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

BACKGROUND: Hansen'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. RESULTS: Here, 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. CONCLUSIONS: Our 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.

The Recovery, Interpretation and Use of Ancient Pathogen Genomes.

The ability to sequence genomes from ancient biological material has provided a rich source of information for evolutionary biology and engaged considerable public interest. Although most studies of ancient genomes have focused on vertebrates, particularly archaic humans, newer technologies allow the capture of microbial pathogens and microbiomes from ancient and historical human and non-human remains. This coming of age has been made possible by techniques that allow the preferential capture and amplification of discrete genomes from a background of predominantly host and environmental DNA. There are now near-complete ancient genome sequences for three pathogens of considerable historical interest - pre-modern bubonic plague (Yersinia pestis), smallpox (Variola virus) and cholera (Vibrio cholerae) - and for three equally important endemic human disease agents - Mycobacterium tuberculosis (tuberculosis), Mycobacterium leprae (leprosy) and Treponema pallidum pallidum (syphilis). Genomic data from these pathogens have extended earlier work by paleopathologists. There have been efforts to sequence the genomes of additional ancient pathogens, with the potential to broaden our understanding of the infectious disease burden common to past populations from the Bronze Age to the early 20th century. In this review we describe the state-of-the-art of this rapidly developing field, highlight the contributions of ancient pathogen genomics to multidisciplinary endeavors and describe some of the limitations in resolving questions about the emergence and long-term evolution of pathogens.

Revisiting the expression signature of pks15/1 unveils regulatory patterns controlling phenolphtiocerol and phenolglycolipid production in pathogenic mycobacteria.

One of the most important and exclusive characteristics of mycobacteria is their cell wall. Amongst its constituent components are two related families of glycosylated lipids, diphthioceranates and phthiocerol dimycocerosate (PDIM) and its variant phenolic glycolipids (PGL). PGL have been associated with cell wall impermeability, phagocytosis, defence against nitrosative and oxidative stress and, intriguingly, biofilm formation. In bacteria from the Mycobacterium tuberculosis complex (MTBC), the biosynthetic pathway of the phenolphthiocerol moiety of PGL depends upon the expression of several genes encoding type I polyketide synthases (PKS), namely ppsA-E and pks15/1 which constitute the PDIM + PGL locus, and that are highly conserved in PDIM/PGL-producing strains. Consensus has not been achieved regarding the genetic organization of pks15/1 locus and knowledge is lacking on its transcriptional signature. Here we explore publicly available datasets of transcriptome data (RNA-seq) from more than 100 MTBC experiments in 40 growth conditions to outline the transcriptional structure and signature of pks15/1, using a differential expression approach to infer the regulatory patterns involving these and related genes. We show that pks1 expression is highly correlated with fadD22, Rv2949c, lppX, fadD29 and, also, pks6 and pks12, with the first three putatively integrating into a polycistronic structure. We evidence dynamic transcriptional heterogeneity within the genes involved in phenolphtiocerol and phenolic glycolipid production, most exhibiting up-regulation upon acidic pH and antibiotic exposure and down-regulation under hypoxia, dormancy, and low/high iron concentration. We finally propose a model based on transcriptome data in which σD positively regulates pks1, pks15 and fadD22, while σB and σE factors exert negative regulation at an upper level.

Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy.

AIMS: Diagnosis of leprosy, a chronic infection caused by Mycobacterium leprae, predominantly depends on clinical manifestations and histopathological analysis, hampering rapid and accurate diagnostics. Our aim was to increase accuracy of leprosy diagnosis by improving M. leprae's DNA detection based on polymerase chain reaction (PCR) technique using new specific primers for the RLEP repetitive sequence. METHODS AND RESULTS: The specific target region, RLEP, of M. leprae's genome was selected based on comparative genomics. After confirming the specificity of this region, using blastn analysis, primers were designed and tested for their in silico specificity. To evaluate the specificity and sensitivity of these primers in vitro, 184 blood samples from patients were used in qPCR. The new primer pair LYON1/LYON2 produced 91% positive samples, whereas the current primer pair LP1/LP2 produced 46%. Specificity and DNA detection limit test were carried out to compare the efficiency of the developed primer pair. The LYON1/LYON2 primer showed 100% specificity, whereas LP1/LP2 showed 64%. The DNA detection limit of LYON1/LYON2 was 10 copies of bacterial genomes per millilitre, whereas LP1/LP2 was 1000 copies of bacterial genomes per millilitre. CONCLUSIONS: In conclusion, the developed LYON1/LYON2 primer pair presented to be a specific and sensitive new molecular marker for the diagnosis of leprosy. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of a specific primer pair for the detection of the M. leprae genome through qPCR technique contributes to a fast, sensitive and specific diagnosis, which is essential to prevent spreading and progression of this disease.

