RESUMEN

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.

Asunto(s)

Aedes/microbiología , Aedes/fisiología , Toxinas Bacterianas/metabolismo , Macrólidos/metabolismo , Mycobacterium ulcerans/patogenicidad , Oviposición/fisiología , Animales , Úlcera de Buruli/microbiología , Femenino

RESUMEN

A research colony of Xenopus (Silurana) tropicalis frogs presented with nodular and ulcerative skin lesions. Additional consistent gross findings included splenomegaly with multiple tan-yellow nodular foci in the spleen and liver of diseased frogs. Copious acid-fast positive bacteria were present in touch impression smears of spleen, skin, and livers of diseased frogs. Histologically, necrotizing and granulomatous dermatitis, splenitis, and hepatitis with numerous acid-fast bacilli were consistently present, indicative of systemic mycobacteriosis. Infrequently, granulomatous inflammation was noted in the lungs, pancreas, coelomic membranes, and rarely reproductive organs. Ultrastructurally, both extracellular bacilli and intracellular bacilli within macrophages were identified. Frogs in the affected room were systematically depopulated, and control measures were initiated. Cultured mycobacteria from affected organs were identified and genetically characterized as Mycobacterium liflandii by polymerase chain reaction amplification of the enoyl reductase domain and specific variable numbers of tandem repeats. In recent years, M. liflandii has had a devastating impact on research frog colonies throughout the United States. This detailed report with ultrastructural description of M. liflandii aids in further understanding of this serious disease in frogs.

Asunto(s)

Infecciones por Mycobacterium no Tuberculosas/veterinaria , Mycobacterium/clasificación , Xenopus , Animales , Brotes de Enfermedades , Femenino , Hemorragia , Hígado/patología , Pulmón/patología , Infecciones por Mycobacterium no Tuberculosas/epidemiología , Infecciones por Mycobacterium no Tuberculosas/patología , Ovario/patología , Páncreas/patología , Bazo/patología

RESUMEN

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.

Asunto(s)

Heterópteros/microbiología , Mycobacterium ulcerans/aislamiento & purificación , África Occidental , Estructuras Animales/microbiología , Animales , Culicidae/microbiología , ADN Bacteriano/genética , Larva/microbiología , Reacción en Cadena de la Polimerasa/métodos

RESUMEN

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 degrees 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 IS2404. 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 IS2404. In contrast to M. ulcerans and conventional M. marinum, mycolactone F-producing mycobacteria are incapable of growth at above 30 degrees 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.

Asunto(s)

Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/toxicidad , Ácidos Grasos Insaturados/biosíntesis , Peces/microbiología , Infecciones por Mycobacterium/metabolismo , Mycobacterium/patogenicidad , Plásmidos/genética , Animales , Apoptosis/inmunología , Toxinas Bacterianas/genética , Toxinas Bacterianas/aislamiento & purificación , Línea Celular , Ácidos Grasos Insaturados/química , Ácidos Grasos Insaturados/toxicidad , Fibroblastos/inmunología , Fibroblastos/microbiología , Fibroblastos/patología , Humanos , Lactonas/química , Lactonas/toxicidad , Macrólidos , Ratones , Datos de Secuencia Molecular , Mycobacterium/genética , Mycobacterium/aislamiento & purificación , Infecciones por Mycobacterium/epidemiología , Necrosis , Virulencia/genética

RESUMEN

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.

Asunto(s)

Toxinas Bacterianas/aislamiento & purificación , Mycobacterium ulcerans/patogenicidad , Micobacterias no Tuberculosas/patogenicidad , Xenopus/microbiología , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Secuencia de Bases , Macrólidos , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Virulencia

RESUMEN

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 microg/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.

