Laboratory Research: Genetics and Cultivation
Gene expression, regulation and pseudogenes in M. leprae
Investigator: Diana Williams, Ph.D .
Fundamental to understanding the pathogenicity of M . leprae is identifying the genes needed for survival, growth and virulence in man.The completion of the M. leprae genome sequence has provided information capable of supporting studies aimed at better understanding M. leprae and the disease it causes.
The genome appears highly degraded and possesses the largest repertoire of pseudogenes compared to other bacteria as well as a large number of deleted genes. This large-scale loss of gene function has resulted in a genome that is occupied by less than 50 percent protein-coding genes and comparative genome analysis has identified deficits in several general cellular metabolic families.
The National Hansen's Disease Program has used the M. leprae genome sequence data, bioinformatics and surrogate genetics in several mycobacterial hosts to study specific M. leprae genes associated with drug resistance, gene regulation, virulence, and growth.
The Program has also used this information, in collaboration with investigators at Colorado State University, to characterize the first M. leprae global DNA microarray, consisting of all open reading frames and pseudogenes.
Dr. Williams' work continues to be at the forefront of understanding M. leprae genetics and has resulted in molecular tests useful for diagnosing Hansen's disease and determining M. leprae 's susceptibility to antileprosy drugs.
Genotyping M. leprae using variable number tandem repeats
Investigators: Tom Gillis, Ph.D and Richard Truman, Ph.D.
Understanding the mode(s) of transmission of leprosy is a major goal of leprosy research. Defining this epidemiologic parameter should help clarify risk of infection and may help direct public health intervention strategies aimed at controlling leprosy.
Mycobacterium leprae is an obligate intracellular pathogen that is widely distributed around the globe. There are no recognized sub-types and the bacillus exhibits little genetic diversity as judged by common DNA sequence structures. The only documented highly variable sequences are associated with variable number tandem repeat (VNTR) sequences distributed throughout the genome.
The National Hansen's Disease Program's primary goal for the VNTR studies is to establish a panel of reliable markers to be used in the field to track M. leprae within endemic communities with the aim of determining transmission links within that community. Initial studies have been focused on establishing standards for assessing VNTR polymorphisms, integrating studies in selected field sites to validate the use of the VNTR marker panel(s) and coordinating the international consortium to implement this kind of testing in sites where leprosy remains a problem. Field studies are set for sites in India, Brazil, the Philippines and Bangladesh.
Mycobacterium leprae cultivation
Investigators: James Krahenbuhl, Ph.D. and Tom Gillis, Ph.D.
Well over a century after its description by Hansen, Mycobacterium leprae has yet to be cultivated in vitro, making it one of the most intractable microorganisms to study.
Experimentally, it can be cultivated reliably only in mouse footpads or the nine-banded armadillo, achievement of significant numbers of organisms requiring 6 months to 2 years, respectively.
The recent completion of the sequence of the M. leprae genome indicates the deletion or partial deletion of multiple genes, notably those involved in respiratory chains, iron acquisition, and catabolic processes. Since these deletions would not necessarily preclude culture medium supplementation with defined growth factors, along with investigators at Colorado State University, the National Hansen's Disease program is designing different media formulations (e.g., different carbon, nitrogen, sulfur and phosphorous sources) taking cues from the M. leprae genome and other biochemical studies and then evaluating those formulations for their growth potential using radiolabeled reporter molecule as an indicator for metabolic activity.
If culture supplements are not sufficient to produce enhanced metabolism leading to sustained cultivation, then newly developed genetic tools are being evaluated for introducing genes into M. leprae with the goal of enhancing metabolism with sustained cultivation potential.
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