Genomics: a Swiss army knife to fight leprosy

Leprosy, a highly disabling and stigmatizing infectious disease, is caused by Mycobacterium leprae and the newly discovered agent, Mycobacterium lepromatosis. Though treatable with antibiotics, leprosy has still not been eradicated, and around 200,000 new cases are reported every year worldwide, mainly in India, Brazil, and Indonesia. Tipping the balance towards leprosy elimination begins with improving our understanding of the pathogenesis and the transmission of the disease, which remains poorly understood. Current research on leprosy is markedly hindered by our incapacity to cultivate the leprosy bacilli on artificial media, as well as by the variation of the clinical forms of the disease. The rise of genomics in the 2000s has helped to get around these problems by opening new ways of studying organisms. Tools were developed to recover enough genetic material for downstream genomic applications; however, none of them is yet suitable for high throughput purposes. In this thesis, we describe an optimized DNA extraction method from skin tissue that allows direct whole-genome sequencing, and enabled us to obtain ~ 250 genome sequences of M. leprae from different geographical locations throughout the world. Firstly, this dataset deepened our insight into the phylogeny of M. leprae, and points to the ancestral strain originating in East Asia and/or Europe. In addition, analysis of more than twenty drug-resistant strains revealed mutations in candidate genes potentially associated with new biological mechanisms such as drug resistance. Moreover, we analysed isolates from restricted geographic areas and from recurrent cases, and show that the distinction between relapse and reinfection with a closely related strain can be made but this remains challenging. The whole genome sequencing of M. lepromatosis was achieved in 2015, and the discovery and use of new specific molecular detection methods allowed us to identify M. lepromatosis in the red squirrel population in the British Isles. In parallel, M. leprae was also discovered in red squirrels on Brownsea Island in the south of England. Though the risk of transmission from animals to humans is not yet clear, the discovery of a new animal reservoir for leprosy bacilli in a non-endemic country raises the question about the existence of other such reservoirs, especially in endemic countries, which could contribute to ongoing transmission. Reliable and sensitive methods for detection of leprosy bacilli are crucial for early diagnosis and monitoring the disease. We show that efficient cell lysis during extraction increases the yield of genetic material recovered from leprosy bacilli and significantly improves the sensitivity of diagnosis by PCR for all leprosy forms. Overall, our results highlight the impact and efficiency of genomics and whole genome sequencing for uncovering new biological mechanisms in unculturable bacteria such as the leprosy bacilli. Our results generated new hypotheses that await testing, and underline the massive potential of omics and bioinformatics for better understanding and fighting the disease

Mycobacterium leprae in Armadillo Tissues from Museum Collections, United States

We examined armadillos from museum collections in the United States using molecular assays to detect leprosy-causing bacilli. We found Mycobacterium leprae bacilli in samples from the United States, Bolivia, and Paraguay; prevalence was 14.8% in nine-banded armadillos. US isolates belonged to subtype 3I-2, suggesting long-term circulation of this genotype

Leprosy in wild chimpanzees

Humans are considered as the main host for Mycobacterium leprae1, the aetiological agent of leprosy, but spillover has occurred to other mammals that are now maintenance hosts, such as nine-banded armadillos and red squirrels2,3. Although naturally acquired leprosy has also been described in captive nonhuman primates4-7, the exact origins of infection remain unclear. Here we describe leprosy-like lesions in two wild populations of western chimpanzees (Pan troglodytes verus) in Cantanhez National Park, Guinea-Bissau and Taï National Park, Côte d'Ivoire, West Africa. Longitudinal monitoring of both populations revealed the progression of disease symptoms compatible with advanced leprosy. Screening of faecal and necropsy samples confirmed the presence of M. leprae as the causative agent at each site and phylogenomic comparisons with other strains from humans and other animals show that the chimpanzee strains belong to different and rare genotypes (4N/O and 2F). These findings suggest that M. leprae may be circulating in more wild animals than suspected, either as a result of exposure to humans or other unknown environmental sources

Origin and spread of leprosy in Suriname. A historical and biomedical study.