Asunto(s)

Toxinas Bacterianas/biosíntesis , Mycobacterium/clasificación , Animales , Toxinas Bacterianas/toxicidad , Cromatografía en Capa Delgada , Medios de Cultivo Condicionados , ADN Ribosómico/análisis , Células L , Macrólidos , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Mycobacterium/genética , Mycobacterium/metabolismo , Mycobacterium ulcerans/metabolismo , Filogenia , ARN Ribosómico 16S/genética , Microbiología del Agua

RESUMEN

Resistance to killing by low pH is a common feature of both Escherichia coli and Shigella flexneri. The most effective E. coli acid resistance system utilizes two isoforms of glutamate decarboxylase encoded by gadA and gadB, and a putative glutamate/gamma-amino butyric acid antiporter encoded by gadC. Expression of the gad system is dependent upon the alternate sigma factor, sigma(s). We confirm that gadA, gadB, and gadC are also all dependent upon sigma(s) for their expression in S. flexneri. -10 sequences similar to the sigma(s)-10 consensus sequence were identified by primer extension in the upstream promoters of all three genes and the transcriptional start points were identical in both E. coli and S. flexneri.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Genes Bacterianos , Regiones Promotoras Genéticas , Shigella flexneri/genética , Shigella flexneri/metabolismo , Factor sigma/metabolismo , Secuencia de Bases , Sitios de Unión/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Farmacorresistencia Bacteriana/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Glutamato Descarboxilasa/genética , Ácido Glutámico/metabolismo , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Shigella flexneri/efectos de los fármacos , Especificidad de la Especie

RESUMEN

Strains of Escherichia coli K-12, O157:H7, and Shigella flexneri grown to stationary phase in complex unbuffered media can survive for several hours at pH 2.5. This stationary-phase acid resistance phenotype is dependent upon the alternate sigma factor sigmas and the supplementation of either glutamate or glutamine in the acidified media used for acid challenge. Acid resistance under these defined conditions can be inhibited by the glutamate analog L-trans-pyrrolidine-2,4-dicarboxylic acid which blocks uptake of glutamate/glutamine by selective inhibition. The gadC gene, encoding an inner membrane antiporter essential for the expression of acid resistance, could not be detected in other family members of the Enterobacteriacae.

Asunto(s)

Proteínas Bacterianas , Ácidos Dicarboxílicos/farmacología , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Inhibidores de la Captación de Neurotransmisores/farmacología , Pirrolidinas/farmacología , Shigella flexneri/efectos de los fármacos , Shigella flexneri/metabolismo , Ácidos/metabolismo , Medios de Cultivo , ADN Bacteriano/análisis , Escherichia coli/genética , Ácido Glutámico/análogos & derivados , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Técnicas In Vitro , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fenotipo , Plásmidos , Shigella flexneri/genética , Factor sigma/metabolismo , Ácido gamma-Aminobutírico/metabolismo

RESUMEN

Resistance to being killed by acidic environments with pH values lower than 3 is an important feature of both pathogenic and nonpathogenic Escherichia coli. The most potent E. coli acid resistance system utilizes two isoforms of glutamate decarboxylase encoded by gadA and gadB and a putative glutamate:gamma-aminobutyric acid antiporter encoded by gadC. The gad system is controlled by two repressors (H-NS and CRP), one activator (GadX), one repressor-activator (GadW), and two sigma factors (sigma(S) and sigma(70)). In contrast to results of previous reports, we demonstrate that gad transcription can be detected in an hns rpoS mutant strain of E. coli K-12, indicating that gad promoters can be initiated by sigma(70) in the absence of H-NS.