Archival, paleopathological and aDNA-based techniques in leprosy research and the case of Father Petrus Donders at the Leprosarium 'Batavia', Suriname.

OBJECTIVE: We assessed whether Petrus Donders (died 1887), a Dutch priest who for 27 years cared for people with leprosy in the leprosarium Batavia, Suriname, had evidence of Mycobacterium (M.) leprae infection. A positive finding of M. leprae ancient (a)DNA would contribute to the origin of leprosy in Suriname. MATERIALS: Skeletal remains of Father Petrus Donders; two additional skeletons excavated from the Batavia cemetery were used as controls. METHODS: Archival research, paleopathological evaluation and aDNA-based testing of skeletal remains. RESULTS: Neither archives nor inspection of Donders skeletal remains revealed evidence of leprosy, and aDNA-based testing for M. leprae was negative. We detected M. leprae aDNA by RLEP PCR in one control skeleton, which also displayed pathological lesions compatible with leprosy. The M. leprae aDNA was genotyped by Sanger sequencing as SNP type 4; the skeleton displayed mitochondrial haplogroup L3. CONCLUSION: We found no evidence that Donders contracted leprosy despite years of intense leprosy contact, but we successfully isolated an archaeological M. leprae aDNA sample from a control skeleton from South America. SIGNIFICANCE: We successfully genotyped recovered aDNA to a M. leprae strain that likely originated in West Africa. The detected human mitochondrial haplogroup L3 is also associated with this geographical region. This suggests that slave trade contributed to leprosy in Suriname. LIMITATIONS: A limited number of skeletons was examined. SUGGESTIONS FOR FURTHER RESEARCH: Broader review of skeletal collections is advised to expand on diversity of the M. leprae aDNA database.

Genomic diversity in Mycobacterium leprae isolates from leprosy cases in South India.

OBJECTIVE: The Objective of this study was to identify the strain diversity of Mycobacterium leprae in terms of SNP types and subtypes stratified as per genomic single nucleotide polymorphisms, in clinical isolates of leprosy patients from a tertiary care leprosy center in South India. Further, the associations of SNP types with clinical outcomes in leprosy were also investigated. METHODS: DNA was extracted from excisional skin biopsies of a total of 172 newly diagnosed untreated leprosy patients from a clinic in Tamil Nadu, in south India, that also serves patients from neighboring states. All the leprosy patients were those who voluntarily reported at the clinic during the study period of one year i.e., 2015. Clinical and histopathological details were collected at diagnosis and leprosy was confirmed through bacteriological smear examination and PCR for M. leprae specific RLEP region. SNP types and subtypes were determined by PCR amplification and Sanger sequencing of PCR products. RESULTS: M. leprae specific RLEP gene amplification was achieved in 160 out of 172 patients. Among 160 specimens 118(73.75%) were type 1 and 42 (26.25%) were type 2 and on subtyping it was noted that 88/160 (55.00%) were 1D, 25/160 (15.62%) 1C, 5/160 (3.12%) 1A, 33/160 (20.62%) 2G and 9/160 (5.62%) were 2H. CONCLUSION: Our results indicated that subtype 1D is predominant in the south Indian population. We also noted 2G, 1C and 1A in the patient sample tested. Additionally we identified subtype 2H for the first time in India.

Molecular studies on ancient M. tuberculosis and M. leprae: methods of pathogen and host DNA analysis.

