provide substrate for M. ulcerans colonization and could act as potential indicators for disease risk. These findings also suggest that M. ulcerans is a widespread environmental bacteria species, but that it is absent or reduced in regions of low disease incidence. A better understanding is needed regarding the mechanistic associations among aquatic plants and M. ulcerans for identifying the mode of transmission of BU disease.

Shigella flexneri grown to stationary phase has the ability to survive for several hours at pH 2.5. This acid resistance, which may contribute to the low infective dose associated with shigellosis, is dependent upon the expression of the stationary-phase-specific sigma factor sigma S. Using random TnphoA and TnlacZ mutagenesis we isolated five acid-sensitive mutants of S. flexneri, which had lost their ability to survive at pH 2.5 for 2 h in vitro. Each transposon insertion with flanking S. flexneri DNA was cloned and sequenced. Database searches indicated that two TnlacZ mutants had an insertion within the hdeA gene, which is the first gene in the hdeAB operon. Acid resistance was restored in one of these mutants by a plasmid carrying the entire hdeAB operon. Further sequence analysis from the remaining TnlacZ and two TnphoA mutants demonstrated that they all had insertions within a previously unidentified open reading frame (ORF), which is directly downstream from the gadB gene. This putative ORF encodes a protein that has homology to a number of inner membrane amino acid antiporters. A 1.8 kb polymerase chain reaction (PCR) product containing this gene was cloned, which was able to restore acid resistance in each mutant. These fusions were induced during entry into late exponential phase and were positively regulated by RpoS. We confirmed that the expression of the acid-resistance phenotype in acidified minimal media was dependent upon the supplementation of glutamic acid and that this glutamate-dependent system was RpoS regulated. Southern hybridization revealed that both the gadC and hdeAB loci are absent in Salmonella. An rpoS deletion mutant of S. flexneri was also constructed to confirm the important role played by this gene in acid resistance. This rpoS- derivative was extremely acid sensitive. Two-dimensional gel electrophoresis of this mutant revealed that it no longer expressed 27 proteins in late log phase that were present in its isogenic parent. These data indicate that the expression of acid resistance in S. flexneri may be multifactorial and involve proteins located at different subcellular locations.

Mycobacterium ulcerans causes Buruli ulcer in humans, a progressive ulcerative epidermal lesion due to the mycolactone toxin produced by the bacterium. Molecular analysis of M. ulcerans reveals it is closely related to Mycobacterium marinum, a pathogen of both fish and man. Molecular evidence from diagnostic PCR assays for the insertion sequence IS2404 suggests an association of M. ulcerans with fish. However, fish infections by M. ulcerans have not been well documented and IS2404 has been found in other mycobacteria. We have thus, employed two experimental approaches to test for M. ulcerans in fish. We show here for the first time that M. ulcerans with or without the toxin does not mount acute or chronic infections in Japanese Medaka "Oryzias latipes" even at high doses. Moreover, M. ulcerans-infected medaka do not exhibit any visible signs of infection nor disease and the bacteria do not appear to replicate over time. In contrast, similar high doses of the wild-type M. marinum or a mycolactone-producing M. marinum "DL" strain are able to mount an acute disease with mortality in medaka. Although these results would suggest that M. ulcerans does not mount infections in fish we have evidence that CLC macrophages from goldfish are susceptible to mycolactones.

