A new paradigm for leprosy diagnosis based on host gene expression

Author summary Despite effective treatment, leprosy is still a significant public health issue in more than 120 countries, with more than 200 000 new cases yearly. The disease is caused mainly by Mycobacterium leprae, a slow-growing bacillus still uncultivable in axenic media. This limitation has hampered basic research into host-pathogen interaction and the development of new diagnostic assays. Currently, leprosy is diagnosed clinically, with no standalone diagnostic assay accurate enough for all clinical forms. Here, we use RNA-seq transcriptome profiling in leprosy lesions and granuloma annulare to identify mRNA biomarkers with potential diagnostic applications. Also, we explored new pathways that can be useful in further understanding the host-pathogen interaction and how the bacteria bypass host immune defenses. We found that IDO1, a gene involved with tryptophan catabolism, is an excellent candidate for distinguishing leprosy lesions from other dermatoses. Additionally, we observed that a previous signature of keratinocyte development and cornification negatively correlates with epithelial-mesenchymal transition genes in the skin, suggesting new ways in which the pathogen may subvert its host to survive and spread throughout the body. Our study identifies new mRNA biomarkers that can improve leprosy diagnostics and describe new insights about host-pathogen interactions in human skin. Transcriptional profiling is a powerful tool to investigate and detect human diseases. In this study, we used bulk RNA-sequencing (RNA-Seq) to compare the transcriptomes in skin lesions of leprosy patients or controls affected by other dermal conditions such as granuloma annulare, a confounder for paucibacillary leprosy. We identified five genes capable of accurately distinguishing multibacillary and paucibacillary leprosy from other skin conditions. Indoleamine 2,3-dioxygenase 1 (IDO1) expression alone was highly discriminatory, followed by TLR10, BLK, CD38, and SLAMF7, whereas the HS3ST2 and CD40LG mRNA separated multi- and paucibacillary leprosy. Finally, from the main differentially expressed genes (DEG) and enriched pathways, we conclude that paucibacillary disease is characterized by epithelioid transformation and granuloma formation, with an exacerbated cellular immune response, while multibacillary leprosy features epithelial-mesenchymal transition with phagocytic and lipid biogenesis patterns in the skin. These findings will help catalyze the development of better diagnostic tools and potential host-based therapeutic interventions. Finally, our data may help elucidate host-pathogen interplay driving disease clinical manifestations.

Genomics: a Swiss army knife to fight leprosy

Charlotte Avanzi

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.

EPFL2018

Highly Reduced Genome of the New Species Mycobacterium uberis, the Causative Agent of Nodular Thelitis and Tuberculoid Scrotitis in Livestock and a Close Relative of the Leprosy Bacilli

Charlotte Avanzi, Andrej Benjak, Stewart Cole

Nodular thelitis is a chronic enzootic infection affecting dairy cows and goats. The causative agent was recently shown to be related to the leprosy-causing bacilli Mycobacterium leprae and Mycobacterium lepromatosis. In this study, the genome of this pathogen was sequenced and analyzed. Phylogenomic analyses confirmed that the pathogen present in nodular thelitis and tuberculoid scrotitis is a distinct species related to the leprosy bacilli and Mycobacterium haemophilum. Because the pathogen was originally isolated from a bovine udder, it was named "Mycobacterium uberis." The genome of "M. uberis" is only 3.12 Mb in length, which represents the smallest mycobacterial genome identified so far but which is close to that of leprosy bacilli in size. The genome contains 1,759 protein-coding genes and 1,081 pseudogenes, indicative of extensive reductive evolution and likely the reason that M. uberis cannot be grown axenically. The pseudogenization and genome reduction in M. uberis seem to have been to some extent independent from the results determined for the genomes of the leprosy bacilli. IMPORTANCE M. uberis is an emerging skin pathogen in dairy animals. Its genome underwent massive reduction and gene decay, leading to a minimal set of genes required for an obligatory intracellular lifestyle, which highly resembles the evolution of the leprosy agents M. leprae and M. lepromatosis. The genomic similarity between M. uberis and the leprosy bacilli can help in identifying key virulence factors of these closely related species or in identifying genes responsible for the distinct differences between thelitis or scrotitis and leprosy with respect to clinical manifestations. Specific DNA markers can now be developed for quick detection of this pathogen.

AMER SOC MICROBIOLOGY2018

Insights from the Genome Sequence of Mycobacterium lepraemurium: Massive Gene Decay and Reductive Evolution

Charlotte Avanzi, Andrej Benjak, Stewart Cole

Mycobacterium lepraemurium is the causative agent of murine leprosy, a chronic, granulomatous disease similar to human leprosy. Due to the similar clinical manifestations of human and murine leprosy and the difficulty of growing both bacilli axenically, Mycobacterium leprae and M. lepraemurium were once thought to be closely related, although it was later suggested that M. lepraemurium might be related to Mycobacterium avium. In this study, the complete genome of M. lepraemurium was sequenced using a combination of PacBio and Illumina sequencing. Phylogenomic analyses confirmed that M. lepraemurium is a distinct species within the M. avium complex (MAC). The M. lepraemurium genome is 4.05 Mb in length, which is considerably smaller than other MAC genomes, and it comprises 2,682 functional genes and 1,139 pseudogenes, which indicates that M. lepraemurium has undergone genome reduction. An error-prone repair homologue of the DNA polymerase III alpha-subunit was found to be nonfunctional in M. lepraemurium, which might contribute to pseudogene formation due to the accumulation of mutations in nonessential genes. M. lepraemurium has retained the functionality of several genes thought to influence virulence among members of the MAC. IMPORTANCE Mycobacterium lepraemurium seems to be evolving toward a minimal set of genes required for an obligatory intracellular lifestyle within its host, a niche seldom adopted by most mycobacteria, as they are free-living. M. lepraemurium could be used as a model to elucidate functions of genes shared with other members of the MAC. Its reduced gene set can be exploited for studying the essentiality of genes in related pathogenic species, which might lead to discovery of common virulence factors or clarify host-pathogen interactions. M. lepraemurium can be cultivated in vitro only under specific conditions and even then with difficulty. Elucidating the metabolic (in) capabilities of M. lepraemurium will help develop suitable axenic media and facilitate genetic studies.

Amer Soc Microbiology2017

Highly Reduced Genome of the New Species Mycobacterium uberis, the Causative Agent of Nodular Thelitis and Tuberculoid Scrotitis in Livestock and a Close Relative of the Leprosy Bacilli

Charlotte Avanzi, Andrej Benjak, Stewart Cole

AMER SOC MICROBIOLOGY2018

Genomics: a Swiss army knife to fight leprosy

Charlotte Avanzi

EPFL2018

Insights from the Genome Sequence of Mycobacterium lepraemurium: Massive Gene Decay and Reductive Evolution

Charlotte Avanzi, Andrej Benjak, Stewart Cole

Amer Soc Microbiology2017