Humans have evolved alongside infectious diseases for millennia. Despite the efforts to reduce their incidence, infectious diseases still pose a tremendous threat to the world population. Fast development of molecular techniques and increasing risk of new epidemics have resulted in several studies that look to the past in order to investigate the origin and evolution of infectious diseases. Tuberculosis and leprosy have become frequent targets of such studies, owing to the persistence of their molecular biomarkers in ancient material and the characteristic skeletal lesions each disease may cause. This review examines the molecular methods used to screen for the presence of M. tuberculosis and M. leprae ancient DNA (aDNA) and their differentiation in ancient human remains. Examples of recent studies, mainly from Europe, that employ the newest techniques of molecular analysis are also described. Moreover, we present a specific approach based on assessing the likely immunological profile of historic populations, in order to further elucidate the influence of M. tuberculosis and M. leprae on historical human populations.

The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae.

Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.

Genome-wide comparison of medieval and modern Mycobacterium leprae.

Leprosy was endemic in Europe until the Middle Ages. Using DNA array capture, we have obtained genome sequences of Mycobacterium leprae from skeletons of five medieval leprosy cases from the United Kingdom, Sweden, and Denmark. In one case, the DNA was so well preserved that full de novo assembly of the ancient bacterial genome could be achieved through shotgun sequencing alone. The ancient M. leprae sequences were compared with those of 11 modern strains, representing diverse genotypes and geographic origins. The comparisons revealed remarkable genomic conservation during the past 1000 years, a European origin for leprosy in the Americas, and the presence of an M. leprae genotype in medieval Europe now commonly associated with the Middle East. The exceptional preservation of M. leprae biomarkers, both DNA and mycolic acids, in ancient skeletons has major implications for palaeomicrobiology and human pathogen evolution.

Comparative genomics analysis of Mycobacterium ulcerans for the identification of putative essential genes and therapeutic candidates.

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is the third most common mycobacterial disease after tuberculosis and leprosy. The present treatment options are limited and emergence of treatment resistant isolates represents a serious concern and a need for better therapeutics. Conventional drug discovery methods are time consuming and labor-intensive. Unfortunately, the slow growing nature of M. ulcerans in experimental conditions is also a barrier for drug discovery and development. In contrast, recent advancements in complete genome sequencing, in combination with cheminformatics and computational biology, represent an attractive alternative approach for the identification of therapeutic candidates worthy of experimental research. A computational, comparative genomics workflow was defined for the identification of novel therapeutic candidates against M. ulcerans, with the aim that a selected target should be essential to the pathogen, and have no homology in the human host. Initially, a total of 424 genes were predicted as essential from the M. ulcerans genome, via homology searching of essential genome content from 20 different bacteria. Metabolic pathway analysis showed that the most essential genes are associated with carbohydrate and amino acid metabolism. Among these, 236 proteins were identified as non-host and essential, and could serve as potential drug and vaccine candidates. Several drug target prioritization parameters including druggability were also calculated. Enzymes from several pathways are discussed as potential drug targets, including those from cell wall synthesis, thiamine biosynthesis, protein biosynthesis, and histidine biosynthesis. It is expected that our data will facilitate selection of M. ulcerans proteins for successful entry into drug design pipelines.

Insights from genomic comparisons of genetically monomorphic bacterial pathogens.

Some of the most deadly bacterial diseases, including leprosy, anthrax and plague, are caused by bacterial lineages with extremely low levels of genetic diversity, the so-called 'genetically monomorphic bacteria'. It has only become possible to analyse the population genetics of such bacteria since the recent advent of high-throughput comparative genomics. The genomes of genetically monomorphic lineages contain very few polymorphic sites, which often reflect unambiguous clonal genealogies. Some genetically monomorphic lineages have evolved in the last decades, e.g. antibiotic-resistant Staphylococcus aureus, whereas others have evolved over several millennia, e.g. the cause of plague, Yersinia pestis. Based on recent results, it is now possible to reconstruct the sources and the history of pandemic waves of plague by a combined analysis of phylogeographic signals in Y. pestis plus polymorphisms found in ancient DNA. Different from historical accounts based exclusively on human disease, Y. pestis evolved in China, or the vicinity, and has spread globally on multiple occasions. These routes of transmission can be reconstructed from the genealogy, most precisely for the most recent pandemic that was spread from Hong Kong in multiple independent waves in 1894.

Whole genome sequence of the rifamycin B-producing strain Amycolatopsis mediterranei S699.

Amycolatopsis mediterranei S699 is an actinomycete that produces an important antibiotic, rifamycin B. Semisynthetic derivatives of rifamycin B are used for the treatment of tuberculosis, leprosy, and AIDS-related mycobacterial infections. Here, we report the complete genome sequence (10.2 Mb) of A. mediterranei S699, with 9,575 predicted coding sequences.

