Dual RNA-Seq of Human Leprosy Lesions Identifies Bacterial Determinants Linked to Host Immune Response.

To understand how the interaction between an intracellular bacterium and the host immune system contributes to outcome at the site of infection, we studied leprosy, a disease that forms a clinical spectrum, in which progressive infection by the intracellular bacterium Mycobacterium leprae is characterized by the production of type I IFNs and antibody production. Dual RNA-seq on patient lesions identifies two independent molecular measures of M. leprae, each of which correlates with distinct aspects of the host immune response. The fraction of bacterial transcripts, reflecting bacterial burden, correlates with a host type I IFN gene signature, known to inhibit antimicrobial responses. Second, the bacterial mRNA:rRNA ratio, reflecting bacterial viability, links bacterial heat shock proteins with the BAFF-BCMA host antibody response pathway. Our findings provide a platform for the interrogation of host and pathogen transcriptomes at the site of infection, allowing insight into mechanisms of inflammation in human disease.

Phylogenetic analysis of vitamin B12-related metabolism in Mycobacterium tuberculosis.

Comparison of genome sequences from clinical isolates of Mycobacterium tuberculosis with phylogenetically-related pathogens Mycobacterium marinum, Mycobacterium kansasii, and Mycobacterium leprae reveals diversity amongst genes associated with vitamin B12-related metabolism. Diversity is generated by gene deletion events, differential acquisition of genes by horizontal transfer, and single nucleotide polymorphisms (SNPs) with predicted impact on protein function and transcriptional regulation. Differences in the B12 synthesis pathway, methionine biosynthesis, fatty acid catabolism, and DNA repair and replication are consistent with adaptations to different environmental niches and pathogenic lifestyles. While there is no evidence of further gene acquisition during expansion of the M. tuberculosis complex, the emergence of other forms of genetic diversity provides insights into continuing host-pathogen co-evolution and has the potential to identify novel targets for disease intervention.

Use of short tandem repeat sequences to study Mycobacterium leprae in leprosy patients in Malawi and India.

BACKGROUND: Inadequate understanding of the transmission of Mycobacterium leprae makes it difficult to predict the impact of leprosy control interventions. Genotypic tests that allow tracking of individual bacterial strains would strengthen epidemiological studies and contribute to our understanding of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Genotyping assays based on variation in the copy number of short tandem repeat sequences were applied to biopsies collected in population-based epidemiological studies of leprosy in northern Malawi, and from members of multi-case households in Hyderabad, India. In the Malawi series, considerable genotypic variability was observed between patients, and also within patients, when isolates were collected at different times or from different tissues. Less within-patient variability was observed when isolates were collected from similar tissues at the same time. Less genotypic variability was noted amongst the closely related Indian patients than in the Malawi series. CONCLUSIONS/SIGNIFICANCE: Lineages of M. leprae undergo changes in their pattern of short tandem repeat sequences over time. Genetic divergence is particularly likely between bacilli inhabiting different (e.g., skin and nerve) tissues. Such variability makes short tandem repeat sequences unsuitable as a general tool for population-based strain typing of M. leprae, or for distinguishing relapse from reinfection. Careful use of these markers may provide insights into the development of disease within individuals and for tracking of short transmission chains.

Microsatellite mapping of Mycobacterium leprae populations in infected humans.

To investigate genetic diversity in a bacterial population, we measured the copy numbers of simple sequence repeats, or microsatellites, in Mycobacterium leprae from patients living in and around Hyderabad, India. Three microsatellite loci containing trinucleotide or dinucleotide repeats were amplified from infected tissues, and the copy numbers were established by sequence analysis. Extensive diversity was observed in a cross-sectional survey of 33 patients, but closely related profiles were found for members of a multicase family likely to share a common transmission source. Sampling of multiple tissues from single individuals demonstrated identical microsatellite profiles in the skin, nasal cavity, and bloodstream but revealed differences at one or more loci for M. leprae present in nerves. Microsatellite mapping of M. leprae represents a useful tool for tracking short transmission chains. Comparison of skin and nerve lesions suggests that the evolution of disease within an individual involves the expansion of multiple distinct subpopulations of M. leprae.

