Buruli ulcer, tuberculosis and leprosy: Exploring the One Health dimensions of three most prevalent mycobacterial diseases: A narrative review.

ΟBJECTIVES: Although Buruli ulcer, tuberculosis, and leprosy are the three most common mycobacterial diseases, One Health dimensions of these infections remain poorly understood. This narrative review aims at exploring the scientific literature with respect to the presence of animal reservoir(s) and other environmental sources for the pathogens of these infections, their role in transmission to humans and the research on/practical implementation of One Health relevant control efforts. METHODS: The literature review was conducted using the online databases PubMed, Scopus, ProQuest and Google Scholar, reviewing articles that were written in English in the last 15 years. Grey literature, published by intergovernmental agencies, was also reviewed. RESULTS: For the pathogen of Buruli ulcer, evidence suggests possums as a possible animal reservoir and thus having an active role in disease transmission to humans. Cattle and some wildlife species are deemed as established animal reservoirs for tuberculosis pathogens, with a non-negligible proportion of infections in humans being of zoonotic origin. Armadillos constitute an established animal reservoir for leprosy pathogens with the transmission of the disease from armadillos to humans being deemed possible. Lentic environments, soil and other aquatic sources may represent further abiotic reservoirs for viable Buruli ulcer and leprosy pathogens infecting humans. Ongoing investigation and implementation of public health measures, targeting (sapro)zoonotic transmission can be found in all three diseases. CONCLUSION: Buruli ulcer, tuberculosis and leprosy exhibit important yet still poorly understood One Health aspects. Despite the microbiological affinity of the respective causative mycobacteria, considerable differences in their animal reservoirs, potential environmental sources and modes of zoonotic transmission are being observed. Whether these differences reflect actual variations between these diseases or rather knowledge gaps remains unclear. For improved disease control, further investigation of zoonotic aspects of all three diseases and formulation of One Health relevant interventions is urgently needed.

A Mycobacterium ulcerans vaccine pilot trial using an accurate low-dose challenge.

A Mycobacterium ulcerans human challenge model has the potential to fundamentally advance our understanding of early human immune responses to infection, while rapidly evaluating vaccines and other therapeutic interventions. Here, using a murine tail infection model, we tested a very well-characterized working cell bank of the proposed challenge isolate M. ulcerans JKD8049 in naïve and Mycobacterium bovis bacille Calmette-Guérin (BCG)-vaccinated BALB/c mice. All 10 naïve mice were successfully infected with 20 colony-forming units (CFU) of M. ulcerans [95% confidence interval (CI) 17-22 CFU] with a mean time to visible lesion of 86 days (95% CI 79-92 days). In the 10 vaccinated mice, there was a significant delay in the mean time to lesion compared to the naïve controls of 24 days (P = 0.0003), but all mice eventually developed ulcerative lesions. This study informs a future human infection model by demonstrating the successful application of the challenge agent in this in vivo model and highlights both the promise and the problems with trying to induce protective immunity against M. ulcerans. IMPORTANCE: In preparation for its proposed use in a controlled human infection model (CHIM), this study reports the successful infection of BALB/c mice using a carefully characterized, low-dose inoculum of Mycobacterium ulcerans JKD8049 (our proposed CHIM strain). We also demonstrate that Mycobacterium bovis bacille Calmette-Guérin delays the onset of disease but cannot alter the course of illness once a lesion becomes apparent. We also validate the findings of previous low-dose challenges that used less accurate methods to determine the inoculum, but our presented methodology is practical, accurate, and anticipated to be reproducible.

Mycobacterium ulcerans challenge strain selection for a Buruli ulcer controlled human infection model.

Critical scientific questions remain regarding infection with Mycobacterium ulcerans, the organism responsible for the neglected tropical disease, Buruli ulcer (BU). A controlled human infection model has the potential to accelerate our knowledge of the immunological correlates of disease, to test prophylactic interventions and novel therapeutics. Here we present microbiological evidence supporting M. ulcerans JKD8049 as a suitable human challenge strain. This non-genetically modified Australian isolate is susceptible to clinically relevant antibiotics, can be cultured in animal-free and surfactant-free media, can be enumerated for precise dosing, and has stable viability following cryopreservation. Infectious challenge of humans with JKD8049 is anticipated to imitate natural infection, as M. ulcerans JKD8049 is genetically stable following in vitro passage and produces the key virulence factor, mycolactone. Also reported are considerations for the manufacture, storage, and administration of M. ulcerans JKD8049 for controlled human infection.