Function-specific accelerations in rates of sequence evolution suggest predictable epistatic responses to reduced effective population size.

Changes in effective population size impinge on patterns of molecular evolution. Notably, slightly deleterious mutations are more likely to drift to fixation in smaller populations, which should typically also lead to an overall acceleration in the rates of evolution. This prediction has been validated empirically for several endosymbiont and island taxa. Here, we first show that rate accelerations are also evident in bacterial pathogens whose recent shifts in virulence make them prime candidates for reduced effective population size: Bacillus anthracis, Bordetella parapertussis, Mycobacterium leprae, Salmonella enterica typhi, Shigella spp., and Yersinia pestis. Using closely related genomes to analyze substitution rate dynamics across six phylogenetically independent bacterial clades, we demonstrate that relative rates of coding sequence evolution are biased according to gene functional category. Notably, genes that buffer against slightly deleterious mutations, such as chaperones, experience stronger rate accelerations than other functional classes at both nonsynonymous and synonymous sites. Although theory predicts altered evolutionary dynamics for buffer loci in the face of accumulating deleterious mutations, to observe even stronger rate accelerations is surprising. We suggest that buffer loci experience elevated substitution rates because the accumulation of deleterious mutations in the remainder of the genome favors compensatory substitutions in trans. Critically, the hyper-acceleration is evident across phylogenetically independent clades, supporting the hypothesis that reductions in effective population size predictably induce epistatic responses in genes that buffer against slightly deleterious mutations.

The MycoBrowser portal: a comprehensive and manually annotated resource for mycobacterial genomes.

In this paper, we present the MycoBrowser portal (http://mycobrowser.epfl.ch/), a resource that provides both in silico generated and manually reviewed information within databases dedicated to the complete genomes of Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum and Mycobacterium smegmatis. A central component of MycoBrowser is TubercuList (http://tuberculist.epfl.ch), which has recently benefited from a new data management system and web interface. These improvements were extended to all MycoBrowser databases. We provide an overview of the functionalities available and the different ways of interrogating the data then discuss how both the new information and the latest features are helping the mycobacterial research communities.

Whole-genome expression analysis of Mycobacterium leprae and its clinical application.

The whole-genome sequence analysis of Mycobacterium leprae, which was completed in 2001, revealed the characteristics of this microbe's genomic structure. Half of the M. leprae genome consists of a limited number of protein-coding genes and the rest comprises non-coding regions and pseudogenes. We performed membrane array and tiling array analyses to analyze the gene-expression profile of the M. leprae genome and found that pseudogenes and non-coding regions were expressed similarly to coding regions at the RNA level. The RNA expressions were confirmed by real-time PCR analysis. Expression of these RNAs in clinical samples showed varying patterns among patients, thus indicating that the analysis of RNA expression patterns, including non-coding regions and pseudogenes, may be useful for understanding the pathological state, prognosis, and assessment of therapeutic progress in leprosy.

[Detection of RNA expression on whole genome analysis of Mycobacterium leprae by tiling array].

Completion of Mycobacterium leprae genome sequence revealed that there are many pseudogenes and non-coding regions, but rather small numbers of protein-coding genes. Although it was thought that pseudogenes and non-coding regions were silent and junk, our previous studies indicated that RNA expression was detected from these regions. To elucidate comprehensive RNA expression pattern on M. leprae whole genome, tiling array was designed and total RNA of M. leprae Thai-53 strain was analyzed. As a result, highly expressed regions were detected among not only the gene regions but also pseudogenes and non-coding regions. Since some of the RNA expression levels were modulated by MDT, evaluation of RNA expression pattern might be a good indicator for the treatment of leprosy.

[Comprehensive analysis of RNA expression of Mycobacterium leprae and clinical and biological significance].