Microsatellite mapping of Mycobacterium leprae populations in infected humans

To investigate genetic diversity in a bacterial population, we measured the copy numbers of simple sequence repeats, or microsatellites, in Mycobacterium leprae from patients living in and around Hyderabad, India. Three microsatellite loci containing trinucleotide or dinucleotide repeats were amplified from infected tissues, and the copy numbers were established by sequence analysis. Extensive diversity was observed in a cross-sectional survey of 33 patients, but closely related profiles were found for members of a multicase family likely to share a common transmission source. Sampling of multiple tissues from single individuals demonstrated identical microsatellite profiles in the skin, nasal cavity, and bloodstream but revealed differences at one or more loci for M. leprae present in nerves. Microsatellite mapping of M. leprae represents a useful tool for tracking short transmission chains. Comparison of skin and nerve lesions suggests that the evolution of disease within an individual involves the expansion of multiple distinct subpopulations of M. leprae.

Futuro de la epidemiología molecular en la lepra

La evidencia actual, aunque limitada, indica que la diversidad genética entre las cepas de M. leprae es muy baja, incluso en comparación con M. tuberculosis. Las variaciones en la cantidad de copias de tandems de repetición cortas, desta can como una importante excepción en este modelo y parecen ser los más prometedores a corto plazo para la epidemiología molecular. Resultará difícil identificar loci polimórficos adicionales y se conseguirá de manera más efectiva generando información secuencial parcial del genoma con más cepas de M. leprae (AU)

Chronic bacterial infections: living with unwanted guests.

Some bacterial pathogens can establish life-long chronic infections in their hosts. Persistence is normally established after an acute infection period involving activation of both the innate and acquired immune systems. Bacteria have evolved specific pathogenic mechanisms and harbor sets of genes that contribute to the establishment of a persistent lifestyle that leads to chronic infection. Persistent bacterial infection may involve occupation of a particular tissue type or organ or modification of the intracellular environment within eukaryotic cells. Bacteria appear to adapt their immediate environment to favor survival and may hijack essential immunoregulatory mechanisms designed to minimize immune pathology or the inappropriate activation of immune effectors.

Three pathways for trehalose biosynthesis in mycobacteria.

Trehalose is present as a free disaccharide in the cytoplasm of mycobacteria and as a component of cell-wall glycolipids implicated in tissue damage associated with mycobacterial infection. To obtain an overview of trehalose metabolism, we analysed data from the Mycobacterium tuberculosis genome project and identified ORFs with homology to genes encoding enzymes from three trehalose biosynthesis pathways previously characterized in other bacteria. Functional assays using mycobacterial extracts and recombinant enzymes derived from these ORFs demonstrated that mycobacteria can produce trehalose from glucose 6-phosphate and UDP-glucose (the OtsA-OtsB pathway) from glycogen-like alpha(1-->4)-linked glucose polymers (the TreY-TreZ pathway) and from maltose (the TreS pathway). Each of the pathways was found to be active in both rapid-growing Mycobacterium smegmatis and slow-growing Mycobacterium bovis BCG. The presence of a disrupted treZ gene in Mycobacterium leprae suggests that this pathway is not functional in this organism. The presence of multiple biosynthetic pathways indicates that trehalose plays an important role in mycobacterial physiology.

Generation and characterization of monoclonal antibodies to the phenolic glycolipid of Mycobacterium leprae

Nine cloned cell lines producing antibodies to the unique phenolic glycolipid of Mycobacterium leprae have been established as a result of fusions with spleens from mice immunized with the glycolipid complexed with methylated bovine serum albumin. One of the antibodies was relatively nonspecific, binding to a related glycolipid from Mycobacterium kansasii, but the remaining antibodies were specific for the M. leprae lipid. Some of the antibodies required the intact (trisaccharide) carbohydrate portion for recognition of the glycolipid antigen, whereas others recognized partially hydrolyzed forms lacking one or two sugar residues. Monoclonal antibodies directed at the terminal saccharide of the glycolipid showed the greatest specificity for M. leprae in enzyme-linked immunoassays. These antibodies brightly labeled whole mycobacteria in indirect immunofluorescence experiments, demonstrating the surface location of M. leprae-specific determinants of the glycolipid antigen. In addition to their use in providing information about the antigenic properties of the phenolic glycolipid, these antibodies have potential applications for elucidating the roles of glycolipid in the pathogenesis of leprosy.