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Mycobacterium ulcerans is found in aquatic ecosystems and causes Buruli ulcer in humans, a neglected but devastating necrotic disease of subcutaneous tissue that is rampant throughout West and Central Africa. Here, we report the complete 5.8-Mb genome sequence of M. ulcerans and show that it comprises two circular replicons, a chromosome of 5632 kb and a virulence plasmid of 174 kb. The plasmid is required for production of the polyketide toxin mycolactone, which provokes necrosis. Comparisons with the recently completed 6.6-Mb genome of Mycobacterium marinum revealed >98% nucleotide sequence identity and genome-wide synteny. However, as well as the plasmid, M. ulcerans has accumulated 213 copies of the insertion sequence IS2404, 91 copies of IS2606, 771 pseudogenes, two bacteriophages, and multiple DNA deletions and rearrangements. These data indicate that M. ulcerans has recently evolved via lateral gene transfer and reductive evolution from the generalist, more rapid-growing environmental species M. marinum to become a niche-adapted specialist. Predictions based on genome inspection for the production of modified mycobacterial virulence factors, such as the highly abundant phthiodiolone lipids, were confirmed by structural analyses. Similarly, 11 protein-coding sequences identified as M. ulcerans-specific by comparative genomics were verified as such by PCR screening a diverse collection of 33 strains of M. ulcerans and M. marinum. This work offers significant insight into the biology and evolution of mycobacterial pathogens and is an important component of international efforts to counter Buruli ulcer.

The roles of nitric oxide (NO) in numerous disease states have generated considerable discussion over the past several years. NO has been labeled as the causative agent in different pathophysiological mechanisms, yet appears to protect against various chemical species such as those generated under oxidative stress. Similarly, NO appears to exert a dichotomy of effects within the multistage model of cancer. Chronic inflammation can lead to the production of chemical intermediates, among them NO, which in turn can mediate damage to DNA. Yet, NO also appears to be critical for the tumoricidal activity of the immune system. Furthermore, NO can also have a multitude of effects on other aspects of tumor biology, including angiogenesis and metastasis. This report will discuss how the chemistry of NO may impact the initiation and progression stages of cancer.

The critical regulatory function of nitric oxide (NO) in many physiologic processes is well established. However, in an aerobic aqueous environment NO is known to generate one or more reactive and potentially toxic nitrogen oxide (NOx) metabolites. This has led to the speculation that mechanisms must exist in vivo by which these reactive intermediates are detoxified, although the nature of these mechanisms has yet to be elucidated. This report demonstrates that among the primary bioorganic products of the reaction of cellular constituents with the intermediates of the NO/O2 reaction are S-nitrosothiol (S-NO) adducts. Anaerobic solutions of NO are not capable of nitrosating cysteine or glutathione, while S-NO adducts of these amino acids are readily formed in the presence of O2 and NO. Investigation of the kinetics for the formation of these S-NO adducts has revealed a rate equation of d[RSNO]/dt = kSNO[NO]2[O2], where kSNO = (6 +/- 2) x 10(6) M-2S-1, a value identical to that for the formation of reactive intermediates in the autoxidation of NO. Competition studies performed with a variety of amino acids, glutathione, and azide have shown that cysteine residues have an affinity for the NOx species that is 3 orders of magnitude greater than that of the nonsulfhydryl amino acids, and > 10(6) times greater than that of the exocyclic amino groups of DNA bases. The dipeptide alanyltyrosine reacts with the intermediates of the NO/O2 reaction with an affinity 150 times less than that of the sulfhydryl-containing compounds. Furthermore, Chinese hamster V79 lung fibroblasts depleted of glutathione display enhanced cytotoxicity on exposure to NO.(ABSTRACT TRUNCATED AT 250 WORDS)

Although the bovine tuberculosis (TB) agent, Mycobacterium bovis, may infect humans and cause disease, long-term epidemiological data indicate that humans represent a spill-over host in which infection with M. bovis is not self-maintaining. Indeed, human-tohuman transmission of M. bovis strains and other members of the animal lineage of the tubercle bacilli is very rare. Here, we report on three mutations affecting the two-component virulence regulation system PhoP/PhoR (PhoPR) in M. bovis and in the closely linked Mycobacterium africanum lineage 6 (L6) that likely account for this discrepancy. Genetic transfer of these mutations into the human TB agent, Mycobacterium tuberculosis, resulted in downregulation of the PhoP regulon, with loss of biologically active lipids, reduced secretion of the 6-kDa early antigenic target (ESAT-6), and lower virulence. Remarkably, the deleterious effects of the phoPR mutations were partly compensated by a deletion, specific to the animal-adapted and M. africanum L6 lineages, that restores ESAT-6 secretion by a PhoPR-independent mechanism. Similarly, we also observed that insertion of an IS6110 element upstream of the phoPR locus may completely revert the phoPR-bovis-associated fitness loss, which is the case for an exceptional M. bovis human outbreak strain from Spain. Our findings ultimately explain the long-term epidemiological data, suggesting that M. bovis and related phoPR-mutated strains pose a lower risk for progression to overt human TB, with major impact on the evolutionary history of TB.evolution | phylogeny | adaptation | zoonosis T uberculosis (TB) is caused by bacilli from the genetically compact Mycobacterium tuberculosis complex (MTBC), which gathers eight defined phylogenetic lineages in addition to the more distantly related Mycobacterium canettii group (1-3): M. tuberculosis sensu-stricto from lineages L1-L4 and L7 form a large group of human-adapted strains responsible for the vast majority of global human TB cases, whereas Mycobacterium africanum lineages (L5, L6), which are restricted to humans from West Africa, are phylogenetically linked with the eighth lineage, comprising the various animal-adapted strains, with Mycobacterium bovis as the most downstream member in the phylogeny (Fig. 1A) (4, 5). Animal strains exhibit a wide host range that includes livestock animals in close contact with humans. Episodes of bovine TB in cattle herds have been reported in 128 of 155 countries during the period 2005-2008 (6). Although the bulk of these episodes is mainly found in developing countries (6), bovine TB remains a major problem even in some industrialized countries, best exemplified by the United Kingdom, which has experienced an important resurgence of bovine TB since the 1980s (7). Because M. bovis and other closely related animal-adapted strains are also capable of causing TB in humans, this situation raises concerns regarding the zoonotic risk. Indeed, human TB cases resulting from M. bovis are estimated to be around 2% worldwide (8), with higher incidence (...

