Introduction Mycolactone is a lipid-like polyketide macrolide virulence factor produced by Mycobacterium ulcerans, the infectious agent of Buruli ulcer (BU) [1], [2]. This progressive, necrotizing, cutaneous lesion is common in West Africa but also found in other regions, including Australia, Asia and South America. Mycolactone is a key factor in BU pathology: possession of a plasmid carrying enzymes involved in mycolactone synthesis is essential for virulence and injection of mycolactone alone can reproduce many characteristics of the infection, including ulceration, necrosis and suppression of immune responses [1], [3]. Mycolactone has been shown to have diverse effects on a range of cells and tissues but a unifying mechanism underlying its pleiotropic actions has remained elusive. In vitro, exposure to pure mycolactone is cytotoxic for many cell lines, but the dose and exposure required is highly variable ([4] and references therein) and primary immune cells (including T-cells, monocytes and macrophages) are considerably more resistant [5], [6]. While early evidence from cell lines implicated G1/G0 growth arrest and apoptosis [7], recent work showed that a more likely mechanism driving cell death in vivo is anoikis due to direct binding of mycolactone to the Wiskott-Aldrich Syndrome Protein (WASP), leading to inappropriate activation of WASP and relocalisation of the actin nucleating complex Arp2/3 [8]. This disrupts the cytoskeleton, altering cell adhesion and migration. Detachment of monolayer cells is a common feature of the mycolactone response and precedes cell death by up to 48 hours. One of the most striking characteristics of BU lesions is an almost complete absence of inflammation despite extensive tissue damage. In ulcerated lesions, where large amounts of mycolactone are produced by foci of extracellular bacilli, inflammatory cell infiltration is limited to the periphery [9]–[11]. Infection is accompanied by alterations in local and systemic immune responses in which mycolactone plays a central role [11]–[14], via direct and indirect effects on T-cells, dendritic cells, monocytes and macrophages [5], [15]–[17]. Mycolactone interferes with T-cell activation, down-regulating expression of the T-cell receptor and reducing IL-2 production in response to activating signals [15], [17], [18]. Lymphocyte homing is also impaired due to suppression of L-selectin and LFA-1 levels, leading to a dramatic depletion of T-cells in peripheral lymph nodes [6]. In monocyte-derived dendritic cells, mycolactone inhibits the production of costimulatory molecules (such as CD40 and CD86). In addition, secretion of various cytokines and chemokines is blocked and mycolactone treated dendritic cells show a reduced ability to activate T-cells [16]. The innate immunity provided by monocytes and macrophages is also suppressed by mycolactone. Tissue resident macrophages normally play a central role in mycobacterial infections. However, M. ulcerans differs from other pathogenic mycobacteria in that, except in very early infection, the vast majority of bacilli are not found within the host macrophage but are located extracellularly. Mycolactone inhibits key macrophage responses such as nitric oxide production and phagocytosis as well as phagosome maturation and acidification [2], [4], [19]. In addition, mycolactone prevents the induction of many proteins essential for driving inflammation, including TNF, other cytokines/chemokines (for example, IL-6, IL-8 and IP-10), and further inflammatory mediators (such as the prostaglandin synthetase Cox-2) [5], [10], [15]. There is good evidence that mycolactone diffuses through the lesion in advance of the proliferating bacilli and the necrotic centre (see for example [20]). Therefore, understanding exactly how this compound mediates its diverse immunosuppressive and cytotoxic effects on cells surrounding the developing lesion is crucial. As outlined above, many of these effects involve loss of expression of specific proteins, both induced and constitutive, such as inflammatory mediators. Consequently, the same molecular mechanism that prevents inflammatory protein production in the macrophage may also explain the inadequate protein production more generally. This makes it an excellent model system with which to examine the basic cell biology of mycolactone function, since the response is inducible by nature and it is therefore straightforward to separate new protein synthesis from