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.

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.

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.

Background: Copper/zinc superoxide dismutase (SOD1) genetic mutants are associated with familial amyotrophic lateral sclerosis (ALS). Mutant proteins form abnormal aggregates. Results: We used imaging of live cells to observe SOD1 proteins harboring mutations associated with ALS. Conclusion: SOD1 mutations impair its dimerization, leading to subsequent aggregation. Significance: Analysis of the SOD1 quaternary structure in living human cells correlates with previous biochemical data.

High-throughput, high-content screening (HT-HCS) of large compound libraries for drug discovery imposes new constraints on image analysis algorithms. Time and robustness are paramount while accuracy is intrinsically statistical. In this article, a fast and fully automated algorithm for cell segmentation is proposed. The algorithm is based on a strong attachment to the data that provide robustness and have been validated on the HT-HCS of large compound libraries and different biological assays. We present the algorithm and its performance, a description of its advantages and limitations, and a discussion of its range of application. ' 2008 International Society for Advancement of CytometryKey terms image analysis; biological image processing; automation; cytometry; object detection; segmentation; high-content screening AUTOMATED fluorescent microscopy and high-performance computing have allowed the emergence of high-content screening (HCS) as a useful tool in the early stages of drug discovery (1-4). The multidimensional information (''high content'' in HCS) allows for the tackling of biological models inaccessible to unidimensional high-throughput screening (HTS). HCS can also measure multiple effects in a single experiment; for example, the effect of a drug on bacteria (virus, receptor, etc.) and its toxicity on the host target (1,4). HCS therefore has a potential to become a risk/delay/ cost reducer for the later stages of drug development. The last few years have seen a huge increase in image acquisition capacity. Automated fluorescent microscopes can now record more than 40,000 images a day (90 Gb/day), and do so for weeks at a time. HCS is therefore truly becoming high throughput. This evolution introduces fundamental differences with former HCS approaches: (i) The number of images acquired during a HT-HCS campaign [%half a million (4)] requires a fully automated image analysis. (ii) For image analysis not to become the bottleneck of HT-HCS, the rate of image acquisition (%2 s per image) imposes the rate of image analysis. (iii) The amount of data forbids visual control. Results have meaning in a statistical way and must be weighted against a statistically based acceptance criteria. (iv) The measure of quality for a HT-HCS algorithm is a trade off between speed on the one hand and accuracy and robustness on the other. PREVIOUS WORKNumerous HT-HCS applications require a stage of cell segmentation. It most often serves as a basis for subsequent operations, more diverse and specific to a biological assay. While manual (5,6) or semiautomatic (7) cell segmentation methods may be used for HCS, the need for speed and repeatability forbids them for HT-HCS. Most of the cell segmentation algorithms commonly used in HCS suffer from one or many drawbacks that make them ill adapted to HT-HCS use. They may be slow when compared with the