Molecular mechanisms underlying the promotion of antibacterial immunity by LC3-associated phagocytosis

• Applicant: Professor Dr. Olaf Utermöhlen, since 4/2021
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 432240111

• Final Report Year: 2024

Final Report Abstract

In light of the increasing resistance of pathogenic bacteria to antibiotics, novel therapeutic approaches are desperately needed. One approach to identify, characterize, develop and implement novel antibacterial therapies is to further the understanding of the antibacterial mechanisms of the immune system. Based on a thorough understandig of molecular and cellular antibacterial immune mechanisms, the aim is to activate or enhance these mechanisms by therapeutic treatments during infections of patients with bacteria. In this research project, non-canonical autophagy pathways of antibacterial defence were characterized in murine macrophages. It is known for a long time that macrophage-like cell lines, bone marrow-derived macrophages (BMDM), and primary tissue macrophages from mice distinctly differ in the type and efficacy of their antimicrobial response mechanisms against the opportunistic pathogen Listeria monocytogenes. Therefore, the two noncanonical autophagy pathways LC3-associated phagocytosis (LAP) and pore-forming toxininduced non-canonical autophagy (PINCA) were characterized side by side in BMDM versus primary macrophages. In non-canonical autophagy, single-membrane vesicles are equipped with Atg8-family proteins such as microtubule-associated protein 1 light chain 3 (MAP1LC3 or shortly LC3). Phagosomes containing the bacterial pathogen Listeria monocytogenes (L.m.) can be targeted by LAP, which substantially contributes to the anti-listerial activity of macrophages. In adjustment to a competitive publication about LAP in infection with L. monocytogenes, PINCA as the second non-canonical autophagy pathway targeting L.m.-containing phagosomes was characterized in detail in this project. This pathway is induced by perforation of the phagosomal membrane by the pore-forming toxin of L.m., listeriolysin O. BMDM exclusively targeted L.m. by PINCA as they completely failed to induce LAP, because of insufficient production of ROS. Similar to LAP, the targeting of L.m.-containing phagosomes by PINCA promoted their fusion with lysosomes. Surprisingly, however, this did not substantially contribute to antilisterial activity of BMDM. Thus, in contrast to LAP, PINCA does not have an obvious antilisterial function, suggesting that the two different non-canonical autophagy pathways targeting L.m. may have discrete functions. The specific functions of PINCA during L.m. infection remain to be elucidated. The insights gained by this project substantially further our understanding of the immune response against pathogenic bacteria. A fundamental understanding of the mechanisms contributing to antibacterial immunity may enable the development of approaches for the immunotherapy of bacterial infections.

Publications

• Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA. Journal of Visualized Experiments(153).
  Herb, Marc; Farid, Alina; Gluschko, Alexander; Krönke, Martin & Schramm, Michael
  
• LC3-associated phagocytosis - The highway to hell for phagocytosed microbes. Seminars in Cell & Developmental Biology, 101, 68-76.
  Herb, Marc; Gluschko, Alexander & Schramm, Michael
  
• The TSPO-NOX1 axis controls phagocyte-triggered pathological angiogenesis in the eye. Nature Communications, 11(1).
  Wolf, Anne; Herb, Marc; Schramm, Michael & Langmann, Thomas
  
• 3D Printing of Antibacterial, Biocompatible, and Biomimetic Hybrid Aerogel-Based Scaffolds with Hierarchical Porosities via Integrating Antibacterial Peptide-Modified Silk Fibroin with Silica Nanostructure. ACS Biomaterials Science & Engineering, 7(9), 4545-4556.
  Karamat-Ullah, Nighat; Demidov, Yan; Schramm, Michael; Grumme, Daniela; Auer, Jaqueline; Bohr, Christoph; Brachvogel, Bent & Maleki, Hajar
  
• Functions of ROS in Macrophages and Antimicrobial Immunity. Antioxidants, 10(2), 313.
  Herb, Marc & Schramm, Michael
  
• Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1. Autophagy, 17(1), 1-382.
  Klionsky, Daniel J.; Abdel-Aziz, Amal Kamal; Abdelfatah, Sara; Abdellatif, Mahmoud; Abdoli, Asghar; Abel, Steffen; Abeliovich, Hagai; Abildgaard, Marie H.; Abudu, Yakubu Princely; Acevedo-Arozena, Abraham; Adamopoulos, Iannis E.; Adeli, Khosrow; Adolph, Timon E.; Adornetto, Annagrazia; Aflaki, Elma; Agam, Galila; Agarwal, Anupam; Aggarwal, Bharat B.; Agnello, Maria ... & Tong, Chun-Kit
  
• Macrophages target Listeria monocytogenes by two discrete non-canonical autophagy pathways. Autophagy, 18(5), 1090-1107.
  Gluschko, Alexander; Farid, Alina; Herb, Marc; Grumme, Daniela; Krönke, Martin & Schramm, Michael
  
• NOX2 Deficiency Permits Sustained Survival of S. aureus in Macrophages and Contributes to Severity of Infection. Frontiers in Immunology, 12.
  Tosetti, Bettina; Ward, Beate; Grumme, Daniela; Herb, Marc; Schramm, Michael; Utermöhlen, Olaf; Heukamp, Lukas C.; Krönke, Martin & Krut, Oleg
  
• Reactive Oxygen Species: Not Omnipresent but Important in Many Locations. Frontiers in Cell and Developmental Biology, 9.
  Herb, Marc; Gluschko, Alexander & Schramm, Michael
  
• When the Phagosome Gets Leaky: Pore-Forming Toxin-Induced Non-Canonical Autophagy (PINCA). Frontiers in Cellular and Infection Microbiology, 12.
  Herb, Marc; Gluschko, Alexander; Farid, Alina & Krönke, Martin