A Novel Mycolactone Toxin
Obtained by Biosynthetic
Hui Hong,[a]Tim Stinear,[b]Jessica Porter,[b]
Caroline Demangel,[c]and Peter F. Leadlay*[a]
Mycolactones are polyketide macrolide toxins produced by the
emerging human pathogen Mycobacterium ulcerans, the causa-
tive agent of the destructive skin disease Buruli ulcer.[1,2]Myco-
lactone appears to be the primary virulence determinant for
the infection,and purified mycolactone has potent cytotoxic,
apoptotic and immunomodulatory properties.[2–4]
The structures of mycolactones A/B ([M+ +Na]+at m/z 765)
have been determined, and their configuration has been con-
firmed by chemical synthesis.[2,5–7]The molecules are Z and E
isomers of a 12-membered macrolactone linked via an ester
bond with a highly unsaturated polyketide chain (Scheme 1).
Further work has revealed the heterogeneity of the toxin from
different geographical sources, with structural variations in the
side chain that could be traced to specific changes in the bio-
synthetic pathway.[8–11]More recently, distinctive mycolactones
with characteristically different side chains have also been iso-
lated from the frog pathogen Mycobacterium liflandii[12,13]and
from several mycobacterial fish pathogens(see below); this
raises the question of which structural features are crucial for
the several effects of mycolactone on human cells.
The genetic basis for mycolactone biosynthesis has been de-
termined.M. ulcerans has a 174 kb megaplasmid harbouring
three genes encoding type I modular polyketide synthases
(PKS). Genes mlsA1 and mlsA2 together encode the PKS for
production of the 12-membered core lactone, while mlsB en-
codes the PKS for the side chain. Three additional genes are
present, MUP_045, MUP_038 and MUP_053. MUP_045 encodes
an enzyme resembling FabH-like type III ketosynthases (KS),
MUP_038 encodes a discrete thioesterase, and MUP_053 en-
codes a cytochrome P450 hydroxylase Cyp140A7 that is
strongly implicated in the catalysis of oxidation at C-12’ of the
side chain. For example, Australian strains of M. ulcerans,
whose virulence plasmid lacks MUP_053, typically produce my-
colactone C, in which the C12’ hydroxy group is missing.We
wished to test whether Cyp140A7 might oxidise an alternative
mycolactone substrate to give rise to additional structural di-
versity by biosynthetic engineering. We now report that when
Cyp140A7 was expressed in the recently discovered fish patho-
gen Mycobacterium marinum DL045, a novel mycolactone (my-
colactone G) was indeed produced from the engineered strain.
Importantly, mycolactone G and other mycolactone variants
were found to differ significantly from mycolactones A/B in
their immunosuppressive activity.
Scheme 1. Structures of mycolactones A/B, C, E, F and G.
[a] Dr. H. Hong, Prof. P. F. Leadlay
Sanger Building, Department of Biochemistry, University of Cambridge
80 Tennis Court Road, Cambridge CB2 1QW (UK)
[b] Dr. T. Stinear, J. Porter
Department of Microbiology, Monash University
Wellington Road, Clayton, 3800 (Australia)
[c] Dr. C. Demangel
Unit? de G?n?tique Mol?culaire Bact?rienne, Institut Pasteur
28 rue du Docteur Roux, 75724 Paris Cedex 15 (France)
Supporting information for this article is available on the WWW under
http://www.chembiochem.org or from the author.
