Common evolutionary origin for the unstable virulence plasmid pMUM found in geographically diverse strains of Mycobacterium ulcerans

The 174-kb virulence plasmid pMUM001 in Mycobacterium ulcerans epidemic strain Agy99 harbors three very large and homologous genes that encode giant polyketide synthases (PKS) responsible for the synthesis of the lipid toxin mycolactone. Deeper investigation of M. ulcerans Agy99 resulted in identification of two types of spontaneous deletion variants of pMUM001 within a population of cells that also contained the intact plasmid. These variants arose from recombination between two 8-kb sections of the same plasmid sequence, resulting in the loss of a 65-kb region bearing two of the three mycolactone PKS genes. Investigation of nine diverse M. ulcerans strains by using PCR and Southern hybridization for eight pMUM001 gene sequences confirmed the presence of pMUM001-like elements (collectively called pMUM) in all M. ulcerans strains. Physical mapping of these plasmids revealed that like M. ulcerans Agy99, three strains had undergone major deletions in their mycolactone PKS loci. Online liquid chromatography-sequential mass spectrometry analysis of lipid extracts confirmed that strains with PKS deletions were unable to produce mycolactone or any related cometabolites. Interstrain comparisons of the plasmid gene sequences revealed greater than 98% nucleotide identity, and the phylogeny inferred from these sequences closely mimicked the phylogeny from a previous multilocus sequence typing study in which chromosomally encoded loci were used, a result that is consistent with the hypothesis that M. ulcerans diverged from the closely related organism Mycobacterium marinum by acquiring pMUM. Our results suggest that pMUM is a defining characteristic of M. ulcerans but that in the absence of purifying selection, deletion of plasmid sequences and a corresponding loss of mycolactone production readily arise.     

Subtractive hybridization reveals a type I polyketide synthase locus specific to Mycobacterium ulcerans

Mycobacterium ulcerans causes Buruli ulcer, the third most prevalent mycobacterial infection of immunocompetent humans after tuberculosis and leprosy. Recent work has shown that the production by M. ulcerans of mycolactone, a novel polyketide, may partly explain the pathogenesis of Buruli ulcer. To search for the genetic basis of virulence in M. ulcerans, we took advantage of the close genetic relationship between M. ulcerans and Mycobacterium marinum by performing genomic suppressive subtractive hybridization of M. ulcerans with M. marinum. We identified several DNA fragments specific to M. ulcerans, in particular, a type I polyketide synthase locus with a highly repetitive modular arrangement. We postulate that this locus is responsible for the synthesis of mycolactone in M. ulcerans.     

Mycobacterium ulcerans toxic macrolide, mycolactone modulates the host immune response and cellular location of M. ulcerans in vitro and in vivo

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 causes minimal pathogenesis and colonization in medaka (Oryzias latipes): an experimental fish model of disease transmission

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.     

Comparative genetic analysis of Mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent divergence

Previous studies of the 16S rRNA genes from Mycobacterium ulcerans and Mycobacterium marinum have suggested a very close genetic relationship between these species (99.6% identity). However, these organisms are phenotypically distinct and cause diseases with very different pathologies. To investigate this apparent paradox, we compared 3,306 nucleotides from the partial sequences of eight housekeeping and structural genes derived from 18 M. ulcerans strains and 22 M. marinum strains. This analysis confirmed the close genetic relationship inferred from the 16S rRNA data, with nucleotide sequence identity ranging from 98.1 to 99.7%. The multilocus sequence analysis also confirmed previous genotype studies of M. ulcerans that have identified distinct genotypes within a geographical region. Single isolates of both M. ulcerans and M. marinum that were shown by the sequence analysis to be the most closely related were then selected for further study. One- and two-dimensional pulsed-field gel electrophoresis was employed to compare the architecture and size of the genome from each species. Genome sizes of approximately 4.4 and 4.6 Mb were obtained for M. ulcerans and M. marinum, respectively. Significant macrorestriction fragment polymorphism was observed between the species. However, hybridization analysis of DNA cleaved with more frequently cutting enzymes identified significant preservation of the flanking sequence at seven of the eight loci sequenced. The exception was the 16S rRNA locus. Two high-copy-number insertion sequences, IS2404 and IS2606, have recently been reported in M. ulcerans, and significantly, these elements are not present in M. marinum. Hybridization of the AseI restriction fragments from M. ulcerans with IS2404 and IS2606 indicated widespread genome distribution for both of these repeated sequences. Taken together, these data strongly suggest that M. ulcerans has recently diverged from M. marinum by the acquisition and concomitant loss of DNA in a manner analogous to the emergence of M. tuberculosis, where species diversity is being driven mainly by the activity of mobile DNA elements.     

