Development of a molecular diagnostic, an animal model and a transposon mutagenesis system for studying fish tuberculosis

Abstract

World wide, 88 million people are predicted to be infected with Mycobacterium tuberculosis (M. tuberculosis) during the current decade, 1990-99, of which 30 million people are expected to die. The slow growth rate and clumping of mycobacterial cells has hindered the application of "conventional" genetic techniques to study the molecular pathogenesis of mycobacterial infections. To overcome the problems encountered in working with M. tuberculosis, M. marinum and the goldfish, Carcassius auratus were developed as a model system for studying mycobacterial pathogenesis. This model system was used to evaluate the virulence of the slowly growing mycobacteria, M. marinum compared to the rapidly growing, M. fortuitum and M. smegmatis. Depending on the dose of mycobacteria inoculated into animals, an acute or a chronic form of the disease develops. With M. marinum, injection of 10 to the 9 or 10 to the 8 cfu per fish induced an acute disease with all animals dying within 17 days postinfection. Inoculation of 10 to the 2 to 10 to the 7 cfu per fish induced a chronic disease with all fish surviving until the end of the experiment (56 days). In contrast, with M. fortuitum, only an inoculum of 10 to the 9 cfu per fish was able to induce an acute form of the disease. Surprisingly, our results showed that M. smegmatis, once considered a nonpathogenic strain of mycobacteria, is pathogenic to fish. Additionally, a genus specific PCR-based protocol was developed to amplify a 924 bp DNA fragment from the 16S rRNA present in all Mycobacteria, followed by restriction enzyme analysis (REA) to identify the species. To develop an efficient protocol for genetic transfer of plasmids to mycobacterial cells, several protocols were tested to and the optimal conditions for M. marinum electroporation. Electrocompetent cells prepared at room temperature from M. marinum cultures grown to late-exponential phase in the presence of ethionamide (1 mug/ml) gave the highest transposition efficiency. Signature-tagged mutagenesis protocol was applied to identify virulence factors in M. marinum. By screening M. marinum mutant library in goldfish, we were able to identify several M. marinum mutants unable to survive in the goldfish. Genetic analysis of such mutants is currently underway.