Sacha J. Pidot

A Major Role for Mammals in the Ecology of Mycobacterium ulcerans

Background Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a destructive skin disease found predominantly in sub-Saharan Africa and south-eastern Australia. The precise mode(s) of transmission and environmental reservoir(s) remain unknown, but several studies have explored the role of aquatic invertebrate species. The purpose of this study was to investigate the environmental distribution of M. ulcerans in south-eastern Australia. Methodology/Principal Findings A range of environmental samples was collected from Point Lonsdale (a small coastal town southwest of Melbourne, Australia, endemic for BU) and from areas with fewer or no reported incident cases of BU. Mycobacterium ulcerans DNA was detected at low levels by real-time PCR in soil, sediment, water residue, aquatic plant biofilm and terrestrial vegetation collected in Point Lonsdale. Higher levels of M. ulcerans DNA were detected in the faeces of common ringtail (Pseudocheirus peregrinus) and common brushtail (Trichosurus vulpecula) possums. Systematic testing of possum faeces revealed that M. ulcerans DNA could be detected in 41% of faecal samples collected in Point Lonsdale compared with less than 1% of faecal samples collected from non-endemic areas (p<0.0001). Capture and clinical examination of live possums in Point Lonsdale validated the accuracy of the predictive value of the faecal surveys by revealing that 38% of ringtail possums and 24% of brushtail possums had laboratory-confirmed M. ulcerans skin lesions and/or M. ulcerans PCR positive faeces. Whole genome sequencing revealed an extremely close genetic relationship between human and possum M. ulcerans isolates. Conclusions/Significance The prevailing wisdom is that M. ulcerans is an aquatic pathogen and that BU is acquired by contact with certain aquatic environments (swamps, slow-flowing water). Now, after 70 years of research, we propose a transmission model for BU in which terrestrial mammals are implicated as reservoirs for M. ulcerans.

Mycolactones: immunosuppressive and cytotoxic polyketides produced by aquatic mycobacteria

Mycolactone structural variants, produced by Mycobacterium ulcerans and related mycobacteria, are caused by rearrangements among the highly homologous domains and modules within the MlsB polyketide megasynthase, that in turn alter the immunosuppressive and cytotoxic potency of these natural products.

Antibiotics from neglected bacterial sources

The current crop of antibiotics in clinical use are either natural products or their derivatives. However, the rise of a multitude of different antibiotic resistant human pathogens has meant that new antibiotics are urgently needed. Unfortunately, the search for new antibiotics from traditional bacterial sources often results in a high rediscovery rate of known compounds and a low chance of identifying truly novel chemical entities. To overcome this, previously unexplored (or under investigated) bacterial sources are being tapped for their potential to produce novel compounds with new activities. Here, we review a number of antibiotic compounds identified from bacteria of the genera Burkholderia, Clostridium, Lysobacter, Pantoea and Xenorhabdus and describe the potential of organisms and their associated metabolites in future drug discovery efforts.

Single Nucleotide Polymorphism Typing of Mycobacterium ulcerans Reveals Focal Transmission of Buruli Ulcer in a Highly Endemic Region of Ghana

Buruli ulcer (BU) is an emerging necrotizing disease of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. While proximity to stagnant or slow flowing water bodies is a risk factor for acquiring BU, the epidemiology and mode of M. ulcerans transmission is poorly understood. Here we have used high-throughput DNA sequencing and comparisons of the genomes of seven M. ulcerans isolates that appeared monomorphic by existing typing methods. We identified a limited number of single nucleotide polymorphisms (SNPs) and developed a real-time PCR SNP typing method based on these differences. We then investigated clinical isolates of M. ulcerans on which we had detailed information concerning patient location and time of diagnosis. Within the Densu river basin of Ghana we observed dominance of one clonal complex and local clustering of some of the variants belonging to this complex. These results reveal focal transmission and demonstrate, that micro-epidemiological analyses by SNP typing has great potential to help us understand how M. ulcerans is transmitted.