Buruli ulcer is a neglected emerging disease that has recently been reported in some countries as the second most frequent mycobacterial disease in humans after tuberculosis. Cases have been reported from at least 32 countries in Africa (mainly west), Australia, Southeast Asia, China, Central and South America, and the Western Pacific. Large lesions often result in scarring, contractual deformities, amputations, and disabilities, and in Africa, most cases of the disease occur in children between the ages of 4–15 years. This environmental mycobacterium, Mycobacterium ulcerans, is found in communities associated with rivers, swamps, wetlands, and human-linked changes in the aquatic environment, particularly those created as a result of environmental disturbance such as deforestation, dam construction, and agriculture. Buruli ulcer disease is often referred to as the “mysterious disease” because the mode of transmission remains unclear, although several hypotheses have been proposed. The above review reveals that various routes of transmission may occur, varying amongst epidemiological setting and geographic region, and that there may be some role for living agents as reservoirs and as vectors of M. ulcerans, in particular aquatic insects, adult mosquitoes or other biting arthropods. We discuss traditional and non-traditional methods for indicting the roles of living agents as biologically significant reservoirs and/or vectors of pathogens, and suggest an intellectual framework for establishing criteria for transmission. The application of these criteria to the transmission of M. ulcerans presents a significant challenge., Author Summary Buruli ulcer (BU) is a serious necrotizing cutaneous infection caused by Mycobacterium ulcerans. It is a neglected emerging disease that has recently been reported in some countries as the second most frequent mycobacterial disease in humans after tuberculosis (TB). Cases have been reported from at least 32 countries in Africa (mainly west), Australia, Southeast Asia, China, Central and South America, and the Western Pacific. BU is a disease found in rural areas located near wetlands (ponds, swamps, marshes, impoundments, backwaters) and slow-moving rivers, especially in areas prone to human-made disturbance and flooding. Despite considerable research on this disease in recent years, the mode of transmission remains unclear, although several hypotheses have been proposed. In this article we review the current state of knowledge on the ecology and transmission of M. ulcerans in Africa and Australia, discuss traditional and non-traditional methods for investigating transmission, and suggest an intellectual framework for establishing criteria for transmission.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe necrotizing skin disease that causes significant morbidity in Africa and Australia. Person-to-person transmission of Buruli ulcer is rare. Throughout Africa and Australia infection is associated with residence near slow-moving or stagnant water bodies. Although M. ulcerans DNA has been detected in over 30 taxa of invertebrates, fish, water filtrate, and plant materials and one environmental isolate cultured from a water strider (Gerridae), the invertebrate taxa identified are not adapted to feed on humans, and the mode of transmission for Buruli ulcer remains an enigma. Recent epidemiological reports from Australia describing the presence of M. ulcerans DNA in adult mosquitoes have led to the hypothesis that mosquitoes play an important role in the transmission of M. ulcerans. In this study we have investigated the potential of mosquitoes to serve as biological or mechanical vectors or as environmental reservoirs for M. ulcerans . Here we show that Aedes aegypti , A. albopictus , Ochlerotatus triseriatus , and Culex restuans larvae readily ingest wild-type M. ulcerans , isogenic toxin-negative mutants, and Mycobacterium marinum isolates and remain infected throughout larval development. However, the infections are not carried over into the pupae or adult mosquitoes, suggesting an unlikely role for mosquitoes as biological vectors. By following M. ulcerans through a food chain consisting of primary (mosquito larvae), secondary (predatory mosquito larva from Toxorhynchites rutilus septentrionalis ), and tertiary ( Belostoma species) consumers, we have shown that M. ulcerans can be productively maintained in an aquatic food web.

A novel mycolactone has been identified from Mycobacterium marinum infecting freshwater fish.

Background Mycobacterium ulcerans disease (Buruli ulcer) is a neglected tropical disease common amongst children in rural West Africa. Animal experiments have shown that tissue destruction is caused by a toxin called mycolactone. Methodology/Principal Findings A molecule was identified among acetone-soluble lipid extracts from M. ulcerans (Mu)-infected human lesions with chemical and biological properties of mycolactone A/B. On thin layer chromatography this molecule had a retention factor value of 0.23, MS analyses showed it had an m/z of 765.6 [M+Na+] and on MS:MS fragmented to produce the core lactone ring with m/z of 429.4 and the polyketide side chain of mycolactone A/B with m/z of 359.2. Acetone-soluble lipids from lesions demonstrated significant cytotoxic, pro-apoptotic and anti-inflammatory activities on cultured fibroblast and macrophage cell lines. Mycolactone A/B was detected in all of 10 tissue samples from patients with ulcerative and pre-ulcerative Mu disease. Conclusions/Significance Mycolactone can be detected in human tissue infected with Mu. This could have important implications for successful management of Mu infection by antibiotic treatment but further studies are needed to measure its concentration., Author Summary Skin infection with bacteria called Mycobacterium ulcerans causes Buruli ulcer, a disease common in West Africa and mainly affecting children. M. ulcerans is the only mycobacterium to cause disease by production of a toxin. This lipid molecule called mycolactone diffuses from the site of infection, killing surrounding cells and, at low concentration, suppressing the immune response. The aim of this study was to show that mycolactone can be detected among lipids extracted from human M. ulcerans lesions in order to study its role in the pathogenesis of M. ulcerans disease. Lipids were extracted from skin biopsies and tested for the presence of mycolactone using thin layer chromatography and mass spectrometry. The extracts were shown to kill cultured cells in a cytotoxicity assay. Mycolactone was detected in both pre-ulcerative and ulcerative forms of the disease and also in lesions during antibiotic treatment but with reduced bioactivity, suggesting a lower concentration compared to untreated lesions. These findings indicate that there is mycolactone in affected skin at all stages of M. ulcerans disease and it could be used as a biomarker for monitoring the clinical response to antibiotic treatment.

BACKGROUND: Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of mycolactone on keratinocytes has not been reported previously and the mechanism of mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS). We have studied the effect of mycolactone in vitro on human keratinocytes--key cells in wound healing--and tested the hypothesis that the cytotoxic effect of mycolactone is mediated by ROS. METHODOLOGY/PRINCIPAL FINDINGS: The effect of mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe(2+), completely prevented mycolactone mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease.

