Membrane perturbing properties of toxin mycolactone from <i>Mycobacterium ulcerans</i>

<div><p>Mycolactone is the exotoxin produced by <i>Mycobacterium ulcerans</i> and is the virulence factor behind the neglected tropical disease Buruli ulcer. The toxin has a broad spectrum of biological effects within the host organism, stemming from its interaction with at least two molecular targets and the inhibition of protein uptake into the endoplasmic reticulum. Although it has been shown that the toxin can passively permeate into host cells, it is clearly lipophilic. Association with lipid carriers would have substantial implications for the toxin’s distribution within a host organism, delivery to cellular targets, diagnostic susceptibility, and mechanisms of pathogenicity. Yet the toxin’s interactions with, and distribution in, lipids are unknown. Herein we have used coarse-grained molecular dynamics simulations, guided by all-atom simulations, to study the interaction of mycolactone with pure and mixed lipid membranes. Using established techniques, we calculated the toxin’s preferential localization, membrane translocation, and impact on membrane physical and dynamical properties. The computed water-octanol partition coefficient indicates that mycolactone prefers to be in an organic phase rather than in an aqueous environment. Our results show that in a solvated membrane environment the exotoxin mainly localizes in the water-membrane interface, with a preference for the glycerol moiety of lipids, consistent with the reported studies that found it in lipid extracts of the cell. The calculated association constant to the model membrane is similar to the reported association constant for Wiskott-Aldrich syndrome protein. Mycolactone is shown to modify the physical properties of membranes, lowering the transition temperature, compressibility modulus, and critical line tension at which pores can be stabilized. It also shows a tendency to behave as a linactant, a molecule that localizes at the boundary between different fluid lipid domains in membranes and promotes inter-mixing of domains. This property has implications for the toxin’s cellular access, T-cell immunosuppression, and therapeutic potential.</p></div

Mycolactone in model lipid membrane.

<p><b>A)</b> All-atom and coarse-grained representations of diC16-PC (cyan and green) and mycolactone (cyan and yellow). The atomistic representation is characterized by an 8-undecenolide region (blue box), the C12-C20 northern fragment (red box), and the pentanoic acid ester southern fragment (yellow box). The CG representation can be described in terms of the head and tail regions. Different bead types (N1-N13) capture the general topology of the CG resolution, according to the definition of MARTINI force field (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005972#sec007" target="_blank">Methods</a>). <b>B)</b> The CG set-up of pure diC16-PC bilayer system in combination with 5% mycolactone. Notice that the simulation box is enclosed by the gray square. <b>C)</b> Cross section snapshot of the equilibrated membrane simulation. For clarity, diC16-PC lipid has been depicted by its head group (orange), the glycerol moiety (red) and the aliphatic tails (dark gray). <b>D)</b> Histograms correspond to probability distribution of two common configurations of mycolactone in lipid bilayer; either at the surface (black) or spanning the bilayer (red). The distributions are calculated as function of the distance between CG beads N1 and N13 for the bilayer system with 5% mycolactone as shown in the set-up of panel B. The dashed lines show the accumulation of population of these two configurations. Insets in both C and D (enclosed in circles) show representative configurations from all atom MD simulations.</p

Mycolactone acts as a linactant.

<p><b>A)</b> Spontaneous Lo (green lipids)-Ld (red lipids) domain formation in a ternary lipid system (diC16-PC, diC18:2-PC and cholesterol) at 4:3:3 lipid ratio. <b>B)</b> The addition of 5% mycolactone, however, decreases the line tension between the domains (see main text). <b>C)</b> Electron density profile along the interface of both domains. Clearly the higher peak corresponding to mycolactone (yellow line) is localized in the interface of both domains. Green and red lines highlight the electron-density profiles for the ordered and disordered lipids (diC16-PC and diC18:2-PC respectively). The white line corresponds to the electron-density of cholesterol within the Lo and Ld domains. <b>D)</b> Radial distribution function of mycolactone with the center of mass of ordered lipids-saturated tails (green line) and disordered lipids-unsaturated tails (red line) clearly show a slight preference for the disordered region, which is expressed by the free energy difference between the red and green (black dashed line). The radial distributions were also split by considering the head <b>(E)</b> and tail <b>(F)</b> regions of mycolactone, suggesting that both regions prefer the Ld region.</p

Mycolactone modifies different physical properties of a lipid membrane.

