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>

Impact of <i>Mycobacterium ulcerans</i> Biofilm on Transmissibility to Ecological Niches and Buruli Ulcer Pathogenesis

Marsollier, Laurent,Brodin, Priscille,Jackson, Mary,Kordulá,ková,, Jana,Tafelmeyer, Petra,Carbonnelle, Etienne,Aubry, Jacques,Milon, Geneviè,ve,Legras, Pierre,André,, Jean-Paul Public Library of Science 2007 PLoS pathogens Vol.3 No.5

<▼1><P>The role of biofilms in the pathogenesis of mycobacterial diseases remains largely unknown. <I>Mycobacterium ulcerans,</I> the etiological agent of Buruli ulcer, a disfiguring disease in humans, adopts a biofilm-like structure in vitro and in vivo, displaying an abundant extracellular matrix (ECM) that harbors vesicles. The composition and structure of the ECM differs from that of the classical matrix found in other bacterial biofilms. More than 80 proteins are present within this extracellular compartment and appear to be involved in stress responses, respiration, and intermediary metabolism. In addition to a large amount of carbohydrates and lipids, ECM is the reservoir of the polyketide toxin mycolactone, the sole virulence factor of M. ulcerans identified to date, and purified vesicles extracted from ECM are highly cytotoxic. ECM confers to the mycobacterium increased resistance to antimicrobial agents, and enhances colonization of insect vectors and mammalian hosts. The results of this study support a model whereby biofilm changes confer selective advantages to M. ulcerans in colonizing various ecological niches successfully, with repercussions for Buruli ulcer pathogenesis.</P></▼1><▼2><P><B>Author Summary</B></P><P><B/></P><P>Mycobacterium ulcerans is the etiologic agent of Buruli ulcer, a necrotic skin disease affecting humans living close to wetlands in tropical countries. This mycobacteria resides in water where it could colonize many ecological niches such as aquatic plants, herbivorous animals, and water bugs. The latter were shown to be able to transmit the bacteria to mammalian hosts. Here, we described that the bacilli could be structured with a thick envelope called the extracellular matrix (ECM). This peculiar coat contains in small vesicles a toxin named mycolactone, the main virulence factor of M. ulcerans. The ECM confers to the mycobacterium increased resistance to antimicrobial agents and plays a role in virulence. Indeed, a bacteria with ECM is more potent for colonization of insect vectors and mammalian hosts compared to bacteria. Unraveling the regulation of the production of the ECM together with the export of mycolactone will be an important step in developing new pharmacological approaches for the treatment of Buruli ulcer, which has been greatly handicapped by the lack of effectiveness of the current antibiotics.</P></▼2>

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 imaging of Mycobacterium tuberculosis-infected macrophages: an in vitro model for tuberculosis drug discovery.

Christophe, Thierry,Ewann, Fanny,Jeon, Hee Kyoung,Cechetto, Jonathan,Brodin, Priscille Future Science 2010 Future medicinal chemistry Vol.2 No.8

<P>Macrophages are reservoirs for replicating mycobacterium during tuberculosis (TB) infections. In this study, small molecules to be developed as anti-tubercular treatments were investigated for their ability to kill intracellular bacteria in in vitro macrophage models. High-content imaging technologies offer a high-throughput method to quantify a drug's ability to inhibit Mycobacterium tuberculosis intracellular invasion and multiplication in host cells. Dedicated image analysis enables the automated quantification of infected macrophages, and compounds that inhibit mycobacteria proliferation can be tested using this method. Furthermore, the implementation of the assay in 384-well microtiter plates combined with automated image acquisition and analysis allows large-scale screening of compound libraries in M. tuberculosis-infected macrophages. Here we describe a high-throughput and high-content workflow and detail its utility for the development of new TB drugs.</P>

Systematic Genetic Nomenclature for Type VII Secretion Systems

Bitter, Wilbert,Houben, Edith N. G.,Bottai, Daria,Brodin, Priscille,Brown, Eric J.,Cox, Jeffery S.,Derbyshire, Keith,Fortune, Sarah M.,Gao, Lian-Yong,Liu, Jun,Gey van Pittius, Nicolaas C.,Pym, Alexand Public Library of Science 2009 PLoS pathogens Vol.5 No.10