A fast, fully automated cell segmentation algorithm for high-throughput and high-content screening

Fenistein, D.,Lenseigne, B.,Christophe, T.,Brodin, P.,Genovesio, A. Wiley Subscription Services, Inc., A Wiley Company 2008 Cytometry. the journal of the International Societ Vol.a73 No.10

<P>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 Cytometry</P>

High Content Screening Identifies Decaprenyl-Phosphoribose 2′ Epimerase as a Target for Intracellular Antimycobacterial Inhibitors

Christophe, Thierry,Jackson, Mary,Jeon, Hee Kyoung,Fenistein, Denis,Contreras-Dominguez, Monica,Kim, Jaeseung,Genovesio, Auguste,Carralot, Jean-Philippe,Ewann, Fanny,Kim, Eun Hye,Lee, Sae Yeon,Kang, S Public Library of Science 2009 PLoS pathogens Vol.5 No.10

<▼1><P>A critical feature of <I>Mycobacterium tuberculosis</I>, 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 <I>M. tuberculosis</I> 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 <I>M. tuberculosis</I>, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of <I>M. tuberculosis</I> 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.</P></▼1><▼2><P><B>Author Summary</B></P><P>Tuberculosis is still a major threat to global health. The disease in humans is caused by a bacterium, <I>Mycobacterium tuberculosis</I>, and treatment of an infected individual requires more than six months of chemotherapy. Because such a long course of treatment is required, compliance is low, which can result in the development of multidrug resistant strains (MDR-TB) and even extremely resistant strains (XDR-TB). Identifying new drug targets and potential lead therapeutic compounds are needed to combat MDR-XDR-TB. We developed a new type of assay based on the visualization of mycobacterium replication within host cells and applied it for the search of compounds that are able to chase the pathogen from its hideout. As a result, we found 20 new series of drug candidates that are effective against the bacilli in its hiding place, potentially addressing a crucial aspect in the resilience of the disease. We also showed that one series of compounds acts by inhibiting a key enzyme required for the synthesis of an essential component from the mycobacterial cell wall that is not targeted by any of the commercially available antituberculosis drugs. Altogether, our results pave the way for development of the next generation of antibacterial agents.</P></▼2>

Mycobacterial Toxin Induces Analgesia in Buruli Ulcer by Targeting the Angiotensin Pathways

Marion, E.,Song, O.R.,Christophe, T.,Babonneau, J.,Fenistein, D.,Eyer, J.,Letournel, F.,Henrion, D.,Clere, N.,Paille, V.,Guerineau, Nathalie C.,Saint Andre, J.P.,Gersbach, P.,Altmann, K.H.,Stinear, T. Cell Press ; MIT Press 2014 Cell Vol.157 No.7

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 (AT<SUB>2</SUB>Rs), 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

Kim, Jiho,Lee, Honggun,Lee, Joo Hyun,Kwon, Do-yoon,Genovesio, Auguste,Fenistein, Denis,Ogier, Arnaud,Brondani, Vincent,Grailhe, Regis American Society for Biochemistry and Molecular Bi 2014 The Journal of biological chemistry Vol.289 No.21

<P>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. <I>In vitro</I> studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and <I>in vivo</I> 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.</P>

High Content Phenotypic Cell-Based Visual Screen Identifies <i>Mycobacterium tuberculosis</i> Acyltrehalose-Containing Glycolipids Involved in Phagosome Remodeling

Brodin, Priscille,Poquet, Yannick,Levillain, Florence,Peguillet, Isabelle,Larrouy-Maumus, Gerald,Gilleron, Martine,Ewann, Fanny,Christophe, Thierry,Fenistein, Denis,Jang, Jichan,Jang, Mi-Seon,Park, Se Public Library of Science 2010 PLoS pathogens Vol.6 No.9

<▼1><P>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 <I>Mycobacterium tuberculosis</I> 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 <I>M. tuberculosis</I>. 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 (<I>fadD28</I>), the ESX-1 secretion system (<I>espL</I>/Rv3880), molybdopterin biosynthesis (<I>moaC1</I> and <I>moaD1</I>), 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.</P></▼1><▼2><P><B>Author Summary</B></P><P>One of the major virulence mechanisms of the tuberculosis bacillus, <I>Mycobacterium tuberculosis</I>, is its ability to resist killing by phagocytic cells of the host immune system, namely the macrophages. Macrophages degrade invading microbes by engulfment inside a vacuole, or phagosome, that progressively acidifies and accumulates hydrolytic properties. <I>M. tuberculosis</I> has the unique ability to block phagosome maturation and acidification. To identify mycobacterial genes involved in phagosome maturation arrest, we developed a novel high-throughput technology based on automated confocal microscopy. We screened a library containing over 11,000 <I>M. tuberculosis</I> mutants, and we could identify 10 mutants that had lost their ability to resist phagosome acidification. Genetic characterization of these mutants revealed that they carried lesions in genes involved in various cell processes, including biogenesis of the cell envelope. In particular, two independent mutants in the same genetic locus showed altered production of two lipids, namely diacyltrehalose (DAT) and sulfoglycolipid (SGL). <I>In vitro</I> experiments showed that SGL can indeed influence phagosome maturation. Our study unravels the role of novel lipid molecules in mycobacterial intracellular parasitism; our approach may be useful to identify virulence genes in other intracellular pathogens, and to identify novel antimicrobials.</P></▼2>