Mycobacterium liflandii outbreak in a research colony of Xenopus (Silurana) tropicalis frogs.

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.

Persistent association of Mycobacterium ulcerans with West African predaceous insects of the family belostomatidae.

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.

Globally distributed mycobacterial fish pathogens produce a novel plasmid-encoded toxic macrolide, mycolactone F.

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.

A newly discovered mycobacterial pathogen isolated from laboratory colonies of Xenopus species with lethal infections produces a novel form of mycolactone, the Mycobacterium ulcerans macrolide toxin.

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.

Analysis of Mycobacterium species for the presence of a macrolide toxin, mycolactone.

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.

Identification of the promoter regions and sigma(s)-dependent regulation of the gadA and gadBC genes associated with glutamate-dependent acid resistance in Shigella flexneri.

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.

The glutamate-dependent acid resistance system of Escherichia coli and Shigella flexneri is inhibited in vitro by L-trans-pyrrolidine-2,4-dicarboxylic acid.

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.

Transcriptional expression of Escherichia coli glutamate-dependent acid resistance genes gadA and gadBC in an hns rpoS mutant.

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.

Uptake and cellular actions of mycolactone, a virulence determinant for Mycobacterium ulcerans.

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.

Heterogeneity of mycolactones produced by clinical isolates of Mycobacterium ulcerans: implications for virulence.

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.

Ultraviolet radiation enhances both the nodular and ulcerative forms of Mycobacterium ulcerans infection in a Crl:IAF(HA)-hrBR hairless guinea pig model of Buruli ulcer disease.

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.