CD8+ T cells specific for a potential HLA-A*0201 epitope from Chlamydophila pneumoniae are present in the PBMCs from infected patients

Infection with the common pathogen Chlamydophila pneumoniae (Cpn, previously Chlamydia pneumoniae) has a high prevalence in patients suffering from arteriosclerosis and may trigger or contribute to heart disease. In mice, CD8-positive T cells are critical for the eradication of the infection and the development of immune memory against Cpn. Although several H2-class I epitopes have been described, no HLA-class I-associated peptides from Cpn are known. In order to define HLA-A*0201 epitopes from Cpn, we focused on the bacterial heat shock proteins (HSP) 60 and 70 which are known to be recognized by the immune system. Using epitope prediction, peptide binding studies and peptide-specific CTLs from HLA-A2 transgenic mice, we could define a potential HSP-70-derived epitope. The study of PBMCs from Cpn-infected individuals using fluorescent MHC tetramers revealed that some patients have CD8+ T cells capable of recognizing the Cpn HSP-70 HLA-A*0201 epitope. Our studies pave the way to the immunomonitoring of the anti-Cpn CTL immune response present in patients suffering from different diseases potentially linked to Cpn or anti-Cpn immunit

MultiBacMam Bimolecular Fluorescence Complementation (BiFC) tool-kit identifies new small-molecule inhibitors of the CDK5-p25 protein-protein interaction (PPI)

Abstract Protein-protein interactions (PPIs) are at the core of virtually all biological processes in cells. Consequently, targeting PPIs is emerging at the forefront of drug discovery. Cellular assays which closely recapitulate native conditions in vivo are instrumental to understand how small molecule drugs can modulate such interactions. We have integrated MultiBacMam, a baculovirus-based mammalian gene delivery tool we developed, with bimolecular fluorescence complementation (BiFC), giving rise to a highly efficient system for assay development, identification and characterization of PPI modulators. We used our system to analyze compounds impacting on CDK5-p25 PPI, which is implicated in numerous diseases including Alzheimer’s. We evaluated our tool-kit with the known inhibitor p5T, and we established a mini-screen to identify compounds that modulate this PPI in dose-response experiments. Finally, we discovered several compounds disrupting CDK5-p25 PPI, which had not been identified by other screening or structure-based methods before

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\u27 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

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

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

Light it up: Highly Efficient Multigene Delivery in Mammalian Cells

Multigene delivery and expression systems are emerging as key technologies for many applications in contemporary biology. We have developed new methods for multigene delivery and expression in eukaryotic hosts for a variety of applications, including production of protein complexes for structural biology and drug development, provision of multicomponent protein biologics and for cell-based assays. We implemented tandem recombineering to facilitate rapid generation of multicomponent gene expression constructs for efficient transformation of mammalian cells, resulting in homogenous cell populations. Analysis of multiple parameters in living cells may require co-expression of fluorescently tagged sensors simultaneously in a single cell, at defined and ideally controlled ratios. Our method enables such applications by overcoming currently limiting challenges. Here, we review recent multigene delivery and expression strategies and their exploits in mammalian cells. We discuss applications in drug discovery assays, interaction studies and biologics production, which may benefit in the future from our novel approach

Evolution of Physico-Chemical and Microbiological Parameters During Large-Scale Coffee-Pulp Silage

International audienc

A novel specific edge effect correction method for RNA interference screenings.

International audienceMOTIVATION: High-throughput screening (HTS) is an important method in drug discovery in which the activities of a large number of candidate chemicals or genetic materials are rapidly evaluated. Data are usually obtained by measurements on samples in microwell plates and are often subjected to artefacts that can bias the result selection. We report here a novel edge effect correction algorithm suitable for RNA interference (RNAi) screening, because its normalization does not rely on the entire dataset and takes into account the specificities of such a screening process. The proposed method is able to estimate the edge effects for each assay plate individually using the data from a single control column based on diffusion model, and thus targeting a specific but recurrent well-known HTS artefact. This method was first developed and validated using control plates and was then applied to the correction of experimental data generated during a genome-wide siRNA screen aimed at studying HIV-host interactions. The proposed algorithm was able to correct the edge effect biasing the control data and thus improve assay quality and, consequently, the hit-selection step