Completion of Mycobacterium leprae genome sequence revealed that there are many pseudogenes and non-coding regions, but rather small numbers of protein-coding genes. This result indicates that M. leprae is a very unique organism, and this future is important to understand the biological nature and/or pathogenicity of M. leprae, which remain unclear. We attempted to find the biological nature of M. leprae by detecting the gene and pseudogene regions transcribed at high level. We detected the genomic regions including pseudogenes and demonstrated that six out of twelve high expression regions were pseudogenes. In addition, its transcription level was changed when M. leprae infects macrophage. RNA was detected from genes, pseudogenes and non-coding regions. The expression levels of these regions were different among patients and a part of them is disappeared just after treatment. These results suggested that RNA derived from pseudogene and non-coding region have some function concerning the infection and/or intracellular parasitism and that the analysis of pseudogene and non-coding region expression pattern of M. leprae is available as a criterion for therapeutic effect and disease type of leprosy, and a prognostic marker.

Simple sequence repeats in different genome sequences of Shigella and comparison with high GC and AT-rich genomes.

Simple sequence repeats (SSRs) are omnipresent in prokaryotes and eukaryotes, and are found anywhere in the genome in both protein encoding and noncoding regions. In present study the whole genome sequences of seven chromosomes (Shigella flexneri 2a str301 and 2457T, Shigella sonnei, Escherichia coli k12, Mycobacterium tuberculosis, Mycobacterium leprae and Staphylococcus saprophyticus) have downloaded from the GenBank database for identifying abundance, distribution and composition of SSRs and also to determine difference between the tandem repeats in real genome and randomness genome (using sequence shuffling tool) of the organisms included in this study. The data obtained in the present study show that: (i) tandem repeats are widely distributed throughout the genomes; (ii) SSRs are differentially distributed among coding and noncoding regions in investigated Shigella genomes; (iii) total frequency of SSRs in noncoding regions are higher than coding regions; (iv) in all investigated chromosomes ratio of Trinucleotide SSRs in real genomes are much higher than randomness genomes and Di nucleotide SSRs are lower; (v) Ratio of total and mononucleotide SSRs in real genome is higher than randomness genomes in E. coli K12, S. flexneri str 301 and S. saprophyticus, while it is lower in S. flexneri str 2457T, S.sonnei and M. tuberculosis and it is approximately same in M. leprae; (vi) frequency of codon repetitions are vary considerably depending on the type of encoded amino acids.

Genome scale portrait of cAMP-receptor protein (CRP) regulons in mycobacteria points to their role in pathogenesis.

cAMP Receptor Protein (CRP)/Fumarate Nitrate Reductase Regulator (FNR) family proteins are ubiquitous regulators of cell stress in eubacteria. These proteins are commonly associated with maintenance of intracellular oxygen levels, redox-state, oxidative and nitrosative stresses, and extreme temperature conditions by regulating expression of target genes that contain regulatory cognate DNA elements. We describe the use of informatics enabled comparative genomics to identify novel genes under the control of CRP regulator in Mycobacterium tuberculosis (M.tb). An inventory of CRP regulated genes and their operon context in important mycobacterial species such as M. leprae, M. avium subsp. paratuberculosis and M. smegmatis and several common genes within this genus including the important cellular functions, mainly, cell-wall biogenesis, cAMP signaling and metabolism associated with such regulons were identified. Our results provide a possible theoretical framework for better understanding of the stress response in mycobacteria. The conservation of the CRP regulated genes in pathogenic mycobacteria, as opposed to non-pathogenic ones, highlights the importance of CRP-regulated genes in pathogenesis.

[Development of a large-scale comparative genome system and its application to the analysis of mycobacteria genomes].

As the number of whole genome sequences available continues to increase rapidly, the raw scale of the sequence data being used in analysis is the first hurdle for comparative genome analysis. When performing whole genome alignments, large-scale rearrangements make it necessary to first find out roughly which short well-conserved segments correspond to what other segments (termed anchors). Successful results have been achieved by adapting tools like BLAT and BLASTZ on a problem-to-problem basis, but the work required to perform a single alignment is considerable. Recently, new programs such as Mauve and Pattern-Hunter can handle slightly larger inputs, but the memory/time requirements for sequences like Human and Chimp X chromosomes are prohibitive for most computational environments. Our novel algorithm, which we have implemented in a program called Murasaki (available at http://murasaki.dna.bio.keio.ac.jp), makes it possible to identify anchors of multiple large sequences on the scale of several hundred megabases (e.g. three mammal chromosomes) in a matter of minutes. We also demonstrate an application of Murasaki to the comparative analysis of multiple mycobacteria genomes.