Pseudogenomic insights into the evolution of Mycobacterium ulcerans.

BACKGROUND: Buruli ulcer (BU) disease, caused by Mycobacterium ulcerans (MU), and characterized by necrotic ulcers is still a health problem in Africa and Australia. The genome of the bacterium has several pseudogenes due to recent evolutionary events and environmental pressures. Pseudogenes are genetic elements regarded as nonessential in bacteria, however, they are less studied due to limited available tools to provide understanding of their evolution and roles in MU pathogenicity. RESULTS: This study developed a bioinformatic pipeline to profile the pseudogenomes of sequenced MU clinical isolates from different countries. One hundred and seventy-two MU genomes analyzed revealed that pseudogenomes of African strains corresponded to the two African lineages 1 and 2. Pseudogenomes were lineage and location specific and African lineage 1 was further divided into A and B. Lineage 2 had less relaxation in positive selection than lineage 1 which may signify different evolutionary points. Based on the Gil-Latorre model, African MU strains may be in the latter stages of evolutionary adaption and are adapting to an environment rich in metabolic resources with a lower temperature and decreased UV radiation. The environment fosters oxidative metabolism and MU may be less reliant on some secondary metabolites. In-house pseudogenomes from Ghana and Cote d'Ivoire were different from other African strains, however, they were identified as African strains. CONCLUSION: Our bioinformatic pipeline provides pseudogenomic insights to complement other whole genome analyses, providing a better view of the evolution of the genome of MU and suggest an adaptation model which is important in understanding transmission. MU pseudogene profiles vary based on lineage and country, and an apparent reduction in insertion sequences used for the detection of MU which may adversely affect the sensitivity of diagnosis.

SIGNIFICANCE: Prevention and treatment of Buruli ulcer is still a problem but large whole genome datasets on M. ulcerans are readily available. However, genomic studies fail to thoroughly investigate pseudogenes to probe evolutionary changes in the bacteria, and this can be attributed to the lack of bioinformatic tools. This work studied pseudogenes in Mycobacterium ulcerans (MU) to understand its adapted niche and evolutionary differences across African strains. Our results posit an MU niche-adapted model important in understanding transmission. Also, MU pseudogene profiles vary based on lineage and country, suggesting their influence on pseudogenization patterns in the genome. We further identify a reduction in insertion sequences that are used for the detection of the bacteria which may affect the sensitivity of diagnosis.

Mosquitoes provide a transmission route between possums and humans for Buruli ulcer in southeastern Australia.

Buruli ulcer, a chronic subcutaneous infection caused by Mycobacterium ulcerans, is increasing in prevalence in southeastern Australia. Possums are a local wildlife reservoir for M. ulcerans and, although mosquitoes have been implicated in transmission, it remains unclear how humans acquire infection. We conducted extensive field survey analyses of M. ulcerans prevalence among mosquitoes in the Mornington Peninsula region of southeastern Australia. PCR screening of trapped mosquitoes revealed a significant association between M. ulcerans and Aedes notoscriptus. Spatial scanning statistics revealed overlap between clusters of M. ulcerans-positive Ae. notoscriptus, M. ulcerans-positive possum excreta and Buruli ulcer cases, and metabarcoding analyses showed individual mosquitoes had fed on humans and possums. Bacterial genomic analysis confirmed shared single-nucleotide-polymorphism profiles for M. ulcerans detected in mosquitoes, possum excreta and humans. These findings indicate Ae. notoscriptus probably transmit M. ulcerans in southeastern Australia and highlight mosquito control as a Buruli ulcer prevention measure.

Genetics in the Host-Mycobacterium ulcerans interaction.

