Mycobacterial toxin induces analgesia in buruli ulcer by targeting the angiotensin pathways.

Mycobacterium ulcerans, the etiological agent of Buruli ulcer, causes extensive skin lesions, which despite their severity are not accompanied by pain. It was previously thought that this remarkable analgesia is ensured by direct nerve cell destruction. We demonstrate here that M. ulcerans-induced hypoesthesia is instead achieved through a specific neurological pathway triggered by the secreted mycobacterial polyketide mycolactone. We decipher this pathway at the molecular level, showing that mycolactone elicits signaling through type 2 angiotensin II receptors (AT2Rs), leading to potassium-dependent hyperpolarization of neurons. We further validate the physiological relevance of this mechanism with in vivo studies of pain sensitivity in mice infected with M. ulcerans, following the disruption of the identified pathway. Our findings shed new light on molecular mechanisms evolved by natural systems for the induction of very effective analgesia, opening up the prospect of new families of analgesics derived from such systems.

Dimerization, oligomerization, and aggregation of human amyotrophic lateral sclerosis copper/zinc superoxide dismutase 1 protein mutant forms in live cells.

More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. In vitro studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and in vivo studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins.

Identification of a novel sulfonamide non-nucleoside reverse transcriptase inhibitor by a phenotypic HIV-1 full replication assay.

Classical target-based, high-throughput screening has been useful for the identification of inhibitors for known molecular mechanisms involved in the HIV life cycle. In this study, the development of a cell-based assay that uses a phenotypic drug discovery approach based on automated high-content screening is described. Using this screening approach, the antiviral activity of 26,500 small molecules from a relevant chemical scaffold library was evaluated. Among the selected hits, one sulfonamide compound showed strong anti-HIV activity against wild-type and clinically relevant multidrug resistant HIV strains. The biochemical inhibition, point resistance mutations and the activity of structural analogs allowed us to understand the mode of action and propose a binding model for this compound with HIV-1 reverse transcriptase.

High content phenotypic cell-based visual screen identifies Mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling.

The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials.

Development of a high-content screening assay to identify compounds interfering with the formation of the hepatitis C virus replication complex.

The hepatitis C virus (HCV) replicates its genome on a membrane-associated replication complex. These complexes are represented by "dot-like" structures on the endoplasmic reticulum when standard fluorescence microscopy techniques are applied. To screen compound libraries for inhibitors interfering with the formation of the HCV replication complex independent of RNA replication, an image-based high-content screening assay was developed utilizing inducible expression of the HCV non-structural proteins NS3-5B in an U2-OS Tet-On cell line. An eGFP was fused to NS5A for the detection of replication complexes. The cell line was tightly regulated and the eGFP insertion within NS5A did not alter polyprotein processing. The NS5AeGFP signal colocalized with other non-structural proteins in "dot-like" structures. Accompanying image analysis tools were developed enabling the detection of changes in replication complex formation. Finally, the addition of a HCV NS3/4A protease inhibitor resulted in a dose-dependent reduction of "dot-like" structures demonstrating the practicability of the assay.

High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors.

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2' epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

Core precession and global modes in granular bulk flow.

We report a novel transition to core precession for granular flows in a split-bottomed shear cell. This transition is related to a qualitative change in the 3D flow structure: For shallow layers of granular material, the shear zones emanating from the split reach the free surface, while for deep layers the shear zones meet below the surface, causing precession. The surface velocities reflect this transition by a change of symmetry. As a function of layer depth, we find that three qualitatively different smooth and robust granular flows can be created in this simple shearing geometry.

Universal and wide shear zones in granular bulk flow.

We present experiments on slow granular flows in a modified (split-bottomed) Couette geometry in which wide and tunable shear zones are created away from the sidewalls. For increasing layer heights, the zones grow wider (apparently without bound) and evolve towards the inner cylinder according to a simple, particle-independent scaling law. After rescaling, the velocity profiles across the zones fall onto a universal master curve given by an error function. We study the shear zones also inside the material as a function of both their local height and the total layer height.

What controls the thickness of wetting layers near bulk criticality?

We study the thickness of wetting layers in the binary-liquid mixture cyclohexane methanol. Far from the bulk critical point, the wetting layer thickness is independent of temperature, resulting from the competition between van der Waals and gravitational forces. Upon approaching the bulk critical temperature [t=(T(c)-T)/T(c)-->0], we observe that the wetting layer thickness diverges as t(-beta) with effective critical exponent beta=0.23+/-0.06. This is characteristic of a broad, intermediate scaling regime for the crossover from van der Waals wetting to critical scaling. We predict beta=beta/3 approximately 0.11, with beta the usual bulk-order parameter critical exponent, showing a small but significant difference with experiment.