Multiplexed Quantitation of Intraphagocyte Mycobacterium tuberculosis Secreted Protein Effectors

Area of Science:

• Microbiology
• Immunology
• Cell Biology

Background:

• Mycobacterium tuberculosis pathogenesis relies on ESX secretion systems.
• ESX systems export diverse protein families (Esx, Esp, PE/PPE) crucial for virulence.
• Studying these proteins is challenging due to sequence homology and immune cross-reactivity.

Purpose of the Study:

• To develop a novel method for studying ESX secretion and regulation patterns.
• To circumvent immune cross-reactivity issues in analyzing mycobacterial effector proteins.
• To investigate the host-mycobacteria interplay and its effect on the intracellular bacterial secretome.

Main Methods:

• Engineered T-cell hybridomas expressing unique fluorescent reporters linked to specific antigen recognition.
• Utilized T-cell receptors (TCRs) to recognize MHC class II epitopes of mycobacterial proteins.
• Developed a polychromatic and multiplexed imaging assay to measure effector secretion in infected host cells.

Main Results:

• Successfully measured the secretion of mycobacterial effectors within host cells.
• Applied the assay to analyze mutants, clinical isolates, and various host cell types.
• Discovered that lung granuloma phagocytes can present mycobacterial antigens via MHC class II.

Conclusions:

• The novel assay effectively measures mycobacterial effector secretion and host-cell interactions.
• This technology provides a powerful tool for exploring the complex host-mycobacteria interplay.
• The findings highlight an unexpected role for granuloma phagocytes in antigen presentation.