Amoxicillin/clavulanate in combination with rifampicin/clarithromycin is bactericidal against Mycobacterium ulcerans.

BACKGROUND: Buruli ulcer (BU) is a skin neglected tropical disease (NTD) caused by Mycobacterium ulcerans. WHO-recommended treatment requires 8-weeks of daily rifampicin (RIF) and clarithromycin (CLA) with wound care. Treatment compliance may be challenging due to socioeconomic determinants. Previous minimum Inhibitory Concentration and checkerboard assays showed that amoxicillin/clavulanate (AMX/CLV) combined with RIF+CLA were synergistic against M. ulcerans. However, in vitro time kill assays (TKA) are a better approach to understand the antimicrobial activity of a drug over time. Colony forming units (CFU) enumeration is the in vitro reference method to measure bacterial load, although this is a time-consuming method due to the slow growth of M. ulcerans. The aim of this study was to assess the in vitro activity of RIF, CLA and AMX/CLV combinations against M. ulcerans clinical isolates by TKA, while comparing four methodologies: CFU enumeration, luminescence by relative light unit (RLU) and optical density (at 600 nm) measurements, and 16S rRNA/IS2404 genes quantification. METHODOLOGY/PRINCIPAL FINDINGS: TKA of RIF, CLA and AMX/CLV alone and in combination were performed against different M. ulcerans clinical isolates. Bacterial loads were quantified with different methodologies after 1, 3, 7, 10, 14, 21 and 28 days of treatment. RIF+AMX/CLV and the triple RIF+CLA+AMX/CLV combinations were bactericidal and more effective in vitro than the currently used RIF+CLA combination to treat BU. All methodologies except IS2404 quantitative PCR provided similar results with a good correlation with CFU enumeration. Measuring luminescence (RLU) was the most cost-effective methodology to quantify M. ulcerans bacterial loads in in vitro TKA. CONCLUSIONS/SIGNIFICANCE: Our study suggests that alternative and faster TKA methodologies can be used in BU research instead of the cumbersome CFU quantification method. These results provide an in vitro microbiological support to of the BLMs4BU clinical trial (NCT05169554, PACTR202209521256638) to shorten BU treatment.

Emergence and spread of Mycobacterium ulcerans at different geographic scales.

The classical lineage of Mycobacterium ulcerans is the most prevalent clonal group associated with Buruli ulcer in humans. Its reservoir is strongly associated with the environment. We analyzed together 1,045 isolates collected from 13 countries on two continents to define the evolutionary history and population dynamics of this lineage. We confirm that this lineage spread over 7,000 years from Australia to Africa with the emergence of outbreaks in distinct waves in the 18th and 19th centuries. In sharp contrast with its global spread over the last century, transmission chains are now mostly local, with little or no dissemination between endemic areas. This study provides new insights into the phylogeography and population dynamics of M. ulcerans, highlighting the importance of comparative genomic analyses to improve our understanding of pathogen transmission. IMPORTANCE: Mycobacterium ulcerans is an environmental mycobacterial pathogen that can cause Buruli ulcer, a severe cutaneous infection, mostly spread in Africa and Australia. We conducted a large genomic study of M. ulcerans, combining genomic and evolutionary approaches to decipher its evolutionary history and pattern of spread at different geographic scales. At the scale of villages in an endemic area of Benin, the circulating genotypes have been introduced in recent decades and are not randomly distributed along the river. On a global scale, M. ulcerans has been spreading for much longer, resulting in distinct and compartmentalized endemic foci across Africa and Australia.

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.

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.

Unlocking Opportunities for Mycobacterium leprae and Mycobacterium ulcerans.

