[R21] Deciphering Phagosomal Membrane Repair Pathways during Mycobacterium tuberculosis Infection Using High-Throughput Single Cell and Single Phagosome Analysis

Ente: National Institute of Allergy and Infectious Diseases

Scadenza: 2028-04-30

Importo max: 234.000 EUR

Paese: US

Descrizione

Phagosomal membrane repair is a cellular process that prevents pathogen escape and host cell death, which are key to the virulence of intracellular pathogens such as Mycobacterium tuberculosis (Mtb). Different effectors and pathways contribute to the repair of the phagosome, such as the ESCRT machinery and an endoplasmic reticulum-dependent membrane repair mechanism. Concomitantly, the induction of damages on the phagosome triggers autophagy, which is also suspected to contribute to the repair of the phagosome. The knowledge of the host cell mechanisms of phagosome damage and repair in Mtb-infected human macrophages is incomplete. Our previous results have shown dynamic recruitment of LC3 to damaged phagosomal membranes, and the loss of Lysoview staining as a readout for membrane damage, indicating active repair processes. Time-lapse microscopy has proven to be an efficient method to directly visualize and decipher membrane damage and repair of the Mycobacterium-Containing Vacuole (MCV). In the first specific aim (SA1) we will expand upon our initial analysis of the spatio-temporal analysis of MCV membrane repair by including novel host cell marker proteins, investigate the process in primary human monocyte-derived macrophages (hMDMs) and develop novel bioinformatics tools to improve image analysis. In SA2 we will perform a targeted genetic screen for host proteins involved in MCV membrane repair via siRNA knock-down in THP-1 cells and the results will be confirmed in hMDMs. SA3 will investigate the phagosomal membrane damage and repair dynamics in response to different mycobacterial species which differ in their virulence factors involved in inducing phagosomal damage. Istituzione: UNIV OF MARYLAND, COLLEGE PARK PI: VOLKER BRIKEN Progetto: 1R21AI193440-01A1

Settori: National Institute of Allergy and Infectious Diseases

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