ChemBioChem 2007, 8, 2043–2047? 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim
We first examined the structure of the mycolactones normal-
ly produced by several recently discovered Mycobacterium ul-
cerans-like mycobacteria isolated from diseased fish.[14,17]Cell
extractsfromeach ofM. marinum
CL240299, M. marinum DL240290 and M. pseudoshottsii L15,
were analysed by LC-MS and LC-MS/MS. These strains all pro-
duced mycolactones with m/z at 723 (major component; my-
colactone F) and m/z 721 (minor component). The MS/MS
spectra of m/z 723 and of m/z 721 from all the fish pathogen
strains checked were identical; this suggests that these strains
produced the same mycolactones. The MS/MS analysis also
showed that the core lactones from these two mycolactone
analogues were identical to the core of all mycolactones dis-
covered so far. High-resolution mass analysis of the m/z 723
and 721 peaks provided the formulae C42H68O8Na and
C42H66O8Na, respectively. By comparison with the formulae
C43H70O8Na and C43H68O8Na of the mycolactone analogues re-
cently reported from M. liflandii,[12–13](m/z 737 and 735, respec-
tively, dubbed mycolactone E), these fish pathogen mycolac-
tones appear to lack a methyl group. Comparison of the MS/
MS spectra of m/z 723 and m/z 737 (Figure 1) revealed that
fragment ions C, D and E, whose identities we have previously
assigned as structures arising from the conjugated double
bonds of the mycolactone side chain (C1’ to C8’),were pres-
ent in both spectra. In addition, both compounds could lose a
120 Da fragment, which has been assigned as 1,3,5-trimethyl-
benzene, from the side chain.These data strongly suggest
that the cumulated double bonds in the side chain are identi-
cal in the two molecules, and that the methyl difference lies in
the distal (hydroxy) part of the side chain. Comparison of the
fragment ions relating to this part of the side chain showed
that instead of an observed loss of 58 and 102 Da in the MS/
MS 737 spectrum (for mycolactone E) there was an observed
loss of 44 and 88 Da in the MS/MS 723 spectrum. This finding
([M+ +Na]+at m/z 737) produced by the frog pathogen, the my-
colactone from the fish pathogen ([M+ +Na]+at m/z 723) has
acetate instead of propionate as its starter unit. An acetate
starter is typical of the mycolactones produced by the human
pathogen M. ulcerans.[2,7,8]Therefore, based on our previously
published structure of mycolactone E,the structure for the
mycolactone from the fish pathogens is that shown in
Figure 1. The same structure has been recently proposed for
this compound by Small and colleaguesand named there as
mycolactone F. Both mycolactone F from the fish pathogen
and mycolactone E from the frog pathogen have one less
CH2=CH2unit in the side chain than mycolactones A/B from
M. ulcerans. The shorter side chain might arise if an entire ex-
tension module (module 4) were missing from the PKS MLSB,
which is responsible for synthesising the side chain of mycolac-
tone A/B,but this remains to be established.
The production of mycolactone F by the fish pathogen
M. marinum DL045 is not accompanied by any significant
amount of the hydroxylated form (expected [M+ +Na]+at m/z
739), which would be produced if a cytochrome P450 acted on
the side chain of mycolactone F. We therefore tested the ability
compared to mycolactone E
Figure 1. MS/MS spectrum of A) mycolactone F at m/z 723 (from the fish pathogen, M. marinum DL045) and B) mycolactone E at m/z 737 (from the frog
pathogen, M. liflandii).
? 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2007, 8, 2043–2047
of the cytochrome P450 Cyp140A7 encoded by MUP_053 from
M. ulcerans to bioconvert mycolactone F into a novel oxidised
mycolactone, when this gene was cloned into M. marinum
DL045. LC-MS analysis of cell pellet extracts from the engi-
neered strain clearly showed that coexpression of the heterolo-
gous cytochrome P450 gave rise to new peaks at m/z 739, 16
mass units higher than mycolactone F (m/z 723), as shown in
Figure 2. LC-MS/MS analysis at m/z 739 resulted in a typical
mycolactone MS/MS spectrum, with characteristic fragment
ions at m/z 429 (ion A) and m/z 333 (ion B), corresponding to
the core lactone and the side chain, respectively (Figure 3).
High-resolution mass analysis confirmed that this novel myco-
lactone is indeed the oxidised product of mycolactone F (des-
ignated as mycolactone G in Table 1).
To identify the exact position of oxidation, deuterium ex-
change and periodate oxidation experiments were performed
on HPLC-purified mycolactone G. If MUP_053 acted in M. mar-
inum DL045 exactly as in M. ulcerans Agy99, a hydroxy group
would be introduced at C10’ of mycolactone F, the position
equivalent to the C12’ in mycolactone A/B. Then, mycolac-
ACHTUNGTRENNUNGtone G should, like mycolactone A/B, have five exchangeable
hydrogens, one more than its precursor mycolactone F, and it
should be cleavable by periodate. However, no periodate
cleavage was observed on mycolactone G under conditions
under which mycolactone A/B was fully converted to its alde-
hyde oxidation product at m/z 675 ([M+ +Na]+). Also, when my-
colactone G was treated with deuterated methanol, only four
instead of five protons were exchanged by deuterium. There-
fore mycolactone G does not bear a hydroxy group at C10’.