The virulence and immunosuppressive activity of Mycobacterium ulcerans is attributed to mycolactone, a macrolide toxin synthesized by the bacteria. We have explored the consequence and mechanism of mycolactone pretreatment of primary human monocytes activated by a wide range of TLR ligands. The production of cytokines (TNF, IL-1β, IL-6, IL-10, and IFN-γ-inducible protein-10), chemokines (IL-8), and intracellular effector molecules (exemplified by cyclooxygenase-2) was found to be powerfully and dose dependently inhibited by mycolactone, irrespective of the stimulating ligand. However, mycolactone had no effect on the activation of signaling pathways that are known to be important in inducing these genes, including the MAPK and NF-κB pathways. Unexpectedly, LPS-dependent transcription of TNF, IL-6, and cyclooxygenase-2 mRNA was found not to be inhibited, implying that mycolactone has a novel mechanism of action and must function posttranscriptionally. We propose that mycolactone mediates its effects by inhibiting the translation of a specific subset of proteins in primary human monocytes. This mechanism is distinct from rapamycin, another naturally occurring immunosuppressive lactone. The current findings also suggest that monocyte-derived cytokine transcript and protein levels may not correlate in Buruli ulcer lesions, and urge caution in the interpretation of RT-PCR data obtained from patient biopsy samples.

Mycobacteriophage L5 is a temperate phage with a broad host range among the fast- and slow-growing mycobacteria such as Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium ulcerans. L5 switches off host protein synthesis during the early stage of lytic growth, as was previously shown by protein expression profiling. Also, lethal genetic elements have been identified in I based on the fact that transformants could not be obtained with these genes. Using an inducible mycobacterial shuttle vector, we have identified three ORFs within an early operon of mycobacteriophage L5 which encode gene products (gp) toxic to the host M. smegmatis when expressed. These ORFs, coding for gp77, gp78 and gp79, presumably function as shut-off genes during early stages of phage replication. There is evidence that cell division is affected by one of the proteins (gp79). The transcription of the cytotoxic polypeptides is directed by a promoter situated in ORF83 and transcription control is achieved through the phage repressor gp71, which is shown by co-expression of this protein. The findings presented here should provide useful tools for the molecular genetics of mycobacteria. Further analysis of these and other mycobacteriophage-derived toxic polypeptides, together with the identification of their cellular targets, might provide a tool for the rapid identification of promising drug targets in emerging and re-emerging mycobacterial pathogens. Refereed/Peer-reviewed

Mycolactone produced by Mycobacterium ulcerans is the toxin responsible for most of the pathology in Buruli ulcer, the cutaneous signature of a complex disease. Although mycolactone cytopathicity is well described in various in vitro and in vivo models, the effect of this molecule on mammalian skeletal muscles has not been addressed. This is particularly surprising since muscle damage is characteristic of severe Buruli ulcer. We have thus investigated the impact of mycolactone on the mouse soleus muscle during degenerative and regenerative phases. Mice were intramuscularly injected with 300 μg of mycolactone and soleus muscles assessed histologically, biochemically and functionally at 7 and 42 days post-injection. Our results show that mycolactone induces local acute and chronic inflammatory responses which are respectively associated with a 65% and 68% decrease in maximal isometric force production (P0) relative to sham injections. In addition, muscle stiffness and total hydroxyproline content rose by 46% and 134% at day 42 relative to sham injections indicating an extensive fibrotic process in injured soleus muscles. Histological observations demonstrate significant muscle necrosis and atrophy with limited signs of regeneration. Together, our data indicate that mycolactone not only induces muscle damage but also prevents muscle regeneration to occur. These results may help to explain why patients with Buruli ulcer, experience muscle weakness and contracture.

Buruli ulcer is a chronic skin disease caused by Mycobacterium ulcerans , which produces a toxic lipid mycolactone. Despite the extensive necrosis and tissue damage, the lesions are painless. This absence of pain prevents patients from seeking early treatment and, as a result, many patients experience severe sequelae, including limb amputation. We have reported that mice inoculated with M. ulcerans show loss of pain sensation and nerve degeneration. However, the molecules responsible for the nerve damage have not been identified. In order to clarify whether mycolactone alone can induce nerve damage, mycolactone A/B was injected to footpads of BALB/c mice. A total of 100 μg of mycolactone induced footpad swelling, redness, and erosion. The von Frey sensory test showed hyperesthesia on day 7, recovery on day 21, and hypoesthesia on day 28. Histologically, the footpads showed epidermal erosion, moderate stromal edema, and moderate neutrophilic infiltration up to day 14, which gradually resolved. Nerve bundles showed intraneural hemorrhage, neutrophilic infiltration, and loss of Schwann cell nuclei on days 7 and 14. Ultrastructurally, vacuolar change of myelin started on day 14 and gradually subsided by day 42, but the density of myelinated fibers remained low. This study demonstrated that initial hyperesthesia is followed by sensory recovery and final hypoesthesia. Our present study suggests that mycolactone directly damages nerves and is responsible for the absence of pain characteristic of Buruli ulcer. Furthermore, mice injected with 200 μg of mycolactone showed pulmonary hemorrhage. This is the first study to demonstrate the systemic effects of mycolactone.