The new world was considered free of leprosy before the arrival of Europeans. In Suriname, historical migration routes suggest that leprosy could have been introduced from West Africa by the slave trade, from Asia by indentured workers, from Europe by the colonizers, and more recently by Brazilian gold miners. Previous molecular studies on environmental and ancient samples suggested a high variability of the strains circulating in the country, possibly resulting from the various migration waves. However, a current overview of such diversity in humans still needs to be explored. The origin and spread of leprosy in Suriname are investigated from a historical point of view and by strain genotyping of Mycobacterium leprae from skin biopsies of 26 patients with multibacillary leprosy using PCR-genotyping and whole-genome sequencing. Moreover, molecular signs of resistance to the commonly used anti-leprosy drugs i.e. dapsone, rifampicin and ofloxacin, were investigated. Molecular detection was positive for M. leprae in 25 out of 26 patient samples, while M. lepromatosis was not found in any of the samples. The predominant M. leprae strain in our sample set is genotype 4P (n=8) followed by genotype 1D-2 (n=3), 4N (n=2), and 4O/P (n=1). Genotypes 4P, 4N, 4O/P are predominant in West Africa and Brazil, and could have been introduced in Suriname by the slave trade from West Africa, and more recently by gold miners from Brazil. The presence of the Asian strains 1D-2 probably reflects an introduction by contract workers from India, China and Indonesia during the late 19th and early 20th century after the abolition of slavery. There is currently no definite evidence for the occurrence of the European strain 3 in the 26 patients. Geoplotting reflects internal migration, and also shows that most patients live in and around Paramaribo. A biopsy of one patient harbored two M. leprae genotypes, 1D-2 and 4P, suggesting co-infection. A mutation in the dapsone resistance determining region offolP1 was detected in two out of 13 strains for which molecular drug susceptibility was obtained, suggesting the circulation of dapsone resistant strain

Red squirrels in the British Isles are infected with leprosy bacilli

Leprosy, caused by infection with Mycobacterium leprae or the recently discovered Mycobacterium lepromatosis, was once endemic in humans in the British Isles. Red squirrels in Great Britain (Sciurus vulgaris) have increasingly been observed with leprosy-like lesions on the head and limbs. Using genomics, histopathology, and serology, we found M. lepromatosis in squirrels from England, Ireland, and Scotland, and M. leprae in squirrels from Brownsea Island, England. Infection was detected in overtly diseased and seemingly healthy animals. Phylogenetic comparisons of British and Irish M. lepromatosis with two Mexican strains from humans show that they diverged from a common ancestor around 27,000 years ago, whereas the M. leprae strain is closest to one that circulated in Medieval England. Red squirrels are thus a reservoir for leprosy in the British Isles

Cell Surface Remodeling of Mycobacterium abscessus under Cystic Fibrosis Airway Growth Conditions.

Understanding the physiological processes underlying the ability of Mycobacterium abscessus to become a chronic pathogen of the cystic fibrosis (CF) lung is important to the development of prophylactic and therapeutic strategies to better control and treat pulmonary infections caused by these bacteria. Gene expression profiling of a diversity of M. abscessus complex isolates points to amino acids being significant sources of carbon and energy for M. abscessus in both CF sputum and synthetic CF medium and to the bacterium undergoing an important metabolic reprogramming in order to adapt to this particular nutritional environment. Cell envelope analyses conducted on the same representative isolates further revealed unexpected structural alterations in major cell surface glycolipids known as the glycopeptidolipids (GPLs). Besides showing an increase in triglycosylated forms of these lipids, CF sputum- and synthetic CF medium-grown isolates presented as yet unknown forms of GPLs representing as much as 10% to 20% of the total GPL content of the cells, in which the classical amino alcohol located at the carboxy terminal of the peptide, alaninol, is replaced with the branched-chain amino alcohol leucinol. Importantly, both these lipid changes were exacerbated by the presence of mucin in the culture medium. Collectively, our results reveal potential new drug targets against M. abscessus in the CF airway and point to mucin as an important host signal modulating the cell surface composition of this pathogen

Ancient genomes reveal a high diversity of Mycobacterium leprae in medieval Europe.