Buruli ulcer is an emerging infectious disease associated with high morbidity and unpredictable outbreaks. It is caused by Mycobacterium ulcerans, a slow-growing pathogen evolutionarily shaped by the acquisition of a plasmid involved in the production of a potent macrolide-like cytotoxin and by genome rearrangements and downsizing. These events culminated in an uncommon infection pattern, whereby M. ulcerans is both able to induce the initiation of the inflammatory cascade and the cell death of its proponents, as well as to survive within the phagosome and in the extracellular milieu. In such extreme conditions, the host is sentenced to rely on a highly orchestrated genetic landscape to be able to control the infection. We here revisit the dynamics of M. ulcerans infection, drawing parallels from other mycobacterioses and integrating the most recent knowledge on its evolution and pathogenicity in its interaction with the host immune response.

Genetic variants in human BCL2L11 (BIM) are associated with ulcerative forms of Buruli ulcer.

Buruli ulcer (BU) is a devastating skin mycobacterial infection characterized by extensive cell death, which was previously suggested to be mediated by Bcl2-like protein 11 (BIM, encoded by the BCL2L11 gene). We here report the association of genetic variants in BCL2L11 with ulcerative forms of the disease in a cohort of 618 Beninese individuals. Our results show that regulation of apoptosis in humans contributes to BU lesions associated with worse prognosis, prompting for further investigation on the implementation of novel methods for earlier identification of at-risk patients.

Genome-wide association study of Buruli ulcer in rural Benin highlights role of two LncRNAs and the autophagy pathway.

Buruli ulcer, caused by Mycobacterium ulcerans and characterized by devastating necrotizing skin lesions, is the third mycobacterial disease worldwide. The role of host genetics in susceptibility to Buruli ulcer has long been suggested. We conduct the first genome-wide association study of Buruli ulcer on a sample of 1524 well characterized patients and controls from rural Benin. Two-stage analyses identify two variants located within LncRNA genes: rs9814705 in ENSG00000240095.1 (P = 2.85 × 10-7; odds ratio = 1.80 [1.43-2.27]), and rs76647377 in LINC01622 (P = 9.85 × 10-8; hazard ratio = 0.41 [0.28-0.60]). Furthermore, we replicate the protective effect of allele G of a missense variant located in ATG16L1, previously shown to decrease bacterial autophagy (rs2241880, P = 0.003; odds ratio = 0.31 [0.14-0.68]). Our results suggest LncRNAs and the autophagy pathway as critical factors in the development of Buruli ulcer.

Human genetics of Buruli ulcer.

Buruli ulcer, the third most common mycobacterial disease worldwide, is caused by Mycobacterium ulcerans and characterized by devastating necrotizing skin lesions. Susceptibility to Buruli ulcer is thought to depend on host genetics, but very few genetic studies have been performed. The identification of a microdeletion on chromosome 8 in a familial form of severe Buruli ulcer suggested a monogenic basis of susceptibility. The role of common host genetic variants in Buruli ulcer development has been investigated in only three candidate-gene studies targeting genes involved in mycobacterial diseases. A recent genome-wide association study suggested a probable role for long non-coding RNAs and strengthened the contribution of autophagy as a major defense mechanism against mycobacteria. In this review, we summarize the history, epidemiological and clinical aspects of Buruli ulcer, focusing particularly on genetic findings relating to susceptibility to this disease. Finally, we discuss exciting new genetic avenues arising, in particular, from studies of mouse models, and the need for different disciplines to work together, to benefit from the extensive work on other mycobacterial diseases, mostly tuberculosis and leprosy. We are convinced that such pooling of effort will lead to the development of efficient novel strategies for combatting Buruli ulcer.

PCR detection of Mycobacterium ulcerans-significance for clinical practice and epidemiology.