In the recent decade, scientific communities have toiled to tackle the emerging burden of drug-resistant tuberculosis (DR-TB) and rapidly growing opportunistic nontuberculous mycobacteria (NTM). Among these, two neglected mycobacteria species of the Acinetobacter family, Mycobacterium leprae and Mycobacterium ulcerans, are the etiological agents of leprosy and Buruli ulcer infections, respectively, and fall under the broad umbrella of neglected tropical diseases (NTDs). Unfortunately, lackluster drug discovery efforts have been made against these pathogenic bacteria in the recent decade, resulting in the discovery of only a few countable hits and majorly repurposing anti-TB drug candidates such as telacebec (Q203), P218, and TB47 for current therapeutic interventions. Major ignorance in drug candidate identification might aggravate the dramatic consequences of rapidly spreading mycobacterial NTDs in the coming days. Therefore, this Review focuses on an up-to-date account of drug discovery efforts targeting selected druggable targets from both bacilli, including the accompanying challenges that have been identified and are responsible for the slow drug discovery. Furthermore, a succinct discussion of the all-new possibilities that could be alternative solutions to mitigate the neglected mycobacterial NTD burden and subsequently accelerate the drug discovery effort is also included. We anticipate that the state-of-the-art strategies discussed here may attract major attention from the scientific community to navigate and expand the roadmap for the discovery of next-generation therapeutics against these NTDs.

Mycobacterium ulcerans-Bordetella trematum chronic tropical cutaneous ulcer: A four-case series, Côte d'Ivoire.

BACKGROUND: Chronic tropical cutaneous ulcers remain a neglected medical condition in West Africa, particularly Buruli ulcer, which is caused by mycolactone cytotoxin-secreting Mycobacterium ulcerans (M. ulcerans). Medical management of this highly debilitating and necrotising skin infection may be modified by colonisation and co-infection of the ulcer by opportunistic and pathogenic microorganisms, which considerably delays and increases the cost of treatment. METHODOLOGY/PRINCIPAL FINDING: We diagnosed chronic tropical cutaneous ulcers in nine patients in Côte d'Ivoire using M. ulcerans-specific PCRs and culturomics. This revealed M. ulcerans in 7/9 ulcer swabs and 5/9 control swabs as well as an additional 122 bacterial species, 32 of which were specific to ulcers, 61 specifics to the controls, and 29 which were shared, adding 40 bacterial species to those previously reported. Whole genome sequencing of four Bordetella trematum (B. trematum) isolates in four Buruli ulcer swabs and no controls indicated cytolethal distending toxins, as confirmed by cytotoxic assay. CONCLUSIONS/SIGNIFICANCE: In four cases of Buruli ulcer in Côte d'Ivoire, B. trematum was a co-pathogen which was resistant to rifampicin and clarithromycin, unmatching M. ulcerans antibiotic susceptibility profile and counteracting the current treatment of Buruli ulcer in West Africa and Australia. Thus, we report here chronic mixed M. ulcerans-B. trematum chronic tropical ulcer as a specific form of Buruli ulcer in West Africa.

Inflammasome-triggered IL-18 controls skin inflammation in the progression of Buruli ulcer.

Buruli ulcer is an emerging chronic infectious skin disease caused by Mycobacterium ulcerans. Mycolactone, an exotoxin produced by the bacterium, is the only identified virulence factor so far, but the functions of this toxin and the mechanisms of disease progression remain unclear. By interfering Sec61 translocon, mycolactone inhibits the Sec61-dependent co-translational translocation of newly synthesized proteins, such as induced cytokines and immune cell receptors, into the endoplasmic reticulum. However, in regard to IL-1ß, which is secreted by a Sec61-independent mechanism, mycolactone has been shown to induce IL-1ß secretion via activation of inflammasomes. In this study, we clarified that cytokine induction, including that of IL-1ß, in infected macrophages was suppressed by mycolactone produced by M. ulcerans subsp. shinshuense, despite the activation of caspase-1 through the inflammasome activation triggered in a manner independent of mycolactone. Intriguingly, mycolactone suppressed the expression of proIL-1ß as well as TNF-α at the transcriptional level, suggesting that mycolactone of M. ulcerans subsp. shinshuense may exert additional inhibitory effect on proIL-1ß expression. Remarkably, constitutively produced IL-18 was cleaved and mature IL-18 was actually released from macrophages infected with the causative mycobacterium. IL-18-deficient mice infected subcutaneously with M. ulcerans exhibited exacerbated skin inflammation during the course of disease progression. On the other hand, IL-1ß controls bacterial multiplication in skin tissues. These results provide information regarding the mechanisms and functions of the induced cytokines in the pathology of Buruli ulcer.

