Transposon Mutagenesis ofMycobacterium marinumIdentifies a Locus Linking Pigmentation and Intracellular Survival

Abstract

Pathogenic mycobacteria survive and replicate within host macrophages, but the molecular mechanisms involved in this necessary step in the pathogenesis of infection are not completely understood. Mycobacterium marinum has recently been used as a model for aspects of the pathogenesis of tuberculosis because of its close genetic relationship to Mycobacterium tuberculosis and because of similarities in the pathology and course of infection caused by this organism in its natural hosts, fish and frogs, with tuberculosis in humans. In order to advance the utility of the M. marinum model, we have developed efficient transposon mutagenesis of the organism by using a Drosophila melanogaster mariner-based transposon. To determine the efficiency of transposition, we have analyzed pigmentation mutants from the transposon mutant library. In addition to insertions in four known genes in the pathway of pigment biosynthesis, two insertions in novel genes were identified in our mutant library. One of these is in a putative inhibitor of the carotenoid biosynthesis pathway. The second unexpected insertion is in an intergenic region between two genes homologous to Rv2603c and Rv2604c of M. tuberculosis. In addition to a pigmentation defect, this mutant showed increased susceptibility to singlet oxygen and grew poorly in murine macrophages. Complementation with M. tuberculosis genomic DNA encompassing Rv2603c to Rv2606c corrected the pigmentation and growth defects of the mutant. These data demonstrate the utility of mariner-based transposon mutagenesis of M. marinum and that M. marinum can be used to study the function of M. tuberculosis genes involved in intracellular survival and replication.

Pigments and Pathogenesis

“…Pigments seem to increase virulence of pathogens by increasing invasiveness, survival in immune cells, and size of local abscesses. In fact in M. marinum pigmentation is linked to a locus important in intracellular survival [22]. Thus the finding that pigments in environmental mycobacteria are produced under conditions of acidity which can be found in the phagosomes of macrophages or the centers of caseating granulomas indicate that pigments may be important for pathogenesis [17].…”

“…Pigment production may aid in the resistance to acidity and help these mycobacteria survive. The promoter driving expression of carotenoid synthesis genes has regions that respond transcriptionally to exposure to light in Mycobacterium marinum [22,23]. In M. smegmatis, exposure to acidity transcriptionally upregulates the homologue of this promoter region [17].…”

Drosophila: a polyvalent model to decipher host–pathogen interactions

“…In both cases, the bacteria escape from the host immune system by hiding in macrophages. Notably, mutants of M. marinum that are impaired in macrophage survival can be complemented using homologous genes from M. tuberculosis [39], suggesting that these two bacteria share similar virulence mechanisms. Recently, Dionne et al [40] showed that injection of M. marinum expressing GFP into flies resulted in the accumulation of bacteria within hemocytes.…”

Physiological role and potential clinical interest of mycobacterial pigments

“…Recently, molecular genetics has allowed the characterization of loci required for pigment synthesis in Mycobacterium tuberculosis. Since 2003, it is known that an Mycobacterium tuberculosis genomic region that encompasses genes Rv2603c to Rv2606c can correct the golden pigmentation phenotype of a Mycobacterium marinum mutant (68).…”