Transfer, stable maintenance and expression of the mycolactone polyketide megasynthase mls genes in a recombination-impaired Mycobacterium marinum

Porter, Jessica L.; Tobias, Nicholas J.; Hong, Hui; Tuck, Kellie L.; Jenkin, Grant A.; Stinear, Timothy P.

doi:10.1099/mic.0.027029-0

Cited by 14 publications

(17 citation statements)

References 29 publications

(42 reference statements)

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“…Similarly, we investigated DNA sequences upstream of mup045 and found a TSP at T207 with a potential SigA promoter element predicted by PoSSuM and confirmed by a loss of GFP expression in M. smegmatis following mutagenesis of the proposed −10 box. The principal mycobacterial sigma factor sigA is utilized by genes expressed during exponential growth [49], thus the data from mlsA/B and mup045 fit well with our previous report that show these genes are constantly expressed during exponential growth in the heterologous host, M. marinum [35].…”

Section: Discussionsupporting

confidence: 82%

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

et al. 2009

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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.

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Section: Discussionsupporting

confidence: 82%

“…The constructs were confirmed to be correct in mycobacteria by Southern hybridization and back transformation to E. coli . Acetone soluble lipids were extracted from recombinant M. ulcerans and analysed by LC-MS for the presence of mycolactones as previously described [35].…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2009

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“…Dormancy of the bacteria was estimated by quantifying the expression of devR, devS, and icl (27)(28)(29), and mycolactone biosynthesis gene expression, that is, mup053, mup045, mup038, mlsA1 (30)(31)(32), and KR (33), was also estimated. Analysis was performed utilizing the DDCt method with the ppk gene as housekeeping gene (34). Primer sequences are presented in Supplemental Fig.…”

Section: Quantitative Pcr Analysismentioning

confidence: 99%

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

Marion

Jarry

Cano

et al. 2016

The Journal of Immunology

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Buruli ulcer, a debilitating disease, is caused by Mycobacterium ulcerans. The incidence of this neglected tropical disease is steadily increasing. As a rule, without treatment, skin ulcers occur and a lengthy healing process may be observed associated with severe functional disabilities. Mouse models are already available to study establishment of lesions or evaluation of therapy but a lack of a suitable animal model, mimicking all clinical stages, in particular the healing process, remains an obstacle to understand the pathophysiology of M. ulcerans infection. M. ulcerans was s.c. inoculated in three consanguine mouse strains, that is, BALB/c and C57BL/6, classically used to study mycobacterial infection, and FVB/N. Strikingly, FVB/N mice, although as sensitive as all other mouse strains with respect to M. ulcerans infection, presented a spontaneous healing after the ulcerative phase despite stable bacterial load, and mycolactone toxin was not detected in the healed tissues. The spontaneous healing process was accompanied by an activation of the innate immune system. The adaptive response initiated by FVB/N mice was not involved in the healing process and did not confer protection against M. ulcerans. Our work highlights the importance of innate immune responses to control M. ulcerans infection. This in vivo model of M. ulcerans infection now paves the way for new avenues of research toward the elucidation of critical stages of this disease, such as the characterization of the regulation of mycolactone production, a better understanding of the pathophysiology of M. ulcerans infection, and the development of new therapeutic strategies.

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“…M. marinum M and M. ulcerans 06-3844 (the latter isolate also referred to as M. marinum 06-3844) [25] were cultured at 30°C in 7H9 Middlebrook broth or 7H10 Middlebrook agar as described [12]. Antibiotics were used at the following final concentrations in mycobacteria: apramycin 50 μg/mL; kanamycin 25 μg/mL; and hygromycin 50 μg/mL.…”

Section: Methodsmentioning

confidence: 99%

“…However, it has never been shown that these genes are sufficient for toxin synthesis. There is only one published report of an experiment to test sufficiency, with the transfer (in two parts) of the 174 kb pMUM001 plasmid that harbours the mls genes to Mycobacterium marinum , a natural producer of many polyketide metabolites and a very close relative of M. ulcerans [12]. This experiment showed that the mls genes were expressed in M. marinum but mycolactones were not detected [12].…”

Section: Introductionmentioning

confidence: 99%

The Cell Wall-Associated Mycolactone Polyketide Synthases Are Necessary but Not Sufficient for Mycolactone Biosynthesis

et al. 2013

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Mycolactones are polyketide-derived lipid virulence factors made by the slow-growing human pathogen, Mycobacterium ulcerans. Three unusually large and homologous plasmid-borne genes (mlsA1: 51 kb, mlsB: 42 kb and mlsA2: 7 kb) encode the mycolactone type I polyketide synthases (PKS). The extreme size and low sequence diversity of these genes has posed significant barriers for exploration of the genetic and biochemical basis of mycolactone synthesis. Here, we have developed a truncated, more tractable 3-module version of the 18-module mycolactone PKS and we show that this engineered PKS functions as expected in the natural host M. ulcerans to produce an additional polyketide; a triketide lactone (TKL). Cell fractionation experiments indicated that this 3-module PKS and the putative accessory enzymes encoded by mup045 and mup038 associated with the mycobacterial cell wall, a finding supported by confocal microscopy. We then assessed the capacity of the faster growing, Mycobacterium marinum to harbor and express the 3-module Mls PKS and accessory enzymes encoded by mup045 and mup038. RT-PCR, immunoblotting, and cell fractionation experiments confirmed that the truncated Mls PKS multienzymes were expressed and also partitioned with the cell wall material in M. marinum. However, this heterologous host failed to produce TKL. The systematic deconstruction of the mycolactone PKS presented here suggests that the Mls multienzymes are necessary but not sufficient for mycolactone synthesis and that synthesis is likely to occur (at least in part) within the mycobacterial cell wall. This research is also the first proof-of-principle demonstration of the potential of this enzyme complex to produce tailored small molecules through genetically engineered rearrangements of the Mls modules.

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Transfer, stable maintenance and expression of the mycolactone polyketide megasynthase mls genes in a recombination-impaired Mycobacterium marinum

Cited by 14 publications

References 29 publications

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

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

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

The Cell Wall-Associated Mycolactone Polyketide Synthases Are Necessary but Not Sufficient for Mycolactone Biosynthesis

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