Mycobacterium marinum infections in humans and tracing of its possible environmental sources

The low frequency of nontuberculous mycobacterial infections, nonspecific symptoms for individual mycobacteria, and the lack of specific identification methods could alter correct diagnosis. This study presents a combined microbiology and molecular-based approach for Mycobacterium?marinum detection in four aquarists with cutaneous mycobacterial infection. Simultaneously, ecology screening for M.?marinum presence in the aquarists' fish tanks was performed. A total of 38 mycobacterial isolates originated from four human patients (n?=?20), aquarium animals (n?=?8), and an aquarium environment (n?= 10). Isolate identification was carried out using 16S?rRNA sequence analysis. A microbiology-based approach, followed by 16S rRNA sequence analysis, was successfully used for detection of M.?marinum in all four patients. Animal and environmental samples were simultaneously examined, and a total of seven mycobacterial species were isolated: Mycobacterium chelonae , Mycobacterium fortuitum , Mycobacterium gordonae , Mycobacterium?kansasii , Mycobacterium mantenii , Mycobacterium marinum , and Mycobacterium?peregrinum . The presence of M.?marinum was proven in the aquarium environments of two patients. Although M.?marinum is described as being present in water, it was detected only in fish.     

Genotyping Mycobacterium ulcerans and Mycobacterium marinum by using mycobacterial interspersed repetitive units

A novel category of variable tandem repeats (VNTR) called mycobacterial interspersed repetitive units (MIRUs) has been identified for Mycobacterium ulcerans (n = 39), M. marinum (n = 27), and one related organism. Fifteen MIRU loci were identified in the genome of M. marinum and were used to genotype M. ulcerans, M. marinum, and an M. marinum-like organism that is considered a possible missing link between M. marinum and M. ulcerans. Seven MIRU loci were polymorphic, and locus-specific PCRs for four of these loci differentiated seven M. ulcerans genotypes, four M. marinum genotypes, and a unique genotype for the missing link organism. The seven M. ulcerans genotypes were related to six different geographic origins of isolates. All isolates from West and Central Africa, including old and recent isolates, belonged to the same genotype, emphasizing the great spatiotemporal homogeneity among African isolates. Unlike the M. ulcerans genotypes, the four M. marinum genotypes could not be clearly related to the geographic origins of the isolates. According to MIRU-VNTR typing, all M. ulcerans and M. marinum isolates of American origin were closely related, suggesting a common American ancestor for these two pathogenic species on the American continents. MIRU typing has significant potential value for discriminating between reoccurrence and reinfection for M. ulcerans disease.     

The phenolic mycoside of Mycobacterium ulcerans: structure and taxonomic implications.

Mycobacterium ulcerans and some pathogenic mycobacterial species elaborate wax A consisting of related long-chain beta-diol components (phthiocerol and related compounds) esterified by multimethyl-branched fatty acids. With the exception of M. ulcerans, wax A-containing mycobacteria also synthesize glycosylated phenol phthiocerol diester and related compounds: the so-called phenolic mycosides. In a deliberate effort to characterize this latter class of compounds in M. ulcerans, 20 strains were examined. Phenolic mycosides were found in two strains. Application of chemical analyses, including one- and two-dimensional NMR spectroscopy, allowed the structural elucidation of glycolipids identified as 3-O-methyl-alpha-L-rhamnosyl phenol phthiocerol diphthioceranate investigators. As phenolic mycosides are highly species-specific molecules, this finding stresses the close phylogenetic link between M. marinum and M. ulcerans. Incidentally, a survey of the mycolate content of M. ulcerans showed that methoxymycolate could not be detected in three strains.     

