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Cell Rep. 2018 Apr 24;23(4):1072-1084. doi: 10.1016/j.celrep.2018.03.125.

Multiplexed Quantitation of Intraphagocyte Mycobacterium tuberculosis Secreted Protein Effectors.

Author information

1
Institut Pasteur, Unit for Integrated Mycobacterial Pathogenomics, CNRS UMR 3525, 25 rue du Dr. Roux, Paris 75015, France.
2
Institut Pasteur, Unit for Molecular Virology and Vaccinology, 28 rue du Dr. Roux, Paris 75015, France.
3
Université de Lille, CNRS UMR 8204, INSERM U1019, CHU Lille, Institut Pasteur de Lille, Center for Infection and Immunity of Lille (CIIL), 1 rue du Professeur Calmette, 59000 Lille, France.
4
Institut Pasteur, Neonatal Immunity Group, Unit for Human Histopathology and Animal Models, 28 rue du Dr. Roux, Paris 75015, France.
5
Institut Pasteur, Unit for Integrated Mycobacterial Pathogenomics, CNRS UMR 3525, 25 rue du Dr. Roux, Paris 75015, France; Sorbonne Universités, UPMC University of Paris 06, CIMI-Paris, AP-HP, Hôpital Pitié-Salpêtrière, CNR-MyRMA, Paris, France.
6
Sorbonne Universités, UPMC University of Paris 06, CIMI-Paris, AP-HP, Hôpital Pitié-Salpêtrière, CNR-MyRMA, Paris, France.
7
University of Pisa, Department of Biology, via S. Zeno 35-39, 56127 Pisa, Italy.
8
Institut Pasteur, Unit for Integrated Mycobacterial Pathogenomics, CNRS UMR 3525, 25 rue du Dr. Roux, Paris 75015, France. Electronic address: laleh.majlessi@pasteur.fr.

Abstract

The pathogenic potential of Mycobacterium tuberculosis largely depends on ESX secretion systems exporting members of the multigenic Esx, Esp, and PE/PPE protein families. To study the secretion and regulation patterns of these proteins while circumventing immune cross-reactions due to their extensive sequence homologies, we developed an approach that relies on the recognition of their MHC class II epitopes by highly discriminative T cell receptors (TCRs) of a panel of T cell hybridomas. The latter were engineered so that each expresses a unique fluorescent reporter linked to specific antigen recognition. The resulting polychromatic and multiplexed imaging assay enabled us to measure the secretion of mycobacterial effectors inside infected host cells. We applied this novel technology to a large panel of mutants, clinical isolates, and host-cell types to explore the host-mycobacteria interplay and its impact on the intracellular bacterial secretome, which also revealed the unexpected capacity of phagocytes from lung granuloma to present mycobacterial antigens via MHC class II.

KEYWORDS:

T-cell hybridomas; bacterial antigen presentation; intracellular bacteria; in vivo antigen presentation; lentiviral vectors; mycobacterial virulence factors; mycobacterium tuberculosis; protein localization; reporter T cells; type VII secretion systems

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