Mycolactone Gene Expression Is Controlled by Strong SigA-Like Promoters with Utility in Studies of Mycobacterium ulcerans and Buruli Ulcer

Abstract

Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans.

Interaction of Mycobacterium ulcerans with Mosquito Species: Implications for Transmission and Trophic Relationships

“…Our findings on M. ulcerans in mosquito larvae are in partial agreement with recent reports from Australia (43). Both studies provide evidence for the survival of M. ulcerans in mosquito larvae.…”

“…In the Australian study, the infection was not studied throughout the mosquito developmental cycle. One difference between our work and that of Tobias et al (43) is that Tobias found that M. ulcerans survived much better in Aedes comptorhynchus mosquito larvae than did a nonpigmented variant of the closely related non-toxin-producing M. marinum species. In contrast, we find that the survival phenotype of WT M. marinum in Aedes aegypti mosquito larvae is identical to that of M. ulcerans; both species survived in high numbers throughout the larval developmental cycle.…”

Investigating the Role of Free-living Amoebae as a Reservoir for Mycobacterium ulcerans

“…Real-time PCR was performed on M. ulcerans strains to estimate cell numbers as previously described [28]. Primers targeting the 16S rRNA gene were used for detection of mycobacteria.…”

FVB/N Mice Spontaneously Heal Ulcerative Lesions Induced by Mycobacterium ulcerans and Switch M. ulcerans into a Low Mycolactone Producer

“…ulcerans strains and inoculation M. ulcerans strain nos. 01G897, JKD8083, and S4018 were originally isolated from patients from French Guiana, Australia, and Benin, respectively (22)(23)(24). Bacterial suspensions were prepared as previously described (10), and then adjusted at 2 3 10 5 acid-fast bacilli/ml to be inoculated (50 ml) into the tail of 6-wk-old females of the consanguine BALB/c, C57BL/6, and FVB/N mouse strains (Charles River Laboratories, Saint-Germain-Nuelles, France).…”