Asunto(s)

Farmacorresistencia Bacteriana/genética , Escherichia coli/efectos de los fármacos , Glutamato Descarboxilasa/metabolismo , Glutamatos/metabolismo , Proteínas de la Membrana/metabolismo , Transcripción Genética , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Glutamato Descarboxilasa/genética , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/genética , Mutación , Factor sigma/genética

RESUMEN

Mycolactone is a macrolide secreted by Mycobacterium ulcerans. Experimental evidence suggests that mycolactone plays a prominent role in the pathogenesis of Buruli ulcer by causing both tissue destruction and immunosuppression. To understand the cell biology of mycolactone activity, we have synthesized the fluorescent mycolactone derivativebodipymycolactone. Although derivatization resulted in a modest decrease in cytopathic activity, the derivatized and native molecules produce identical phenotypes in cultured cells. Confocal microscopy of bodipymycolactone added to cultured fibroblasts, shows that it is localized to the cytosol. Bodipymycolactone fails to bind to the cell membrane and is excluded from the nucleus. Uptake is both nonsaturable and noncompetitive with excess mycolactone, consistent with passive diffusion of this toxin through the cell membrane. These facts, combined with the inability of signal transduction inhibitors to inhibit mycolactone cytopathicity point towards the presence of an cytosolic target for mycolactone.A dose dependent increase in intracellular calcium levels at occurs upon mycolactone exposure, but chelation of intracellular calcium alters neither the cytopathicity nor the caspase induction profile of treated cells. Mitochondrial polarization is maintained in treated cells for up to 3 days arguing that the rise in intracellular calcium levels may be a result of cytoskeletal remodeling.

Asunto(s)

Toxinas Bacterianas/metabolismo , Mycobacterium ulcerans/patogenicidad , Factores de Virulencia/metabolismo , Toxinas Bacterianas/clasificación , Toxinas Bacterianas/toxicidad , Calcio/metabolismo , Caspasas/análisis , Caspasas/metabolismo , Células Cultivadas , Fibroblastos/enzimología , Fibroblastos/metabolismo , Macrólidos , Mitocondrias/metabolismo , Estructura Molecular , Infecciones por Mycobacterium no Tuberculosas/microbiología

RESUMEN

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.

Asunto(s)

Toxinas Bacterianas/clasificación , Toxinas Bacterianas/toxicidad , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium ulcerans/patogenicidad , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/aislamiento & purificación , Línea Celular , Cromatografía en Capa Delgada , Fibroblastos , Humanos , Macrólidos , Espectrometría de Masas , Ratones , Virulencia

RESUMEN

BACKGROUND: Ultraviolet radiation (UV) pre-exposure enhances intracellular mycobacterial infections, however, its effect upon the pathogenesis of the extracellular Mycobacterium ulcerans parasite had not been previously examined. The hypothesis tested was that UV pre-exposure enhances both the nodular and ulcerative forms of M. ulcerans infection in the Crl:IAF(HA)-hrBR hairless guinea pig. METHODS: Groups of five animals were exposed to total cumulative UV doses of 0 (control), 3 or 30 kJ/m2 followed 3 days later by subcutaneous infection with 3 x 10(4) CFU of M. ulcerans in order to induce the nodular form of the disease. The resultant nodules were then measured for the next 22 days. The experiment was then repeated using intradermal infection with 2 x 10(6) CFU in order to induce the ulcerative form of the disease. The resultant ulcers were measured for the next 30 days. In both experiments, the animals were tested for delayed-type hypersensitivity (DTH) reactivity to Burulin-S as a marker of the onset of the reactive phase of the disease. RESULTS: Following low inoculum subcutaneous infection, distinct, well-demarcated, subcutaneously situated skin nodules were present at infected skin sites between 7 and 22 days post-infection. Between days 14 and 21, the mean nodule diameters of the UV irradiated groups were significantly (P < 0.03) greater than that of the control group. UV pre-exposure resulted in significant (P < 0.035) suppression of DTH responses to Burulin-S challenge. High inoculum intradermal infection resulted in the development of ulcerative lesions. Between 10 and 30 days post-infection, the mean lesion diameters and mean ulcer development times of UV irradiated groups were significantly (P < 0.05) greater than those of the controls. However, UV irradiation did not affect DTH responses to Burulins in the high inoculum experiment. In both experiments, the lesions were histologically consistent with human Buruli ulcer disease. These results demonstrate that UV pre-exposure results in enhanced M. ulcerans infection in the hairless guinea pig model of Buruli ulcer disease and suggest that UV exposure may be a relevant factor in the pathogenesis of human forms of the disease.