Detailed comparisons between the MS/MS spectra of mycolac-
ACHTUNGTRENNUNGtone G (m/z 739) and of mycolactone F (m/z 723; Figure 3)
showed that both peaks gave rise to ion C (at m/z 565) and
ion D (at m/z 579); this suggests that the C1’–C7’ part of the
side chain of mycolactone G is the same as that of mycolac-
ACHTUNGTRENNUNGtone F.[11,13]In contrast, the 120 Da fragment, which corre-
sponds to the loss of 1,3,5-trimethylbenzene from the side
chain, was missing in the MS/MS of mycolactone G; this sug-
gests that the four cumulated double bonds required to
permit loss of substituted benzene from the side chainare
not present. This change also resulted in the absence of frag-
ment ion E at m/z 605.Compared to the MS/MS spectrum of
mycolactone F, the spectrum of mycolactone G (Figure 3) also
showed three new fragment ions at m/z 581, 625 and 711.
High-resolution MS/MS analysis on mycolactone G (m/z 739;
see the Supporting Information) showed that it could lose a
CO molecule to form the fragment ion at m/z 711, a strong in-
dication of an aldehyde group in the side chain. Also, MS3on
the fragment ion at m/z 651, arising from the loss of (C4H8O2,
b-OH-n-butyraldehyde), showed that mycolactone G could
lose C4H8O2from either the core or the side chain, thus indicat-
ing that (like mycolactone F) mycolactone G contains the 1,3-
dihydroxyl C4 unit (C11’-C14’) intact at the end of the side
chain. Therefore, the only possible position that could be oxi-
dised and converted to an aldehyde group is the methyl
branch at C8’. An aldehyde at this position also fits with the
observation of two unique fragment ions at m/z 581 and 625
in the MS/MS spectrum of 739, produced via a hemiacetal in-
termediate (see Supporting Information).
Figure 2. LC-MS analysis of lipid extracts from A) and B) M. marinum DL045; C) and D) M. marinum DL045 in which cytochrome P450 Cyp140A7 is expressed.
The conversion to mycolactone G is signalled by its [M+ +Na]+at m/z 739 compared to mycolactone F ([M+ +Na]+at m/z 723).
ChemBioChem 2007, 8, 2043–2047 ? 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim
To confirm the novel structure of mycolactone G, the puri-
fied compound was treated either with NaBH4 or with O-
treatment with NaBH4, a peak at m/z 741 was observed that
coeluted with the remaining unreacted mycolactone G (m/z
739; data not shown). Fractions containing m/z 741 were col-
lected, lyophilised and analysed by using Fourier transform ion
cyclotron resonance (FTICR) mass spectrometry. The FTICR data
confirmed that m/z 741 was indeed the reduced form of m/z
739 (see Supporting Information). The lyophilised sample was
also treated with deuterated methanol, upon which, as expect-
ed, the reduced mycolactone G at m/z 741 shifted to m/z 746
by exchanging five deuterons, while the remaining unreacted
mycolactone G at m/z 739 shifted to m/z 743 with four ex-
changed deuterons (see the Supporting Information). When
purified mycolactone G was treated with PFBHA, a pentafluoro-
benzyloxime (PFBO) derivative at m/z 934 was observed by LC-
MS; its formula was further confirmed by high-resolution MS
analysis (see the Supporting Information). On the basis of this
evidence taken together, we propose that mycolactone G has
the structure shown in Scheme 1. The relative and absolute
configurations of mycolactones A/B[5–7]and Chave been es-
tablished by total synthesis. For mycolactones E, F and G this
remains to be established. We initially expected that hydroxyl-
ation by the heterologously expressed cytochrome P450
would occur on mycolactone F at C10’, the position equivalent
hydroxylACHTUNGTRENNUNGation of this substrate was found to take place at the
neighbouring methyl branch at C8’, presumably because of
small alterations in the geometry of the enzyme–substrate in-
The biological activities of mycolactone variants were com-
pared in assays of cell cytotoxicity and cytokine production by
human lymphocytes. None of the variants induced detectable
levels of apoptosis, as determined by phosphatidylserine expo-
sure, in Jurkat T-cells incubated with up to 1 mgmL?1mycolac-
tone for 24 h (data not shown). However, when T-cells were ac-
tivated with phorbol 12-myristate-3-acetate (PMA)/ionomycin
after exposure to mycolactone, the production of interleukin
(IL)-2 was repressed in a dose-dependent way by each of the
mycolactones. However, the mycolactone structural variants
differed markedly in their immunosuppressive activity. Myco-
(Scheme 1).Intriguingly, the
Figure 3. MS/MS spectrum of A) mycolactone G at m/z 739 and B) mycolactone F at m/z 723.