Characterization of photochromogenic Mycobacterium spp. from Chesapeake Bay striped bass Morone saxatilis

A large diversity of Mycobacterium spp. has been isolated from striped bass Morone saxatilis in Chesapeake Bay, USA. The new species M. shottsii and M. pseudoshottsii are the dominant isolates, while the classical fish pathogen M. marinum is found much less frequently. M. fortuitum and M. chelonae, other Mycobacterium spp. known to commonly infect fishes, have not yet been aseptically isolated from striped bass within Chesapeake Bay. While M. pseudoshottsii and M. shottsii have been phenotypically and genotypically characterized, other less common mycobacterial isolates have not. In the present study, we describe 17 photochromogenic isolates from Chesapeake Bay striped bass using phenotypic characterization and multilocus sequencing of 16S rRNA, hsp65 and rpoB genes. Genetic characterization reveals that these isolates are related to widely divergent portions of the mycobacterial phylogeny; however, some interesting trends are observed, such as a majority of isolates (10/17) belonging to the M. simiae-related grouping. Five additional isolates were assigned to the slow-growing mycobacteria (including 2 identified as M. marinum), while 2 are clearly shown to belong genetically to the fast-growing mycobacteria.     

Evaluation of VNTR typing for the identification of Mycobacterium ulcerans in environmental samples from Victoria, Australia

Reliable molecular detection of Mycobacterium ulcerans in environmental samples is essential to study the ecology and transmission of this important human pathogen. Variable number tandem repeat (VNTR) typing is a valuable method for distinguishing M. ulcerans isolates from different geographic regions and for distinguishing M. ulcerans from other members of the Mycobacterium marinum/M. ulcerans complex, but its application to environmental samples has not yet been evaluated systematically. This study compares the sensitivity and specificity of PCR detection of 13 VNTR loci to determine the best loci for the analysis of environmental samples. This study demonstrates that VNTR typing using selected loci can be a useful addition to established molecular methods for detecting M. ulcerans in the environment and highlights some of the issues encountered when using molecular methods to detect microorganisms in environmental samples. When applied to environmental samples collected from an endemic region in Victoria, Australia, VNTR typing confirmed that the strain of M. ulcerans being detected was indistinguishable from the strain causing disease in humans in that region.     

The Cell Wall-Associated Mycolactone Polyketide Synthases Are Necessary but Not Sufficient for Mycolactone Biosynthesis

Mycolactones are polyketide-derived lipid virulence factors made by the slow-growing human pathogen, Mycobacterium ulcerans. Three unusually large and homologous plasmid-borne genes (mlsA1: 51 kb, mlsB: 42 kb and mlsA2: 7 kb) encode the mycolactone type I polyketide synthases (PKS). The extreme size and low sequence diversity of these genes has posed significant barriers for exploration of the genetic and biochemical basis of mycolactone synthesis. Here, we have developed a truncated, more tractable 3-module version of the 18-module mycolactone PKS and we show that this engineered PKS functions as expected in the natural host M. ulcerans to produce an additional polyketide; a triketide lactone (TKL). Cell fractionation experiments indicated that this 3-module PKS and the putative accessory enzymes encoded by mup045 and mup038 associated with the mycobacterial cell wall, a finding supported by confocal microscopy. We then assessed the capacity of the faster growing, Mycobacterium marinum to harbor and express the 3-module Mls PKS and accessory enzymes encoded by mup045 and mup038. RT-PCR, immunoblotting, and cell fractionation experiments confirmed that the truncated Mls PKS multienzymes were expressed and also partitioned with the cell wall material in M. marinum. However, this heterologous host failed to produce TKL. The systematic deconstruction of the mycolactone PKS presented here suggests that the Mls multienzymes are necessary but not sufficient for mycolactone synthesis and that synthesis is likely to occur (at least in part) within the mycobacterial cell wall. This research is also the first proof-of-principle demonstration of the potential of this enzyme complex to produce tailored small molecules through genetically engineered rearrangements of the Mls modules.     

Differentiation Between Mycobacterium kansasii and Mycobacterium marinum by Gas-Liquid Chromatographic Analysis of Cellular Fatty Acids

Comparison of the cellular fatty acids of 10 strains of Mycobacterium marinum and 35 strains of Mycobacterium kansasii revealed similarities within each species but differences between these two photochromogenic mycobacteria. A branched-chain fatty acid characteristic of M. kansasii was found in trace amounts in 2 of the 10 strains of M. marinum.     

Biofilm formation and biocide susceptibility testing of Mycobacterium fortuitum and Mycobacterium marinum

The ability of non-tuberculous mycobacteria to form biofilms may allow for their increased resistance to currently used biocides in medical and industrial settings. This study examines the biofilm growth of Mycobacterium fortuitum and Mycobacterium marinum, using the MBEC trade mark assay system, and compares the susceptibility of planktonic and biofilm cells to commercially available biocides. With scanning electron microscopy, both M. fortuitum and M. marinum form biofilms that are morphologically distinct. Biocide susceptibility testing suggested that M. fortuitum biofilms displayed increased resistance over their planktonic state. This is contrasted with M. marinum biofilms, which were generally as or more susceptible over their planktonic state.     

