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.
Highlights
Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU) an emerging but neglected disease found predominantly in tropical regions of the world and with an increasing incidence in West and Central Africa [1,2]
There are many unanswered questions surrounding BU, regarding the role of mycolactone in disease and how M. ulcerans is transmitted to humans
Using a variety of experimental approaches, including green fluorescent protein (GFP) as a reporter of gene expression, we have identified strong promoters that drive transcription of the mycolactone genes in M. ulcerans
Summary
Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU) an emerging but neglected disease found predominantly in tropical regions of the world and with an increasing incidence in West and Central Africa [1,2]. Substantial tissue damage often necessitates surgery [4]. The social and economic burden of BU can be severe, in impoverished rural regions of West Africa where the prevalence of BU is sometimes higher than that of the two most significant mycobacterial diseases, leprosy and tuberculosis. Cases of BU are usually clustered around swamps and slow-flowing water and while the mode of transmission of M. ulcerans is unknown, evidence to date suggests, fish [5], snails [6] and certain carnivorous aquatic insects [7,8] can all harbour the bacterium. Recent studies in Australia suggest mosquitoes may play a role in transmission [9,10]
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