Mycobacterium ulcerans not detected by PCR on human skin in Buruli ulcer endemic areas of south eastern Australia.

INTRODUCTION: Mycobacterium ulcerans (MU) causes Buruli ulcer (Buruli), a geographically restricted infection that can result in skin loss, contracture and permanent scarring. Lesion-location maps compiled from more than 640 cases in south eastern Australia suggest biting insects are likely involved in transmission, but it is unclear whether MU is brought by insects to humans or if MU is already on the skin and inoculation is an opportunistic event that need not be insect dependent. METHODS: We validated a PCR swab detection assay and defined its dynamic range using laboratory cultured M. ulcerans and fresh pigskin. We invited volunteers in Buruli-endemic and non-endemic areas to sample their skin surfaces with self-collected skin swabs tested by IS2404 quantitative PCR. RESULTS: Pigskin validation experiments established a limit-of-detection of 0.06 CFU/cm2 at a qPCR cycle threshold (Ct) of 35. Fifty-seven volunteers returned their self-collected kits of 4 swabs (bilateral ankles, calves, wrists, forearms), 10 from control areas and 47 from endemic areas. Collection was timed to coincide with the known peak-transmission period of Buruli. All swabs from human volunteers tested negative (Ct ≥35). CONCLUSIONS: M. ulcerans was not detected on the skin of humans from highly Buruli endemic areas.

Behavioral interplay between mosquito and mycolactone produced by Mycobacterium ulcerans and bacterial gene expression induced by mosquito proximity.

Mycolactone is a cytotoxic lipid metabolite produced by Mycobacterium ulcerans, the environmental pathogen responsible for Buruli ulcer, a neglected tropical disease. Mycobacterium ulcerans is prevalent in West Africa, particularly found in lentic environments, where mosquitoes also occur. Researchers hypothesize mosquitoes could serve as a transmission mechanism resulting in infection by M. ulcerans when mosquitoes pierce skin contaminated with M. ulcerans. The interplay between the pathogen, mycolactone, and mosquito is only just beginning to be explored. A triple-choice assay was conducted to determine the host-seeking preference of Aedes aegypti between M. ulcerans wildtype (MU, mycolactone active) and mutant (MUlac-, mycolactone inactive). Both qualitative and quantitative differences in volatile organic compounds' (VOCs) profiles of MU and MUlac- were determined by GC-MS. Additionally, we evaluated the interplay between Ae. aegypti proximity and M. ulcerans mRNA expression. The results showed that mosquito attraction was significantly greater (126.0%) to an artificial host treated with MU than MUlac-. We found that MU and MUlac produced differential profiles of VOCs associated with a wide range of biological importance from quorum sensing (QS) to human odor components. RT-qPCR assays showed that mycolactone upregulation was 24-fold greater for MU exposed to Ae. aegypti in direct proximity. Transcriptome data indicated significant induction of ten chromosomal genes of MU involved in stress responses and membrane protein, compared to MUlac- when directly having access to or in near mosquito proximity. Our study provides evidence of possible interkingdom interactions between unicellular and multicellular species that MU present on human skin is capable of interreacting with unrelated species (i.e., mosquitoes), altering its gene expression when mosquitoes are in direct contact or proximity, potentially impacting the production of its VOCs, and consequently leading to the stronger attraction of mosquitoes toward human hosts. This study elucidates interkingdom interactions between viable M. ulcerans bacteria and Ae. aegypti mosquitoes, which rarely have been explored in the past. Our finding opens new doors for future research in terms of disease ecology, prevalence, and pathogen dispersal outside of the M. ulcerans system.

Buruli ulcer - A neglected tropical disease in the Barwon region of Victoria, Australia: An emerging public health threat with local and national ramifications.