Early trafficking events of Mycobacterium ulcerans within Naucoris cimicoides

The severe skin-destructive disease caused by Mycobacterium ulcerans, named Buruli ulcer, is the third most important mycobacterial disease in humans after tuberculosis and leprosy. Recently we demonstrated that M. ulcerans could colonize the salivary glands of the water bug, Naucoris cimicoides. In this study, we report that M. ulcerans may be delivered from the digested prey aspirate to the coelomic cavity via a unique headspace, the head capsule (HC). During the infected meal, we observed that M. ulcerans clusters adhered to the stylets that were retracted in the HC at the end of the meal. M. ulcerans was able to translocate from the HC to the coelomic cavity where it is phagocytosed by the plasmatocytes. These cells are subverted as shuttle cells and deliver M. ulcerans to the salivary glands. At this early stage of its parasitic life style, two other important features of M. ulcerans can be documented: first, mycolactone is not required for translocation of M. ulcerans into the HC, in contrast to the next step, colonization of the salivary glands; second, M. ulcerans clusters bind a member of the serpin protein family present in the salivary gland homogenate.     

VNTR analysis differentiates Mycobacterium ulcerans and IS2404 positive mycobacteria

In recent years, numerous IS2404 positive mycobacteria have been identified, compromising the detection of Mycobacterium ulcerans. In this study, variable number of tandem repeats (VNTR) analysis was successfully applied on cultures and tissue specimens to differentiate all currently known IS2404 positive mycobacteria from M. ulcerans.     

Comparative pathogenesis of Mycobacterium marinum and Mycobacterium tuberculosis

A thorough understanding of Mycobacterium tuberculosis pathogenesis in humans has been elusive in part because of imperfect surrogate laboratory hosts, each with its own idiosyncrasies. Mycobacterium marinum is the closest genetic relative of the M. tuberculosis complex and is a natural pathogen of ectotherms. In this review, we present evidence that the similar genetic programmes of M. marinum and M. tuberculosis and the corresponding host immune responses reveal a conserved skeleton of Mycobacterium host–pathogen interactions. While both species have made niche-specific refinements, an essential framework has persisted. We highlight genetic comparisons of the two organisms and studies of M. marinum in the developing zebrafish. By pairing M. marinum with the simplified immune system of zebrafish embryos, many of the defining mechanisms of mycobacterial pathogenesis can be distilled and investigated in a tractable host/pathogen pair.     

Mycobacterium marinum: the generalization and specialization of a pathogenic mycobacterium

Mycobacterium marinum is being used increasingly as a model for understanding pathogenic mycobacteria. However, recently discovered differences between M. marinum and M. tuberculosis suggest that adaptation to specialized niches is reflected in unique strategies of pathogenesis. This review emphasizes the areas in which studying M. marinum has made contributions to the understanding of tuberculosis, as well as the potential for using characteristics unique to M. marinum for understanding general issues of host-pathogen interactions.     

Comprehensive proteome analysis of Mycobacterium ulcerans and quantitative comparison of mycolactone biosynthesis

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rapidly emerging human disease in which mycolactone, a cytotoxic and immunosuppressive macrocyclic polyketide, is responsible for massive skin destruction. The genome sequencing of M. ulcerans has recently been accomplished (http://genolist.pasteur.fr/BuruList/) enabling the first proteome study of this important human pathogen. Here, we present a comprehensive proteome analysis of different subcellular fractions and culture supernatant of in vitro grown M. ulcerans. By a combination of gel-based and gel-free techniques for protein and peptide separation with subsequent analysis by MS, we identified 1074 different proteins, corresponding to 25% of the protein-coding DNA sequence. Interestingly, new information was obtained about central metabolism and lipid biosynthesis, and as many as 192 conserved hypothetical proteins were found. Comparative analysis of the wild-type strain and an isogenic mycolactone-deficient mutant, by 2-DE and iTRAQ labeling of the cytoplasmic fraction, revealed differences in the expression profiles of proteins involved in lipid metabolism and information pathways, as well as stress responses.