Deciphering the genetic basis for polyketide variation among mycobacteria producing mycolactones

BackgroundMycolactones are immunosuppressive and cytotoxic polyketides, comprising five naturally occurring structural variants (named A/B, C, D, E and F), produced by different species of very closely related mycobacteria including the human pathogen, Mycobacterium ulcerans. In M. ulcerans strain Agy99, mycolactone A/B is produced by three highly homologous type I polyketide megasynthases (PKS), whose genes (mlsA1: 51 kb, mlsA2: 7.2 kb and mlsB: 42 kb) are found on a 174 kb plasmid, known as pMUM001.ResultsWe report here comparative genomic analysis of pMUM001, the complete DNA sequence of a 190 kb megaplasmid (pMUM002) from Mycobacterium liflandii 128FXT and partial sequence of two additional pMUM replicons, combined with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. These data reveal how PKS module and domain differences affecting MlsB correlate with the production of mycolactones E and F. For mycolactone E these differences from MlsB in M. ulcerans Agy99 include replacement of the AT domain of the loading module (acetate to propionate) and the absence of an entire extension module. For mycolactone F there is also a reduction of one extension module but also a swap of ketoreductase domains that explains the characteristic stereochemistry of the two terminal side-chain hydroxyls, an arrangement unique to mycolactone FConclusionThe mycolactone PKS locus on pMUM002 revealed the same large, three-gene structure and extraordinary pattern of near-identical PKS domain sequence repetition as observed in pMUM001 with greater than 98.5% nucleotide identity among domains of the same function. Intra- and inter-strain comparisons suggest that the extreme sequence homogeneity seen among the mls PKS genes is caused by frequent recombination-mediated domain replacement. This work has shed light on the evolution of mycolactone biosynthesis among an unusual group of mycobacteria and highlights the potential of the mls locus to become a toolbox for combinatorial PKS biochemistry.

The NanI and NanJ Sialidases of Clostridium perfringens Are Not Essential for Virulence

ABSTRACT The essential toxin in Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis is alpha-toxin, although other toxins and extracellular enzymes may also be involved. In many bacterial pathogens extracellular sialidases are important virulence factors, and it has been suggested that sialidases may play a role in gas gangrene. C. perfringens strains have combinations of three different sialidase genes, two of which, nanI and nanJ, encode secreted sialidases. The nanI and nanJ genes were insertionally inactivated by homologous recombination in derivatives of sequenced strain 13 and were shown to encode two functional secreted sialidases, NanI and NanJ. Analysis of these derivatives showed that NanI was the major sialidase in this organism. Mutation of nanI resulted in loss of most of the secreted sialidase activity, and the residual activity was eliminated by subsequent mutation of the nanJ gene. Only a slight reduction in the total sialidase activity was observed in a nanJ mutant. Cytotoxicity assays using the B16 melanoma cell line showed that supernatants containing NanI or overexpressing NanJ enhanced alpha-toxin-mediated cytotoxicity. Finally, the ability of nanI, nanJ, and nanIJ mutants to cause disease was assessed in a mouse myonecrosis model. No attenuation of virulence was observed for any of these strains, providing evidence that neither the NanI sialidase nor the NanJ sialidase is essential for virulence.

Genome mining for ribosomally synthesized and post-translationally modified peptides (RiPPs) in anaerobic bacteria

BackgroundRibosomally synthesized and post-translationally modified peptides (RiPPs) are a diverse group of biologically active bacterial molecules. Due to the conserved genomic arrangement of many of the genes involved in their synthesis, these secondary metabolite biosynthetic pathways can be predicted from genome sequence data. To date, however, despite the myriad of sequenced genomes covering many branches of the bacterial phylogenetic tree, such an analysis for a broader group of bacteria like anaerobes has not been attempted.ResultsWe investigated a collection of 211 complete and published genomes, focusing on anaerobic bacteria, whose potential to encode RiPPs is relatively unknown. We showed that the presence of RiPP-genes is widespread among anaerobic representatives of the phyla Actinobacteria, Proteobacteria and Firmicutes and that, collectively, anaerobes possess the ability to synthesize a broad variety of different RiPP classes. More than 25% of anaerobes are capable of producing RiPPs either alone or in conjunction with other secondary metabolites, such as polyketides or non-ribosomal peptides.ConclusionAmongst the analyzed genomes, several gene clusters encode uncharacterized RiPPs, whilst others show similarity with known RiPPs. These include a number of potential class II lanthipeptides; head-to-tail cyclized peptides and lactococcin 972-like RiPP. This study presents further evidence in support of anaerobic bacteria as an untapped natural products reservoir.