baseline levels. We have previously shown that inducible inflammatory mediator production is inhibited by a post-transcriptional mechanism, since mycolactone does not modulate the LPS-dependent activation of ERK, JNK, p38 MAPK or NFκB and induced levels of mRNA are maintained or even enhanced [5]. However there is no significant decrease in total protein synthesis, nor are phosphorylation patterns of Akt, p70S6K, eIF4E and eIF2α changed; a finding confirmed in another model system, Jurkat T-cells [17]. In the current manuscript we demonstrate conclusively that mycolactone does not selectively inhibit translation as predicted [2], [5], and instead blocks co-translational translocation into the ER. This leads to the rapid degradation of mislocalised proteins in the cytosol and hence loss of detectable expression. We show that the production of nearly all new glycosylated and secreted proteins ceases following mycolactone exposure, not only in macrophages but in fibroblasts, epithelial and endothelial cells. This mechanism therefore provides the necessary explanation for many of the pleiotropic effects of this unique molecule and accounts for much of the underlying disease pathology. Results Mycolactone does not inhibit the translation of proinflammatory mRNAs In order to establish the dose of synthetic mycolactone A/B required to completely inhibit the production of TNF in RAW264.7 cells, we carried out a dose response (Fig. 1A). It was determined that the effective dose was 125 ng/ml, and this also prevented LPS-dependent Cox-2 production without affecting cell viability (Fig. S1A). This dose is marginally higher than required for inhibition of TNF production by natural mycolactone A/B in primary human macrophages (Fig. S1B), probably reflecting the known variation in sensitivity between different cell types, preparations of mycolactone (natural vs. synthetic) and/or target activities (immunosuppressive vs. cytotoxic). We then performed polysome profiling of macrophages to investigate whether mycolactone selectively inhibits the translation of inflammatory mediators. This technique allows the association of TNF, IL-6 and Cox-2 transcripts with actively translating polysomes in various experimental conditions to be assessed. RAW264.7 cells were used because, in preliminary experiments, the low mRNA yields and high RNase content of primary human monocytes and macrophages precluded the use of these cells (data not shown). The post-transcriptional mechanism of mycolactone-dependent inhibition of cytokine production observed in primary cells is conserved in this cell line (Fig. S1C, performed as a control experiment for all profiles obtained). 10.1371/journal.ppat.1004061.g001 Figure 1 Mycolactone does not change the polysomal association of proinflammatory mRNAs. A. RAW264.7 cells were incubated for 1+/−various concentrations of mycolactone (MYC as indicated), 0.5 µg/ml Actinomycin D (Act D) or 0.0125% DMSO then stimulated or not with LPS for 4 hr. Supernatant TNF levels were measured by ELISA (mean±SEM of triplicate assays). B–D. RAW264.7 cells were incubated for 1 hr+/−125 ng/ml mycolactone (MYC) then stimulated or not with LPS. After 4 hrs cells were harvested and lysed in the presence of CHX. B and C. Polysomes were separated on a 10–50% sucrose gradient and the profiles measured by absorbance at 254 nm. Note the increase in the 60S peak and reduced height of the polysome peaks in MYC and LPS+MYC samples. RNA was purified from gradient fractions and transcripts were detected by Northern blotting using full coding region cDNA probes for the genes indicated. D. Signal intensity was quantified by ImageJ analysis of non-saturated phosphorscreen images. Values are presented as percentage of total signal for control (dashed line), LPS (solid black line) and LPS+MYC (red line) cells. All are representative of 3 independent experiments. PABP; poly A-tract binding protein. Mycolactone exposure was found to consistently cause a change in the shape of the polysome profiles, associated with an increase in the size of the 60S peak and change in the profile in the area associated with heavy polysomes in both unstimulated (Fig. 1B; MYC) and stimulated (Fig. 1C; LPS+MYC) cells. However, these changes occurred gradually over several hours, while the inhibition of TNF production is manifest as little as 20 min after LPS addition (data not shown). This suggests it may be a secondary, rather than primary, effect. Mycolactone alone