Michael Käser

Genomic Diversity and Evolution of Mycobacterium ulcerans Revealed by Next-Generation Sequencing

Mycobacterium ulcerans is the causative agent of Buruli ulcer, the third most common mycobacterial disease after tuberculosis and leprosy. It is an emerging infectious disease that afflicts mainly children and youths in West Africa. Little is known about the evolution and transmission mode of M. ulcerans, partially due to the lack of known genetic polymorphisms among isolates, limiting the application of genetic epidemiology. To systematically profile single nucleotide polymorphisms (SNPs), we sequenced the genomes of three M. ulcerans strains using 454 and Solexa technologies. Comparison with the reference genome of the Ghanaian classical lineage isolate Agy99 revealed 26,564 SNPs in a Japanese strain representing the ancestral lineage. Only 173 SNPs were found when comparing Agy99 with two other Ghanaian isolates, which belong to the two other types previously distinguished in Ghana by variable number tandem repeat typing. We further analyzed a collection of Ghanaian strains using the SNPs discovered. With 68 SNP loci, we were able to differentiate 54 strains into 13 distinct SNP haplotypes. The average SNP nucleotide diversity was low (average 0.06–0.09 across 68 SNP loci), and 96% of the SNP locus pairs were in complete linkage disequilibrium. We estimated that the divergence of the M. ulcerans Ghanaian clade from the Japanese strain occurred 394 to 529 thousand years ago. The Ghanaian subtypes diverged about 1000 to 3000 years ago, or even much more recently, because we found evidence that they evolved significantly faster than average. Our results offer significant insight into the evolution of M. ulcerans and provide a comprehensive report on genetic diversity within a highly clonal M. ulcerans population from a Buruli ulcer endemic region, which can facilitate further epidemiological studies of this pathogen through the development of high-resolution tools.

Optimized method for preparation of DNA from pathogenic and environmental mycobacteria

ABSTRACT Genomic studies on pathogenic and environmental mycobacteria are of growing interest for understanding of their evolution, distribution, adaptation, and host-pathogen interaction. Since most mycobacteria are slow growers, material from in vitro cultures is usually scarce. The robust mycobacterial cell wall hinders both experimental cell lysis and efficient DNA extraction. Here, we compare elements of several DNA preparation protocols and describe a method that is economical and practical and reliably yields large amounts—usually 10-fold increased compared to earlier protocols—of highly pure genomic DNA for sophisticated downstream applications. This method was optimized for cultures of a variety of pathogenic and environmental mycobacterial species and proven to be suitable for direct mycobacterial DNA extraction from infected insect specimens.

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.

Ongoing genome reduction in Mycobacterium ulcerans.

M. ulcerans is adapting to a more stable environment.

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.

Large Sequence Polymorphisms Unveil the Phylogenetic Relationship of Environmental and Pathogenic Mycobacteria Related to Mycobacterium ulcerans

ABSTRACT Mycolactone is an immunosuppressive cytotoxin responsible for the clinical manifestation of Buruli ulcer in humans. It was believed to be confined to its etiologic agent, Mycobacterium ulcerans. However, the identification of other mycolactone-producing mycobacteria (MPMs) in other species, including Mycobacterium marinum, indicated a more complex taxonomic relationship. This highlighted the need for research on the biology, evolution, and distribution of such emerging and potentially infectious strains. The reliable genetic fingerprinting analyses presented here aim at both the unraveling of phylogenetic relatedness and of dispersal between environmental and pathogenic mycolactone producers and the identification of genetic prerequisites that enable lateral gene transfer of such plasmids. This will allow for the identification of environmental reservoirs of virulence plasmids that encode enzymes required for the synthesis of mycolactone. Based on dynamic chromosomal loci identified earlier in M. ulcerans, we characterized large sequence polymorphisms for the phylogenetic analysis of MPMs. Here, we identify new insertional-deletional events and single-nucleotide polymorphisms that confirm and redefine earlier strain differentiation markers. These results support other data showing that all MPMs share a common ancestry. In addition, we found unique genetic features specific for M. marinum strain M, the genome sequence strain which is used widely in research.

Independent Loss of Immunogenic Proteins in Mycobacterium ulcerans Suggests Immune Evasion