Mycobacterium ulcerans and other mycolactone-producing mycobacteria should be considered a single species

The nomenclature of Mycobacterium ulcerans has become confused with the discovery that other mycobacteria that are not necessarily associated with Buruli ulcer also produce the lipid toxin mycolactone. These mycobacteria—collectively known as mycolactone-producing mycobacteria (MPM)—have been given a variety of species names, including Mycobacterium shinshuense, Mycobacterium pseudoshottsii, Mycobacterium marinum, and Mycobacterium “liflandii”. Here we highlight the fact that all MPM share sufficient phenotypic and genotypic characteristics such that they should all be formally recognised as M. ulcerans and not separate species. Renaming all MPM as M. ulcerans is taxonomically correct and will resolve the confusion that is prevalent in the field and will assist political and financial advocacy for Buruli ulcer. Defining a bacterial species has become an increasingly difficult task, particularly when bacteria exhibit different phenotypes but are genetically very closely related. Genomics has shown us very clearly that subtle genetic differences between bacteria can result in impressive phenotypic differences. It is not surprising that the expansion of bacterial genomics has led to a reassessment of the taxonomy of many bacterial species. Such is the case with M. ulcerans, M. marinum, and other closely associated mycobacteria. M. ulcerans and M. marinum are genetically related species that cause quite different human skin diseases. M. ulcerans causes Buruli ulcer, a disease characterised by chronic and severe skin ulcers. The bacterium produces a lipid toxin called mycolactone, replicates slowly (doubling time >48 h) [1], and is apigmented. In contrast, M. marinum causes relatively minor granulomatous skin lesions, often referred to as “fish tank granulomas”, has a doubling time of 6–11 h, and produces bright yellow pigments when exposed to light. Despite their widely different phenotypes, genome comparisons have shown that these species share over 4,000 genes with 98.3% average DNA sequence identity [2]. However, there are also some important genetic differences between them. DNA–DNA hybridisation (DDH) analysis confirmed their status as distinct species, as inter-species relative hybridisation ratios (RBR) were less than 40% [3], [4]. The low RBR is explained by a number of features unique to M. ulcerans, such as the presence of a large virulence plasmid (pMUM) required for mycolactone production, and multiple copies of the insertion sequence element IS2404 that itself accounts for 6% of the M. ulcerans genome [2], [5]. Mycobacteria isolated recently from humans, fish, and frogs around the world (including Japan, the Mediterranean Sea, the Red Sea, Belgium, and the United States) have been variously called M. shinshuense, M. marinum, M. pseudoshottsii, or given unofficial names such as M. “liflandii” [6]–[10]. Subsequent studies have used the collective term MPM when describing M. ulcerans and these bacteria, as they all produce a form of mycolactone [5], . Phylogenetic studies of more than 50 M. ulcerans, other MPM, and M. marinum strains, based on multi-locus sequence analysis (MLSA) of chromosomal and pMUM sequences and studies of large DNA InDel polymorphisms, indicate that all MPM have likely evolved from a common M. marinum progenitor [5], [11], [12] and have then diverged again into two distinct lineages, with both lineages bearing strains that cause Buruli ulcer [5], [13] (Figure 1). Figure 1 Overview of the evolution and principal species-defining features of Mycobacterium ulcerans as established by multi-locus sequence and genome deletion analysis. The new species assignations for MPM have not considered their genomic context and have been based on variable phenotypic characteristics (such as colony morphology and in vitro growth rates) and limited, monophyletic rRNA, hsp65, or rpoB analyses, which have shown these mycobacteria have a few unique nucleotide sequences when compared to a small number of allele sequences in GenBank. However, more complex and time-consuming DDH analyses, which, together with 16S rRNA sequencing, are the prescribed methods for defining a prokaryotic species [14], were not performed in these studies. In the only study to utilise DDH to investigate the relationship between recently described MPM and M. marinum, Yip et al. (2007) showed that MPM have an RBR of 88%–100% when