did not influence the quantity or location of TNF mRNA (not shown) and LPS stimulation in itself did not induce any gross changes to the polysome profiles (Fig. 1C). In each profile, poly-A tract binding protein (PABP) and β-actin are used as control transcripts that confirm the location of unformed ribosomes and polysomes, respectively (Fig. 1C). While unstimulated RAW264.7 cells expressed very little TNF mRNA, as expected, LPS stimulation led to increased abundance of TNF, IL-6 and Cox-2 mRNAs and their location moved so that a higher proportion of the mRNAs were in the polysomal fractions (Fig. 1C, compare ‘control’ and ‘LPS’ - and quantified in Fig. 1D), due to the known translational derepression that occurs following stimulation [21]. Again, as expected, neither the location of β-actin (known to be mycolactone insensitive [5]) or PABP were affected by mycolactone (Fig. 1B and D). However, in stark contrast to expectations, mycolactone had no effect on the polysomal association of any of the three inflammatory transcripts; all remained in heavy-polysomal fractions (Fig. 1B, compare ‘LPS’ and LPS+MYC’). When quantitated, the distribution of the mRNAs was very similar in the presence and absence of mycolactone (Fig. 1D). We confirmed this unexpected finding in a number of ways. First, the localisation of these transcripts was assessed at various times after LPS stimulation to investigate whether the findings were influenced by the kinetics of the LPS response or time of mycolactone exposure (>1 hr), but this was found not to be the case (data not shown). Second, we examined the effects of short term exposure to two translation-inhibiting drugs on polysome profiles (Fig. 2A and B). Puromycin (PURO) causes premature termination and ribosomal release from translating mRNAs, whereas homoharringtonine (HH) prevents translation initiation leading to ribosome run-off of translating mRNAs) [22], [23]. Neither drug influenced the production of TNF or its inhibition by mycolactone (Fig. S2), but both caused a change in the profiles obtained from LPS stimulated cells, with HH being the more efficient (Fig. 2A). As expected, there was a concomitant change in the location of β-actin mRNA to monosomes (HH) or lighter polysomes (PURO) (Fig. 2B, left panel LPS, compare the black with the blue or green lines respectively). Cox-2 and TNF mRNAs also both moved into lighter polysomal fractions, confirming that our experimental system was sensitive to inhibition of translation. 10.1371/journal.ppat.1004061.g002 Figure 2 Proinflammatory mRNAs are actively translating in the presence of mycolactone. RAW264.7 cells were incubated for 1+/−125 ng/ml mycolactone (MYC), then stimulated with LPS. A and B. After 4 hr 100 µg/ml puromycin (PURO) or 5 µM homoharringtonine (HH) were added for 3 min, then CHX was added before lysis and separation of polysomes on a 10–20% sucrose gradient. LPS (solid black line), LPS+PURO (solid green line) and LPS+HH (dotted blue line). A. RNA profiles measured by absorbance at 254 nm. B. Quantitation of specific mRNAs purified from each fraction and analysed by Northern blotting. Signal intensity was quantified by ImageJ analysis of non-saturated phosphorscreen images. Values are presented as percentage of total signal. C and D. Cytosolic and digitonin-resistant membrane fractions were prepared from treated cells as described. C. Western blot of cell fractions (0.5×105 cell equivalents/lane). GCS1; glucosidase I (∼92 kDa), GAPDH (∼40 kDa). D. total RNA was used as a template in qRT-PCR absolute quantitation assays and is presented as % total RNA for each gene (mean±SEM). All data representative of 3 independent experiments. When the action of these drugs on mycolactone treated cells was assessed, it could be seen that, while mycolactone altered the profiles but not the location of β-actin, Cox-2 or TNF transcripts as before (Fig. 2A and 2B, black lines), the response to PURO and HH was the same in the absence and presence of mycolactone. For Cox-2, PURO caused a similar ∼2-fraction shift (Fig. 2B, green lines), whereas HH causes a similar ∼1-fraction shift (Fig. 2B, blue lines) in both untreated and mycolactone treated cells. It is interesting to note that the shift in the Cox-2 peaks following HH treatments were smaller than that seen for β-actin, suggesting that Cox-2 is being translated more slowly (compare the blue lines in Fig. 2B, LPS, Actin and Cox-2). Both drugs had a less marked effect on TNF but