Introduction: Buruli ulcer (BU) is a neglected disease which has been reported from mostly impoverished, remote rural areas from 35 countries worldwide. BU affects skin, subcutaneous tissue, and bones, and may cause massive tissue destruction and life-long disabilities if not diagnosed and treated early. Without laboratory confirmation diagnostic and treatment errors may occur. This review describes the application of IS2404 PCR, the preferred diagnostic test, in the area of individual patient management and clinico-epidemiological studies. Areas covered: A Medline search included publications on clinical sample collection, DNA extraction, and PCR detection formats of the past and present, potential and limitations of clinical application, as well as clinico-epidemiological studies. Expert commentary: A global network of reference laboratories basically provides the possibility for PCR confirmation of 70% of all BU cases worldwide as requested by the WHO. Keeping laboratory confirmation on a constant level requires continuous outreach activities. Among the potential measures to maintain sustainability of laboratory confirmation and outreach activities are decentralized or mobile diagnostics available at point of care, such as IS2404-based LAMP, which complement the standard IS2404-based diagnostic tools available at central level.

Human genetics of mycobacterial disease.

Mycobacterial diseases are caused by members of the genus Mycobacterium, acid-fast bacteria characterized by the presence of mycolic acids within their cell walls. Claiming almost 2 million lives every year, tuberculosis (TB) is the most common mycobacterial disease and is caused by infection with M. tuberculosis and, in rare cases, by M. bovis or M. africanum. The second and third most common mycobacterial diseases are leprosy and buruli ulcer (BU), respectively. Both diseases affect the skin and can lead to permanent sequelae and deformities. Leprosy is caused by the uncultivable M. leprae while the etiological agent of BU is the environmental bacterium M. ulcerans. After exposure to these mycobacterial species, a majority of individuals will not progress to clinical disease and, among those who do, inter-individual variability in disease manifestation and outcome can be observed. Susceptibility to mycobacterial diseases carries a human genetic component and intense efforts have been applied over the past decades to decipher the exact nature of the genetic factors controlling disease susceptibility. While for BU this search was mostly conducted on the basis of candidate genes association studies, genome-wide approaches have been widely applied for TB and leprosy. In this review, we summarize some of the findings achieved by genome-wide linkage, association and transcriptome analyses in TB disease and leprosy and the recent genetic findings for BU susceptibility.

Microdeletion on chromosome 8p23.1 in a familial form of severe Buruli ulcer.

Buruli ulcer (BU), the third most frequent mycobacteriosis worldwide, is a neglected tropical disease caused by Mycobacterium ulcerans. We report the clinical description and extensive genetic analysis of a consanguineous family from Benin comprising two cases of unusually severe non-ulcerative BU. The index case was the most severe of over 2,000 BU cases treated at the Centre de Dépistage et de Traitement de la Lèpre et de l'Ulcère de Buruli, Pobe, Benin, since its opening in 2003. The infection spread to all limbs with PCR-confirmed skin, bone and joint infections. Genome-wide linkage analysis of seven family members was performed and whole-exome sequencing of both patients was obtained. A 37 kilobases homozygous deletion confirmed by targeted resequencing and located within a linkage region on chromosome 8 was identified in both patients but was absent from unaffected siblings. We further assessed the presence of this deletion on genotyping data from 803 independent local individuals (402 BU cases and 401 BU-free controls). Two BU cases were predicted to be homozygous carriers while none was identified in the control group. The deleted region is located close to a cluster of beta-defensin coding genes and contains a long non-coding (linc) RNA gene previously shown to display highest expression values in the skin. This first report of a microdeletion co-segregating with severe BU in a large family supports the view of a key role of human genetics in the natural history of the disease.

Multiple Introductions and Recent Spread of the Emerging Human Pathogen Mycobacterium ulcerans across Africa.

Buruli ulcer (BU) is an insidious neglected tropical disease. Cases are reported around the world but the rural regions of West and Central Africa are most affected. How BU is transmitted and spreads has remained a mystery, even though the causative agent, Mycobacterium ulcerans, has been known for more than 70 years. Here, using the tools of population genomics, we reconstruct the evolutionary history of M. ulcerans by comparing 165 isolates spanning 48 years and representing 11 endemic countries across Africa. The genetic diversity of African M. ulcerans was found to be restricted due to the bacterium's slow substitution rate coupled with its relatively recent origin. We identified two specific M. ulcerans lineages within the African continent, and inferred that M. ulcerans lineage Mu_A1 existed in Africa for several hundreds of years, unlike lineage Mu_A2, which was introduced much more recently, approximately during the 19th century. Additionally, we observed that specific M. ulcerans epidemic Mu_A1 clones were introduced during the same time period in the three hydrological basins that were well covered in our panel. The estimated time span of the introduction events coincides with the Neo-imperialism period, during which time the European colonial powers divided the African continent among themselves. Using this temporal association, and in the absence of a known BU reservoir or-vector on the continent, we postulate that the so-called "Scramble for Africa" played a significant role in the spread of the disease across the continent.