Mycobacterium ulcerans (MU) is known to be endemic in heavily touristed coastal regions of Victoria and is the cause of Buruli ulcer (BU) disease. The incidence, severity and geographic spread of MU infection/BU disease is increasing, including metropolitan Victorian suburbs. While the specifics of disease transmission and effective prevention strategies remain uncertain, severe complications can be mitigated by health systems that provide vigilant population surveillance to underpin early recognition, early specialist involvement and definitive treatment for the individual. Current theories regarding disease transmission and 'best practice' (or best guess) prevention and mitigation measures are presented herein. Opportunities to improve the health system response to this emerging public health threat are identified. It is incumbent upon all healthcare providers, including ED clinicians, to contribute by familiarising themselves with the established and emerging areas of endemicity of MU infection and the array of BU clinical presentations.

Type-I interferons promote innate immune tolerance in macrophages exposed to Mycobacterium ulcerans vesicles.

Buruli ulcer is a chronic infectious disease caused by Mycobacterium ulcerans. The pathogen persistence in host skin is associated with the development of ulcerative and necrotic lesions leading to permanent disabilities in most patients. However, few of diagnosed cases are thought to resolve through an unknown self-healing process. Using in vitro and in vivo mouse models and M. ulcerans purified vesicles and mycolactone, we showed that the development of an innate immune tolerance was only specific to macrophages from mice able to heal spontaneously. This tolerance mechanism depends on a type I interferon response and can be induced by interferon beta. A type I interferon signature was further detected during in vivo infection in mice as well as in skin samples from patients under antibiotics regiment. Our results indicate that type I interferon-related genes expressed in macrophages may promote tolerance and healing during infection with skin damaging pathogen.

Alternative boronic acids in the detection of Mycolactone A/B using the thin layer chromatography (f-TLC) method for diagnosis of Buruli ulcer.

BACKGROUND: Mycobacterium ulcerans is the causative agent of Buruli ulcer. The pathology of M. ulcerans disease has been attributed to the secretion of a potent macrolide cytotoxin known as mycolactone which plays an important role in the virulence of the disease. Mycolactone is a biomarker for the diagnosis of BU that can be detected using the fluorescent-thin layer chromatography (f-TLC) technique. The technique relies on the chemical derivatization of mycolactone A/B with 2-naphthylboronic acid (BA) which acts as a fluorogenic chemosensor. However, background interferences due to co-extracted human tissue lipids, especially with clinical samples coupled with the subjectivity of the method call for an investigation to find an alternative to BA. METHODS: Twenty-six commercially available arylboronic acids were initially screened as alternatives to BA using the f-TLC experiment. UV-vis measurements were also conducted to determine the absorption maximum spectra of mycolactone A/B and myco-boronic acid adducts followed by an investigation of the fluorescence-enhancing ability of the boronate ester formation between mycolactone A/B and our three most promising boronic acids (BA15, BA18, and BA21). LC-MS technique was employed to confirm the adduct formation between mycolactone and boronic acids. Furthermore, a comparative study was conducted between BA18 and BA using 6 Polymerase Chain Reaction (PCR) confirmed BU patient samples. RESULTS: Three of the boronic acids (BA15, BA18, and BA21) produced fluorescent band intensities superior to BA. Complexation studies conducted on thin layer chromatography (TLC) using 0.1 M solution of the three boronic acids and various volumes of 10 ng/µL of synthetic mycolactone ranging from 1 µL - 9 µL corresponding to 10 ng - 90 ng gave similar results with myco-BA18 adduct emerging with the most visibly intense fluorescence bands. UV-vis absorption maxima (λmax) for the free mycolactone A/B was observed at 362 nm, and the values for the adducts myco-BA15, myco-BA18, and myco-BA21 were at 272 nm, 270 nm, and 286 nm respectively. The comparable experimental λmax of 362 nm for mycolactone A/B to the calculated Woodward-Fieser value of 367 nm for the fatty acid side chain of mycolactone A/B demonstrate that even though 2 cyclic boronates were formed, only the boronate of the southern side chain with the chromophore was excited by irradiation at 365 nm. Fluorescence experiments have demonstrated that coupling BA18 to mycolactone A/B along the 1,3-diols remarkably enhanced the fluorescence intensity at 537 nm. High-Resolution Mass Spectrometer (HR-MS) was used to confirm the formation of the myco-BA15 adduct. Finally, f-TLC analysis of patient samples with BA18 gave improved BA18-adduct intensities compared to the original BA-adduct. CONCLUSION: Twenty-six commercially available boronic acids were investigated as alternatives to BA, used in the f-TLC analysis for the diagnosis of BU. Three (3) of them BA15, BA18, and BA21 gave superior fluorescence band intensity profiles. They gave profiles that were easier to interpret after the myco-boronic acid adduct formation and in experiments with clinical samples from patients with BA18 the best. BA18, therefore, has been identified as a potential alternative to BA and could provide a solution to the challenge of background interference of co-extracted human tissue lipids from clinical samples currently associated with the use of BA.