a movement of the peak of mRNA recovery to lower fractions could be seen that was not prevented by mycolactone. This shows that all of the tested mRNAs are undergoing active translation in both the presence and absence of mycolactone and are not stalled on the ribosomes. Finally, in an independent approach, the cellular localisation of proinflammatory mRNAs in the presence of mycolactone was also examined. Since TNF and IL-6 are secreted proteins and Cox-2, is ER-resident and contains a signal peptide, their actively-translating, nascent polypeptide chains should be directly associated with the ER due to the interaction of the signal peptide with the signal recognition particle (SRP) and Sec61 complex [24]. Cells were selectively permeabilised with digitonin to separate the cytosolic and digitonin-resistant ER membrane fractions. Western blotting showed the presence GAPDH protein in the cytosol while the ER-resident protein glucosidase I (GCS1) was confined to the membrane fraction (Fig. 2C). As seen by others, GAPDH mRNA was fairly evenly distributed between cytosolic and membrane fractions [25], [26], but the mRNAs for TNF, Cox-2 and IL-6 were all predominantly in the membrane fraction, even in the presence of mycolactone, indicating sufficient synthesis had occurred to allow signal peptide recognition (Fig. 2D). This data also strongly argues against an inhibition of proinflammatory mRNA translation as the mechanism underlying the loss of protein production due to mycolactone. Inhibition of the 26S proteasome allows the cellular expression of pro-TNF and Cox-2 in the presence of mycolactone As proinflammatory protein synthesis is maintained in the absence of detectable protein levels, mechanisms by which these proteins might be targeted for degradation by the cell were investigated. Degradation by the 26S proteasome seemed a likely candidate. However, examining this experimentally is complex for inflammatory mediators since their transcriptional activation requires proteasome-dependent degradation of IκBα [27]. Cells were therefore stimulated with LPS for 2 hrs prior to addition of the proteasome inhibitor (PSI) for an additional 2 hrs. This did not decrease LPS-dependent production of TNF in cell supernatants (Fig. 3A) indicating that this experimental design was satisfactory. 10.1371/journal.ppat.1004061.g003 Figure 3 Mycolactone causes degradation of TNF and Cox-2 by the 26S proteasome in the cytosol. RAW264.7 cells were incubated +/−125 ng/ml mycolactone (MYC) or 5 µg/ml tunicamycin (TUN) for 1 hr and stimulated with LPS for 4 hrs. In certain samples 5 µM PSI was added 2 hrs after the LPS stimulation. This allowed time for the proteasome-dependent activation of NFκB required for transcriptional activation to occur before proteasome activity was inhibited (see text). A. Supernatant TNF levels from cells treated as above, measured by ELISA (mean±SEM). B. Western blot of cell lysates (0.5×106 cell equivalents/lane), the dotted line separates samples with and without PSI treatment for ease of interpretation. C. Signal intensity was quantified by ImageJ analysis of non-saturated blots and normalised to LPS+TUN (not shown). Values are mean intensity for each lane±SEM of 3 independent experiments. For Cox-2 this is the unglycosylated mol wt band only (UG Cox-2). *, P 1.0) and low (normalised intensity <1.0) abundance, respectively B. Pixel intensity was determined using ImageJ software and normalised according to the positive control signals. Values represent the mean of duplicate spots ± range. The asterisk (*) represents proteins that are produced constitutively by RAW264.7 cells C. Raw data for the arrays; reference spots (used to orient the array and to normalise between arrays) are boxed. Proteins produced by LPS stimulated cells are indicated for reference only; orange; strong intensity, yellow, weaker intensity. Note that expression is relative between treatments and differences in intensity between proteins are not necessarily quantitative. D. RAW264.7 cells were incubated +/−125 ng/ml mycolactone for 1 hr then stimulated with LPS for overnight. TNF and MIP-1α levels were measured by ELISA. (TIF) Click here for additional data file. Information S1 Extended experimental procedures. Contains a full list of reagents, primers and antibodies as well as detailed protocols including those for polysome preparation, digitonin permeabilisation and ConA precipitation. (PDF) Click here for additional data file.