ABSTRACT The highly immunogenic mycobacterial proteins ESAT-6, CFP-10, and HspX represent potential target antigens for the development of subunit vaccines and immunodiagnostic tests. Recently, the complete genome sequence revealed the absence of these coding sequences in Mycobacterium ulcerans, the causative agent of the emerging human disease Buruli ulcer. Genome reduction and the acquisition of a cytopathic and immunosuppressive macrolide toxin plasmid are regarded as crucial for the emergence of this pathogen from its environmental progenitor, Mycobacterium marinum. Earlier, we have shown the evolution of M. ulcerans into two distinct lineages. Here, we show that while the genome of M. marinum M contains two copies of the esxB-esxA gene cluster at different loci (designated MURD4 and MURD152), both copies are deleted from the genome of M. ulcerans strains belonging to the classical lineage. Members of the ancestral lineage instead retained some but disrupted most functional MURD4 or MURD152 copies, either by newly identified genomic insertion-deletion events or by conversions of functional genes to pseudogenes via point mutations. Thus, the esxA (ESAT-6), esxB (CFP-10), and hspX genes are located in hot-spot regions for genomic variation where functional disruption seems to be favored by selection pressure. Our detailed genomic analyses have identified a variety of independent genomic changes that have led to the loss of expression of functional ESAT-6, CFP-10, and HspX proteins. Loss of these immunodominant proteins helps the bacteria bypass the hosts immunological response and may represent part of an ongoing adaptation of M. ulcerans to survival in host environments that are screened by immunological defense mechanisms.

Risk factors for buruli ulcer in ghana-a case control study in the suhum-kraboa-coaltar and akuapem South districts of the eastern region

Background Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans. Its exact mode of transmission is not known. Previous studies have identified demographic, socio-economic, health and hygiene as well as environment related risk factors. We investigated whether the same factors pertain in Suhum-Kraboa-Coaltar (SKC) and Akuapem South (AS) Districts in Ghana which previously were not endemic for BU. Methods We conducted a case control study. A case of BU was defined as any person aged 2 years or more who resided in study area (SKC or AS District) diagnosed according to the WHO clinical case definition for BU and matched with age- (+/−5 years), gender-, and community controls. A structured questionnaire on host, demographic, environmental, and behavioural factors was administered to participants. Results A total of 113 cases and 113 community controls were interviewed. Multivariate conditional logistic regression analysis identified presence of wetland in the neighborhood (OR = 3.9, 95% CI = 1.9–8.2), insect bites in water/mud (OR = 5.7, 95% CI = 2.5–13.1), use of adhesive when injured (OR = 2.7, 95% CI = 1.1–6.8), and washing in the Densu river (OR = 2.3, 95% CI = 1.1–4.96) as risk factors associated with BU. Rubbing an injured area with alcohol (OR = 0.21, 95% CI = 0.008–0.57) and wearing long sleeves for farming (OR = 0.29, 95% CI = 0.14–0.62) showed protection against BU. Conclusion This study identified the presence of wetland, insect bites in water, use of adhesive when injured, and washing in the river as risk factors for BU; and covering limbs during farming as well as use of alcohol after insect bites as protective factors against BU in Ghana. Until paths of transmission are unraveled, control strategies in BU endemic areas should focus on these known risk factors.

Single nucleotide polymorphisms on the road to strain differentiation in Mycobacterium ulcerans.

ABSTRACT The genomic fine-typing of strains of Mycobacterium ulcerans, the causative agent of the emerging human disease Buruli ulcer, is difficult due to the clonal population structure of geographical lineages. Although large sequence polymorphisms (LSPs) resulted in the clustering of patient isolates originating from across the globe, differentiation of strains within continents using conventional typing methods is very limited. In this study, we analyzed M. ulcerans LSP haplotype-specific insertion sequence elements among 83 M. ulcerans strains and identified single nucleotide polymorphisms (SNPs) that differentiate between regional strains. This is the first genetic discrimination based on SNPs of M. ulcerans strains from African countries where Buruli ulcer is endemic, resulting in the highest geographic resolution of genotyping so far. The findings support the concept of genome-wide SNP analyses as tools to study the epidemiology and evolution of M. ulcerans at a local level.

Differential Gene Repertoire in Mycobacterium ulcerans Identifies Candidate Genes for Patho-Adaptation

Background Based on large genomic sequence polymorphisms, several haplotypes belonging to two major lineages of the human pathogen Mycobacterium ulcerans could be distinguished among patient isolates from various geographic origins. However, the biological relevance of insertional/deletional diversity is not understood. Methodology Using comparative genomics, we have investigated the genes located in regions of difference recently identified by DNA microarray based hybridisation analysis. The analysed regions of difference comprise ∼7% of the entire M. ulcerans genome. Principal Findings Several different mechanisms leading to loss of functional genes were identified, ranging from pseudogenization, caused by frame shift mutations or mobile genetic element interspersing, to large sequence polymorphisms. Four hot spot regions for genetic instability were unveiled. Altogether, 229 coding sequences were found to be differentially inactivated, constituting a repertoire of coding sequence variation in the rather monomorphic M. ulcerans. Conclusions/Significance The differential gene inactivation patterns associated with the M. ulcerans haplotypes identified candidate genes that may confer enhanced adaptation upon ablation of expression. A number of gene conversions confined to the classical lineage may contribute to particular virulence of this group comprising isolates from Africa and Australia. Identification of this spectrum of anti-virulence gene candidates expands our understanding of the pathogenicity and ecology of the emerging infectious disease Buruli ulcer.