Two-component regulatory signal transduction systems are important elements of the adaptative response of prokaryotes to a variety of environmental stimuli. Disruption of PhoP-PhoR in Mycobacterium tuberculosisdramatically attenuates virulence, implying that this system directly and/or indirectly coordinates the expression of important virulence factors whose identity remains to be established. Interestingly, in knockingout the PhoP-PhoR two-component system in M. tuberculosis Mt103, dramatic changes in the colonial morphology, cording properties, and reactivity of the mutant strain to the basic dye neutral red, all intrinsic properties of tubercle bacilli known to correlate with virulence, were noted. Because deficiencies in the ability of the mutant to form serpentine cords and stain with the dye are likely the results of alterations of its cell envelope composition, we undertook to analyze the lipid content of phoP and phoP-phoR mutants constructed in two different strains of M. tuberculosis. Our results indicate that PhoP coordinately and positively regulates the synthesis of methyl-branched fatty acid-containing acyltrehaloses known to be restricted to pathogenic species of the M. tuberculosis complex, namely diacyltrehaloses, polyacyltrehaloses, and sulfolipids. Evidence is also provided that PhoP but not PhoR is required for the production of these lipids. This work represents an important step toward the functional characterization of PhoP-PhoR and the understanding of complex lipid synthesis in M. tuberculosis.Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is one of the leading causes of mortality due to a single infectious agent (1). In the tubercle bacillus as in other prokaryotes, two-component signal transduction systems are important elements of the adaptative response to a variety of stimuli (2). So far, of the 11 paired two-component systems, 5 unpaired response regulators and 2 unpaired protein sensors that M. tuberculosis possesses, the two-component system PhoP-PhoR is the one whose disruption was shown to affect the most dramatically the ability of M. tuberculosis to replicate in cellular and animal models (3). Interestingly, PhoP shows high similarity to the PhoP response regulator of Salmonella enterica serovar typhimurium, which senses Mg 2ϩ starvation and controls the expression of at least 40 genes, among which some encoding important virulence determinants (4). Further supporting the concept that PhoP is important for virulence and transmissibility of tubercle bacilli, a multidrug-resistant strain of Mycobacterium bovis (strain B) responsible for large tuberculosis outbreaks in Spain was found to carry an IS6110 insertion in the promoter region of phoP causing a strong up-regulation of the expression of this gene (5). To date, the stimuli sensed by the sensor histidine kinase PhoR and the genes controlled by the DNA-binding response regulator PhoP are not known. The identification of the environmental signals regulating PhoP-PhoR and the characterization of t...

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SummaryMycobacterium tuberculosis infects one-third of the world's population and causes two million deaths annually. The unusually low permeability of its cell wall contributes to the ability of M. tuberculosis to grow within host macrophages, a property required for pathogenesis of infection. Mycobacterium marinum is an established model for discovering genes involved in mycobacterial infection. Mycobacterium marinum mutants with transposon insertions in the b b b bketoacyl-acyl carrier protein synthase B gene ( kasB ) grew poorly in macrophages, although growth in vitro was unaffected. Detailed analyses by thin-layer chromatography, nuclear magnetic resonance (NMR), matrix-assisted laser desorption/ionization time-offlight mass spectrometry, infrared spectroscopy, and chemical degradations showed that the kasB mutants synthesize mycolic acids that are 2-4 carbons shorter than wild type; the defect was localized to the proximal portion of the meromycolate chain. In addition, these mutants showed a significant ( ~ 30%) reduction in the abundance of keto-mycolates, with a slight compensatory increase of both a a a a -and methoxymycolates. Despite these small changes in mycolate length and composition, the kasB mutants exhibited strikingly altered cell wall permeability, leading to a marked increase in susceptibility to lipophilic antibiotics and the host antimicrobial molecules defensin and lysozyme. The abnormalities of the kasB mutants were fully complemented by expressing M. tuberculosis kasB , but not by the closely related gene kasA . These studies identify kasB as a novel target for therapeutic intervention in mycobacterial diseases.