compared to M. ulcerans strains from Africa and Australia and only 15%–60% RBR when compared with a genetically diverse range of nonmycolactone-producing M. marinum strains [5] (Table 1). Furthermore, the analysis of large sequence polymorphisms down to the exact nucleotide breakpoints also showed clear clustering of strains that have been assigned different species names, rendering these assignments inadequate [11]. Table 1 The Key Characteristics That Define Mycobacterium ulcerans. A species is defined as “...a category that circumscribes a (preferably) genomically coherent group of individual isolates/strains sharing a high degree of similarity in (many) independent features, comparatively tested under highly standardized conditions” [15]. In practice, a prokaryotic species is considered to be a group of strains (including the type strain) that is characterised by a certain degree of phenotypic consistency, showing greater than 97% 16S rRNA gene-sequence identity and greater than 70% DDH [16]. If these criteria are applied to the MPM, all of which are “genomically coherent” as revealed by MLSA and InDel analysis, have >98% 16S rRNA identity to M. ulcerans, >70% DDH, possess pMUM plasmids, contain IS2404, and make mycolactone, they can clearly be considered as variants of the same species, namely M. ulcerans. It is on this solid genetic and phenotypic basis that we propose all MPM should be considered strains of M. ulcerans. Furthermore, we suggest that characteristics such as growth rate, colony morphology, pigment production, enzymatic activity, antibiotic susceptibility, and pathogenicity are useful traits for characterizing a particular mycobacterium, but are too sensitive for reliably defining a new taxon. Defining mycobacteria that satisfy our proposed diagnostic criteria as outlined in Table 1 as M. ulcerans will greatly simplify the nomenclature and alleviate confusion. It does not matter that under this revised naming scheme some strains of M. ulcerans will not be associated with human disease. Indeed, many MPM, such as M. pseudoshottsii, have only been associated with disease in animals other than humans; however, they still present the same consistent genetic signatures to assign them as strains of M. ulcerans. Furthermore, the extent of M. ulcerans recovered from humans to also cause disease in other animals, including koalas, possums, cats, and horses, is now being realised [17], [18]. These factors demonstrate how pathogenicity or host range of a bacterium is not a useful parameter for defining a species. Reclassifying all MPM as M. ulcerans is more than an academic exercise. It will also highlight both the large geographic distribution and broad host range of this organism. Advocacy for a neglected tropical disease is not helped with confusion about the name of the causative organism. For example, renaming M. shinshuense to M. ulcerans would assist efforts to raise awareness about Buruli ulcer in Japan. Similarly, highlighting the fact that M. ulcerans is found around the world, including Europe and the US, can only help promote research in this field and encourage broader community interest in Buruli ulcer.

Mycolactone gene expression is controlled by strong SigA-like promoters with utility in studies of Mycobacterium ulcerans and Buruli ulcer.

Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans.

Discovery of Clostrubin, an Exceptional Polyphenolic Polyketide Antibiotic from a Strictly Anaerobic Bacterium

Genome mining of the strictly anaerobic bacterium Clostridium beijerinckii, an industrial producer of solvents, revealed the presence of several cryptic gene clusters for secondary metabolite biosynthesis. To unearth its metabolic potential, a C. beijerinckii strain was cultured under various conditions, which led to the discovery of a deep purple pigment. This novel metabolite, named clostrubin (1), was isolated and its structure was fully elucidated. The pentacyclic polyphenol features a benzo[a]tetraphene ring topology that is unprecedented for natural products. Stable-isotope labeling experiments showed that 1 is an aromatic polyketide that folds in a noncanonical manner to form the unusual perifused ring system. In addition to being the first reported polyketide from an anaerobic bacterium, 1 is a potent antibiotic with pronounced activity against various pathogenic bacteria, such as MRSA, VRE, and mycobacteria, with minimum inhibitory concentrations (MIC) of 0.12-0.97 μM.