From Bacterial Toxin to Therapeutic Agent: The Unexpected Fate of Mycolactone.

"Recognizing a surprising fact is the first step towards discovery." This famous quote from Louis Pasteur is particularly appropriate to describe what led us to study mycolactone, a lipid toxin produced by the human pathogen Mycobacterium ulcerans. M. ulcerans is the causative agent of Buruli ulcer, a neglected tropical disease manifesting as chronic, necrotic skin lesions with a "surprising" lack of inflammation and pain. Decades after its first description, mycolactone has become much more than a mycobacterial toxin. This uniquely potent inhibitor of the mammalian translocon (Sec61) helped reveal the central importance of Sec61 activity for immune cell functions, the spread of viral particles and, unexpectedly, the viability of certain cancer cells. We report in this review the main discoveries that marked our research into mycolactone, and the medical perspectives they opened up. The story of mycolactone is not over and the applications of Sec61 inhibition may go well beyond immunomodulation, viral infections, and oncology.

PPE Barcoding Identifies Biclonal Mycobacterium ulcerans Buruli Ulcer, Côte d'Ivoire.

Mycobacterium ulcerans, an environmental opportunistic pathogen, causes necrotic cutaneous and subcutaneous lesions, named Buruli ulcers, in tropical countries. PCR-derived tests used to detect M. ulcerans in environmental and clinical samples do not allow one-shot detection, identification, and typing of M. ulcerans among closely related Mycobacterium marinum complex mycobacteria. We established a 385-member M. marinum/M. ulcerans complex whole-genome sequence database by assembling and annotating 341 M. marinum/M. ulcerans complex genomes and added 44 M. marinum/M. ulcerans complex whole-genome sequences already deposited in the NCBI database. Pangenome, core genome, and single-nucleotide polymorphism (SNP) distance-based comparisons sorted the 385 strains into 10 M. ulcerans taxa and 13 M. marinum taxa, correlating with the geographic origin of strains. Aligning conserved genes identified one PPE (proline-proline-glutamate) gene sequence to be species and intraspecies specific, thereby genotyping the 23 M. marinum/M. ulcerans complex taxa. PCR sequencing of the PPE gene correctly genotyped nine M. marinum/M. ulcerans complex isolates among one M. marinum taxon and three M. ulcerans taxa in the African taxon (T2.4). Further, successful PPE gene PCR sequencing in 15/21 (71.4%) swabs collected from suspected Buruli ulcer lesions in Côte d'Ivoire exhibited positive M. ulcerans IS2404 real-time PCR and identified the M. ulcerans T2.4.1 genotype in eight swabs and M. ulcerans T2.4.1/T2.4.2 mixed genotypes in seven swabs. PPE gene sequencing could be used as a proxy for whole-genome sequencing for the one-shot detection, identification, and typing of clinical M. ulcerans strains, offering an unprecedented tool for identifying M. ulcerans mixed infections. IMPORTANCE We describe a new targeted sequencing approach that characterizes the PPE gene to disclose the simultaneous presence of different variants of a single pathogenic microorganism. This approach has direct implications on the understanding of pathogen diversity and natural history and potential therapeutic implications when dealing with obligate and opportunistic pathogens, such as Mycobacterium ulcerans presented here as a prototype.