Genomic Characterization ofMycobacterium lepraeto Explore Transmission Patterns Identifies New Subtype in Bangladesh

Mycobacterium leprae, the causative agent of leprosy, is an unculturable bacterium with a considerably reduced genome (3.27 Mb) compared to homologues mycobacteria from the same ancestry. In 2001, the genome ofM. lepraewas first described and subsequently four genotypes (1-4) and 16 subtypes (A-P) were identified providing means to study global transmission patterns for leprosy. In order to understand the role of asymptomatic carriers we investigatedM. lepraecarriage as well as infection in leprosy patients (n= 60) and healthy household contacts (HHC;n= 250) from Bangladesh using molecular detection of the bacterial element RLEP in nasal swabs (NS) and slit skin smears (SSS). In parallel, to studyM. lepraegenotype distribution in Bangladesh we explored strain diversity by whole genome sequencing (WGS) and Sanger sequencing. In the studied cohort in Bangladesh,M. lepraeDNA was detected in 33.3% of NS and 22.2% of SSS of patients with bacillary index of 0 whilst in HHC 18.0% of NS and 12.3% of SSS were positive. The majority of theM. lepraestrains detected in this study belonged to genotype 1D (55%), followed by 1A (31%). Importantly, WGS allowed the identification of a newM. lepraegenotype, designated 1B-Bangladesh (14%), which clustered separately between the 1A and 1B strains. Moreover, we established that the genotype previously designated 1C, is not an independent subtype but clusters within the 1D genotype. Intraindividual differences were present between theM. lepraestrains obtained including mutations in hypermutated genes, suggesting mixed colonization/infection or in-host evolution. In summary, we observed thatM. lepraeis present in asymptomatic contacts of leprosy patients fueling the concept that these individuals contribute to the current intensity of transmission. Our data therefore emphasize the importance of sensitive and specific tools allowing post-exposure prophylaxis targeted atM. leprae-infected or -colonized individuals.

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

Genomics and the chemotherapy of leprosy

Stewart Cole

The information deduced from the genome sequence of Mycobacterium leprae is of immense value for the chemotherapy of leprosy. Knowing the complete set of genes, enzymes and proteins allows us to understand why some drugs are without effect whereas others are fully active. It may also enable better use to be made of existing drugs, such as beta-lactams, and opens new avenues for the development of novel compounds. M. leprae is relatively susceptible to a wide range of drugs, unlike the highly related tubercle bacillus, and several new multidrug regimens are in clinical trials. Genomics provides a number of possible explanations for this broader susceptibility as some of the genes encoding enzymes involved in antibiotic inactivation have decayed whereas the number of transporters available to contribute to drug efflux is considerably lower than in Mycobacterium tuberculosis. Several leads for new drug targets have been uncovered.

2002

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

Charlotte Avanzi, Andrej Benjak, Stewart Cole

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