Studying ancient DNA allows us to retrace the evolutionary history of human pathogens, such as Mycobacterium leprae, the main causative agent of leprosy. Leprosy is one of the oldest recorded and most stigmatizing diseases in human history. The disease was prevalent in Europe until the 16th century and is still endemic in many countries with over 200,000 new cases reported annually. Previous worldwide studies on modern and European medieval M. leprae genomes revealed that they cluster into several distinct branches of which two were present in medieval Northwestern Europe. In this study, we analyzed 10 new medieval M. leprae genomes including the so far oldest M. leprae genome from one of the earliest known cases of leprosy in the United Kingdom-a skeleton from the Great Chesterford cemetery with a calibrated age of 415-545 C.E. This dataset provides a genetic time transect of M. leprae diversity in Europe over the past 1500 years. We find M. leprae strains from four distinct branches to be present in the Early Medieval Period, and strains from three different branches were detected within a single cemetery from the High Medieval Period. Altogether these findings suggest a higher genetic diversity of M. leprae strains in medieval Europe at various time points than previously assumed. The resulting more complex picture of the past phylogeography of leprosy in Europe impacts current phylogeographical models of M. leprae dissemination. It suggests alternative models for the past spread of leprosy such as a wide spread prevalence of strains from different branches in Eurasia already in Antiquity or maybe even an origin in Western Eurasia. Furthermore, these results highlight how studying ancient M. leprae strains improves understanding the history of leprosy worldwide

Mycobacterium leprae diversity and population dynamics in medieval Europe from novel ancient genomes.

Funder: Max-Planck SocietyFunder: St John’s College, CambridgeFunder: Fondation Raoul FollereauFunder: University of Zurich’s University Research Priority Program “Evolution in Action: From Genomes to Ecosystems”Funder: the Senckenberg Centre for Human Evolution and Palaeoenvironment (S-HEP) at the University of TübingenBackgroundHansen's disease (leprosy), widespread in medieval Europe, is today mainly prevalent in tropical and subtropical regions with around 200,000 new cases reported annually. Despite its long history and appearance in historical records, its origins and past dissemination patterns are still widely unknown. Applying ancient DNA approaches to its major causative agent, Mycobacterium leprae, can significantly improve our understanding of the disease's complex history. Previous studies have identified a high genetic continuity of the pathogen over the last 1500 years and the existence of at least four M. leprae lineages in some parts of Europe since the Early Medieval period.ResultsHere, we reconstructed 19 ancient M. leprae genomes to further investigate M. leprae's genetic variation in Europe, with a dedicated focus on bacterial genomes from previously unstudied regions (Belarus, Iberia, Russia, Scotland), from multiple sites in a single region (Cambridgeshire, England), and from two Iberian leprosaria. Overall, our data confirm the existence of similar phylogeographic patterns across Europe, including high diversity in leprosaria. Further, we identified a new genotype in Belarus. By doubling the number of complete ancient M. leprae genomes, our results improve our knowledge of the past phylogeography of M. leprae and reveal a particularly high M. leprae diversity in European medieval leprosaria.ConclusionsOur findings allow us to detect similar patterns of strain diversity across Europe with branch 3 as the most common branch and the leprosaria as centers for high diversity. The higher resolution of our phylogeny tree also refined our understanding of the interspecies transfer between red squirrels and humans pointing to a late antique/early medieval transmission. Furthermore, with our new estimates on the past population diversity of M. leprae, we gained first insights into the disease's global history in relation to major historic events such as the Roman expansion or the beginning of the regular transatlantic long distance trade. In summary, our findings highlight how studying ancient M. leprae genomes worldwide improves our understanding of leprosy's global history and can contribute to current models of M. leprae's worldwide dissemination, including interspecies transmissions