<p><b>A)</b> gel-liquid transition temperature for a pure diC16-PC lipid bilayer (black line) and when combined with 5% mycolactone (red line). The transition is given as the change in area per lipid. As previously referenced[<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005972#pcbi.1005972.ref040" target="_blank">40</a>], in the pure diC16-PC membrane, our data shows that gel-liquid transition occurs at ~295 K and with a mean area per lipid of 0.6 nm². However, the addition of mycolactone drops this temperature by ~ 5 K. <b>B)</b> Pure diC16-PC lipid bilayer under 60 mN/m surface tension. The membrane thins, but no pore formation is observed. <b>C)</b> The addition of 5% mycolactone reduces its resistance to stretching, leading to the formation of a pore with the concomitant rupture of the bilayer at 55 mN/m surface tenstion. Once formed pores were observed to be stabilized at ~ 20 mN/m tension. Pore formation was observed in several independent simulations.</p

Summary of the CG MD simulations carried out in this study.

<p>Summary of the CG MD simulations carried out in this study.</p

Tunable Riboregulator Switches for Post-transcriptional Control of Gene Expression

Until recently, engineering strategies for altering gene expression have focused on transcription control using strong inducible promoters or one of several methods to knock down wasteful genes. Recently, synthetic riboregulators have been developed for translational regulation of gene expression. Here, we report a new modular synthetic riboregulator class that has the potential to finely tune protein expression and independently control the concentration of each enzyme in an engineered metabolic pathway. This development is important because the most straightforward approach to altering the flux through a particular metabolic step is to increase or decrease the concentration of the enzyme. Our design includes a <i>cis</i>-repressor at the 5′ end of the mRNA that forms a stem-loop helix, occluding the ribosomal binding sequence and blocking translation. A <i>trans</i>-expressed activating-RNA frees the ribosomal-binding sequence, which turns on translation. The overall architecture of the riboregulators is designed using Watson–Crick base-pairing stability. We describe here a <i>cis</i>-repressor that can completely shut off translation of antibiotic-resistance reporters and a <i>trans</i>-activator that restores translation. We have established that it is possible to use these riboregulators to achieve translational control of gene expression over a wide dynamic range. We have also found that a targeting sequence can be modified to develop riboregulators that can, in principle, independently regulate translation of many genes. In a selection experiment, we demonstrated that by subtly altering the sequence of the <i>trans</i>-activator it is possible to alter the ratio of the repressed and activated states and to achieve intermediate translational control

Neutron and Atomic Resolution X‑ray Structures of a Lytic Polysaccharide Monooxygenase Reveal Copper-Mediated Dioxygen Binding and Evidence for N‑Terminal Deprotonation

A 1.1 Å resolution, room-temperature X-ray structure and a 2.1 Å resolution neutron structure of a chitin-degrading lytic polysaccharide monooxygenase domain from the bacterium <i>Jonesia denitrificans</i> (<i>Jd</i>LPMO10A) show a putative dioxygen species equatorially bound to the active site copper. Both structures show an elongated density for the dioxygen, most consistent with a Cu­(II)-bound peroxide. The coordination environment is consistent with Cu­(II). In the neutron and X-ray structures, difference maps reveal the N-terminal amino group, involved in copper coordination, is present as a mixed ND₂ and ND^–, suggesting a role for the copper ion in shifting the p<i>K</i>_a of the amino terminus

Precise Genomic Riboregulator Control of Metabolic Flux in Microbial Systems

Engineered microbes can be used for producing value-added chemicals from renewable feedstocks, relieving the dependency on nonrenewable resources such as petroleum. These microbes often are composed of synthetic metabolic pathways; however, one major problem in establishing a synthetic pathway is the challenge of precisely controlling competing metabolic routes, some of which could be crucial for fitness and survival. While traditional gene deletion and/or coarse overexpression approaches do not provide precise regulation, cis-repressors (CRs) are RNA-based regulatory elements that can control the production levels of a particular protein in a tunable manner. Here, we describe a protocol for a generally applicable fluorescence-activated cell sorting technique used to isolate eight subpopulations of CRs from a semidegenerate library in Escherichia coli, followed by deep sequencing that permitted the identification of 15 individual CRs with a broad range of protein production profiles. Using these new CRs, we demonstrated a change in production levels of a fluorescent reporter by over two orders of magnitude and further showed that these CRs are easily ported from E. coli to Pseudomonas putida. We next used four CRs to tune the production of the enzyme PpsA, involved in pyruvate to phosphoenolpyruvate (PEP) conversion, to alter the pool of PEP that feeds into the shikimate pathway. In an engineered P. putida strain, where carbon flux in the shikimate pathway is diverted to the synthesis of the commodity chemical cis,cis-muconate, we found that tuning PpsA translation levels increased the overall titer of muconate. Therefore, CRs provide an approach to precisely tune protein levels in metabolic pathways and will be an important tool for other metabolic engineering efforts