Asunto(s)

Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium ulcerans/efectos de la radiación , Enfermedades Cutáneas Bacterianas/microbiología , Úlcera Cutánea/microbiología , Rayos Ultravioleta , Animales , Modelos Animales de Enfermedad , Femenino , Cobayas , Hipersensibilidad Tardía , Pruebas Intradérmicas , Masculino , Infecciones por Mycobacterium no Tuberculosas/patología , Mycobacterium ulcerans/inmunología , Mycobacterium ulcerans/patogenicidad , Enfermedades Cutáneas Bacterianas/patología , Úlcera Cutánea/patología , Factores de Tiempo

RESUMEN

Mycolactones are polyketide toxins produced by Mycobacterium ulcerans, the causative agent of the tropical skin disease known as Buruli ulcer. Development of novel therapeutic agents from mycolactones has been hindered by the difficulty of producing sufficient amounts of material. Here, we describe the successful adaptation of M. ulcerans to suspension cultivation and the development of a fed-batch fermentation process that was scaled up to 150 l. In addition to producing mycolactones A and B, a number of new mycolactone-related compounds were also observed.

Asunto(s)

Toxinas Bacterianas/biosíntesis , Mycobacterium ulcerans/metabolismo , Toxinas Bacterianas/química , Macrólidos , Estructura Molecular , Factores de Tiempo

RESUMEN

We have found the dye LDS-751 to bind almost exclusively to mitochondria when incubated with viable, nucleated cells. Treatment of cells with the nuclear stain acridine orange and LDS-751 revealed little colocalization when the cells were examined by confocal microscopy. Staining with the dye rhodamine 123, which is known to bind polarized mitochondria, was virtually identical to the pattern observed with LDS-751. This staining pattern was observed to be consistent over a range of 0.02-20 microg/ml LDS-751 and was consistent between both fibroblasts and monocytes. Depolarization of mitochondria with the mitochondrial depolarizing agents phenyl arsine oxide and carbonyl cyanide m-chlorophenylhydrazone (CCCP) dramatically reduced both LDS-751 staining, and rhodamine 123 fluorescence. Taken together, these results suggest that LDS-751 is excluded from the nucleus and binds the polarized membranes of mitochondria. Given this, interpretation of LDS-751 fluorescence as being indicative of nuclear status, as is commonly done to discriminate between leukocytes and erythrocytes, is unwarranted and may lead to erroneous conclusions if mitochondria become depolarized upon processing.

Asunto(s)

Colorantes Fluorescentes , Mitocondrias/ultraestructura , Coloración y Etiquetado/métodos , Animales , Arsenicales , Carbonil Cianuro m-Clorofenil Hidrazona , Línea Celular , Digitonina , Potenciales de la Membrana , Ratones , Microscopía Confocal , Monocitos/ultraestructura , Compuestos Orgánicos , Rodamina 123

RESUMEN

An adipose cell (SW872) model was developed to observe cellular necrosis and apoptosis upon Mycobacterium ulcerans infection and treatment with mycobacterial exudate. Apoptosis was likely due to secreted proteins, while necrosis was likely due to mycolactone. Our data suggest that additional factors in M. ulcerans may be involved in Buruli ulcer pathogenesis.