Table 1. Comparison of molecular formulae and number of exchangeable protons in mycolactone F from fish pathogen and the engineered
Metabolite I.D. Metabolite [M+ +Na]+
Formula Observed mass Error (ppm)n[a]
Mycolactone F (from M. marinum DL045)
Mycolactone G (from M. marinum DL045 plus MUP_053)
[a] Number of deuterons after exchange.
? 2007 Wiley-VCH Verlag GmbH&Co. KGaA, WeinheimChemBioChem 2007, 8, 2043–2047
lactones A/B proved to be the most potent inhibitor of IL-2 Download full-text
production, thus suggesting that the presence of a hydroxyl
group on C12’ is critical for immunosuppression. Mycolactones
C, E and F showed lower inhibitory effects, while mycolac-
ACHTUNGTRENNUNGtone G was the least active of all (Figure 4).
Microbiological methods: The plasmid pJKD2888 was constructed
in Escherichia coli DH10B by ligating a 1828 bp PCR fragment that
included the 1314 bp cyp140A7 (MUP_053) gene and 514 bp of up-
stream sequence into the unique XbaI site of the mycobacteria/
E. coli shuttle vector pMV261.M. ulcerans strain Agy99 was used
as a source of template DNA, and the PCR was primed by using
the oligonucleotides MUP450F 5’-ggtctagatacacccttaccgcgacagt-3’
and MUP450R 5’-ggtctagaagccagtggcattgtcagat-3’. Electrocompe-
tent Mycobacterium marinum DL045 cells were prepared as de-
scribedand transformed with 5 mg of either pJKD2888 or the
empty vector. Electroporated bacteria were incubated overnight in
Middlebrook 7H9 medium (1 mL) at 308C then plated onto Middle-
brook 7H10 agar, containing kanamycin (25 mgmL?1). Incubation at
308C was continued for 14 days. Kanamycin-resistant colonies
were selected and subcultured into 7H9 medium (500 mL) and in-
cubated at 308C for a further 14 days. Mycobacteria were con-
firmed as harbouring the correct plasmids by back transformation
to E. coli followed by PCR, restriction enzyme digestion and DNA
sequencing of the recovered plasmid. The preparation of cell ex-
tracts for mycolactone analysis was performed as described.
LC-MS analysis: LC-MS and LC-MS/MS analyses were carried out
on a Finnigan LCQ instrument as previously described.High-res-
olution MS and MS/MS analyses were performed on a LTQ Orbitrap
instrument (Thermo Scientific, San Jose, CA, USA) and on a BioA-
pex II (4.7 Tesla) FTICR mass spectrometer (Bruker Daltonics, Billeri-
ca, MA, USA).
Assay of biological activity: The human T-cell line Jurkat (sub-
clone E6.1) was cultured in RPMI medium with 10% foetal calf
serum, l-glutamine (2 mm), penicillin (100 IUmL?1) and streptomy-
cin (100 mgmL?1). Cells were incubated in microtitre plates (5?105
cells per mL) with mycolactone preparations (0 to 500 ngmL?1) for
6 h, then activatedwith PMA (25 ngmL?1)
(500 ngmL?1; both from Calbiochem, La Jolla, CA) for 16 h. Culture
supernatants were assayed for IL-2 by ELISA (R&D, Minneapolis,
We gratefully acknowledge the financial support of the National
Health and Medical Research Council of Australia (T.S.) and of
the Wellcome Trust (079241/z/06/z; H.H. and P.F.L.).
Keywords: Buruli ulcer · cytochromes · genetic engineering ·
Mycobacterium ulcerans · mycolactones
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Received: July 23, 2007
Published online on September 28, 2007
Figure 4. Differential immunosuppressive activity of mycolactone variants.
IL-2 concentrations were measured in culture supernatants of Jurkat Tcells
activated after treatment with mycolactones or mycolactone mixtures:~:
AB, ^: AB+30% C (AB+C),~: C+30% AB (C+AB), &: E, *: F, *: G, or ^:
ethanol as solvent control. The data are the mean ?SD of duplicate meas-
ACHTUNGTRENNUNGurements, and are representative of two independent experiments.
ChemBioChem 2007, 8, 2043–2047? 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim