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>

Effect of Maternal Immune Status on Responsiveness of Bacillus Calmette-Gurin Vaccination in Mouse Neonates

Choi, Jong Su,Kim, Ryang Yeo,Rho, Semi,Ewann, Fanny,Mielcarek, Nathalie,Song, Man Ki,Czerkinsky, Cecil,Kim, Jae-Ouk Korea Centers for Disease Control and Prevention 2012 Osong Public Health and Research Persptectives Vol.3 No.2

<P><B>Objectives</B></P><P>Bacillus Calmette-Guérin (BCG) vaccination has proven to be efficient in immunologically naïve infants; however, it has not been investigated that maternal natural exposure to <I>Mycobacterium</I> and/or BCG vaccine could influence the characteristics of immune responses to BCG in newborns. In this study, we analyzed whether the maternal immune status to <I>M tuberculosis </I>(<I>M tb</I>) can affect neonatal immunity to BCG using a mouse model.</P><P><B>Methods</B></P><P>Neonates were obtained from mice that were previously exposed to live BCG, to live <I>M avium</I>, or to heat-killed <I>M tb</I> H37Rv, and from naïve control mothers. One week after birth, the neonates were divided into two subgroups: one group immunized with live BCG via the subcutaneous route and the other group of neonates sham-treated. Interferon-gamma (IFNγ) secretion in response to <I>in vitro </I>stimulation with heat-killed BCG or purified protein derivative (PPD) was examined. Protection against <I>M tb</I> infection was evaluated by challenging mice nasally with live <I>M tb</I> H37Rv followed by counting colonies from spleen and lung homogenates.</P><P><B>Results</B></P><P>BCG-immunized neonates showed increased IFNγ secretion in response to heat-killed BCG or PPD. All mice in BCG-immunized neonates subgroups showed reduced bacterial burden (colony forming unit) in the lungs when compared with control naive neonate mice. However, no statistically significant difference was observed when comparing BCG-immunized mice born from mothers previously exposed to <I>M avium</I> or immunized with either heat-killed H37Rv or live BCG and mice born from naïve mothers.</P><P><B>Conclusion</B></P><P>The maternal immune status to <I>M tb</I> does not appear to impact on the immunogenicity of BCG vaccine in their progeny in our experimental conditions</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>

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>