Asunto(s)

Adipocitos/microbiología , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Mycobacterium ulcerans/metabolismo , Adipocitos/citología , Adipocitos/efectos de los fármacos , Tejido Adiposo/citología , Apoptosis , Toxinas Bacterianas/farmacología , Línea Celular , Medios de Cultivo/farmacología , Humanos , Macrólidos , Mycobacterium ulcerans/patogenicidad , Necrosis

Asunto(s)

Toxinas Bacterianas/química , Toxinas Bacterianas/aislamiento & purificación , Cinética , Macrólidos , Mycobacterium ulcerans/química , Resonancia Magnética Nuclear Biomolecular , Estereoisomerismo

Asunto(s)

Toxinas Bacterianas/química , Macrólidos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Estereoisomerismo

RESUMEN

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a tropical ulcerative skin disease. One of the most intriguing aspects of this disease is the presence of extensive tissue damage in the absence of an acute inflammatory response. We recently purified and characterized a macrolide toxin, mycolactone, from M. ulcerans. Injection of this molecule into guinea pig skin reproduced cell death and lack of acute inflammatory response similar to that seen following the injection of viable bacteria. We also showed that mycolactone causes a cytopathic effect on mouse fibroblast L929 cells that is characterized by cytoskeletal rearrangements and growth arrest within 48 h. However, these results could not account for the extensive cell death which occurs in Buruli ulcer. The results presented here demonstrate that L929 and J774 mouse macrophage cells die via apoptosis after 3 to 5 days of exposure to mycolactone. Treatment of cells with a pan-caspase inhibitor can inhibit mycolactone-induced apoptosis. We demonstrate that injection of mycolactone into guinea pig skin results in cell death via apoptosis and that the extent of apoptosis increases as the lesion progresses. These results may help to explain why tissue damage in Buruli ulcer is not accompanied by an acute inflammatory response.

Asunto(s)

Apoptosis/efectos de los fármacos , Toxinas Bacterianas/toxicidad , Mycobacterium ulcerans/patogenicidad , Úlcera Cutánea/etiología , Animales , Línea Celular , ADN Bacteriano/análisis , Femenino , Cobayas , Etiquetado Corte-Fin in Situ , Macrólidos , Ratones

RESUMEN

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-kappa B activation. Surprisingly, a synergy was observed between the factor and phorbol ester-directed NF-kappa B activation. The factor had no effect on IL-1- or LPS-induced NF-kappa B activity, indicating selective activity of the factor. The factor did not inhibit the degradation of I kappa B alpha 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.

Asunto(s)

Toxinas Bacterianas/farmacología , Citocinas/antagonistas & inhibidores , Inmunosupresores/farmacología , Monocitos/inmunología , Mycobacterium ulcerans/inmunología , FN-kappa B/antagonistas & inhibidores , Linfocitos T/inmunología , Animales , Apoptosis/inmunología , Citocinas/biosíntesis , Exotoxinas/farmacología , Cobayas , Humanos , Inmunidad Innata/efectos de los fármacos , Interleucina-10/antagonistas & inhibidores , Interleucina-10/biosíntesis , Células L , Lípidos/fisiología , Ratones , Monocitos/metabolismo , FN-kappa B/fisiología , Solubilidad , Linfocitos T/metabolismo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

A Mycobacterium marinum promoter, designated G13, was isolated from a promoter-trap library as a constitutive producer of the mutant green fluorescent protein. Sequence analysis, primer extension analysis, and computer promoter prediction analysis indicate that the G13 promoter is very similar to Escherichia coli consensus sigma 70 promoters. Expression of the green fluorescent protein from the G13 promoter in M. marinum is, however, up to 40 times higher than that seen from the mycobacterial hsp60 promoter during exponential growth. Further, expression from this promoter does not appear to affect the growth of the organism in culture media or in macrophages. The strong expression of the G13 promoter allows it to be developed as a useful molecular tool for high level expression of markers in vitro.

Asunto(s)

ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/metabolismo , Mycobacterium marinum/genética , Mycobacterium smegmatis/metabolismo , Regiones Promotoras Genéticas/genética , Factor sigma/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/biosíntesis , Macrófagos/microbiología , Ratones , Datos de Secuencia Molecular , Mycobacterium marinum/crecimiento & desarrollo , Mycobacterium marinum/metabolismo , ARN Bacteriano/genética , Análisis de Secuencia de ADN , Factor sigma/metabolismo