Genetic Susceptibility and Predictors of Paradoxical Reactions in Buruli Ulcer.

INTRODUCTION: Buruli ulcer (BU) is the third most frequent mycobacterial disease in immunocompetent persons after tuberculosis and leprosy. During the last decade, eight weeks of antimicrobial treatment has become the standard of care. This treatment may be accompanied by transient clinical deterioration, known as paradoxical reaction. We investigate the incidence and the risks factors associated with paradoxical reaction in BU. METHODS: The lesion size of participants was assessed by careful palpation and recorded by serial acetate sheet tracings. For every time point, surface area was compared with the previous assessment. All patients received antimicrobial treatment for 8 weeks. Serum concentration of 25-hydroxyvitamin D, the primary indicator of vitamin D status, was determined in duplex for blood samples at baseline by a radioimmunoassay. We genotyped four polymorphisms in the SLC11A1 gene, previously associated with susceptibility to BU. For testing the association of genetic variants with paradoxical responses, we used a binary logistic regression analysis with the occurrence of a paradoxical response as the dependent variable. RESULTS: Paradoxical reaction occurred in 22% of the patients; the reaction was significantly associated with trunk localization (p = .039 by Χ(2)), larger lesions (p = .021 by Χ(2)) and genetic factors. The polymorphisms 3'UTR TGTG ins/ins (OR 7.19, p < .001) had a higher risk for developing paradoxical reaction compared to ins/del or del/del polymorphisms. CONCLUSIONS: Paradoxical reactions are common in BU. They are associated with trunk localization, larger lesions and polymorphisms in the SLC11A1 gene.

Genetic Variation in Autophagy-Related Genes Influences the Risk and Phenotype of Buruli Ulcer.

INTRODUCTION: Buruli ulcer (BU) is a severe necrotizing human skin disease caused by Mycobacterium ulcerans. Clinically, presentation is a sum of these diverse pathogenic hits subjected to critical immune-regulatory mechanisms. Among them, autophagy has been demonstrated as a cellular process of critical importance. Since microtubules and dynein are affected by mycolactone, the critical pathogenic exotoxin produced by M. ulcerans, cytoskeleton-related changes might potentially impair the autophagic process and impact the risk and progression of infection. OBJECTIVE: Genetic variants in the autophagy-related genes NOD2, PARK2 and ATG16L1 has been associated with susceptibility to mycobacterial diseases. Here, we investigated their association with BU risk, its severe phenotypes and its progression to an ulcerative form. METHODS: Genetic variants were genotyped using KASPar chemistry in 208 BU patients (70.2% with an ulcerative form and 28% in severe WHO category 3 phenotype) and 300 healthy endemic controls. RESULTS: The rs1333955 SNP in PARK2 was significantly associated with increased susceptibility to BU [odds ratio (OR), 1.43; P = 0.05]. In addition, both the rs9302752 and rs2066842 SNPs in NOD2 gee significantly increased the predisposition of patients to develop category 3 (OR, 2.23; P = 0.02; and OR 12.7; P = 0.03, respectively, whereas the rs2241880 SNP in ATG16L1 was found to significantly protect patients from presenting the ulcer phenotype (OR, 0.35; P = 0.02). CONCLUSION: Our findings indicate that specific genetic variants in autophagy-related genes influence susceptibility to the development of BU and its progression to severe phenotypes.