No abstract

Pathogen interactions with immune cells like macrophages contribute to pathogenesis, yet the mechanisms underlying these interactions remain largely undefined. For emerging respiratory pathogens, like Mycobacterium abscessus , understanding these host-pathogen interactions is important to fully understand disease progression.

The interactions between a host cell and a pathogen can dictate disease outcomes and are important targets for host-directed therapies.Mycobacterium abscessus(Mab) is a highly antibiotic resistant, rapidly growing non-tuberculous mycobacterium that infects patients with chronic lung diseases. Mab can infect host immune cells, such as macrophages, which contribute to its pathogenesis. However, our understanding of initial host-Mab interactions remains unclear. Here, we developed a functional genetic approach to define these host-Mab interactions by coupling a Mab fluorescent reporter with a genome-wide knockout library in murine macrophages. We used this approach to conduct a forward genetic screen to define host genes that contribute to the uptake of Mab by macrophages. We identified known regulators of phagocytosis, such as the integrin ITGB2, and uncovered a key requirement for glycosaminoglycan (sGAG) synthesis for macrophages to efficiently take up Mab. CRISPR-Cas9 targeting of three key sGAG biosynthesis regulators, Ugdh, B3gat3 and B4galt7 resulted in reduced uptake of both smooth and rough Mab variants by macrophages. Mechanistic studies suggest that sGAGs function upstream of pathogen engulfment and are required for the uptake of Mab, but notEscherichia colior latex beads. Further investigation found that the loss of sGAGs reduced the surface expression, but not the mRNA expression, of key integrins suggesting an important role for sGAGs in modulating surface receptor availability. Together, these studies globally define and characterize important regulators of macrophage-Mab interactions and are a first step to understanding host genes that contribute to Mab pathogenesis and disease.

Phagocytosis by macrophages is critical to eradicate invading pathogens. Phagocytosis is regulated by parallel networks that control the binding and uptake of particles, subsequent lysosome delivery, and activation of downstream innate immune responses. However, whether distinct host pathways drive unique responses to targeted substrates within the lung environment remains unknown. Mycobacterium abscessus (MAB) is a rapidly growing non-tuberculous mycobacterium that infects patients with chronic lung diseases, like cystic fibrosis. While interactions between MAB and macrophages contribute to its pathogenesis, how macrophages bind, phagocytose, and control MAB remains entirely unclear. We hypothesize that understanding key MAB-macrophage interactions will identify critical host targets that can be inhibited to prevent disease. To test this hypothesis, using a genome-wide knockout library of immortalized bone marrow derived macrophages, we completed a forward genetic screen to identify host pathways that contribute to fluorescent MAB uptake. Our results show glycosaminoglycan synthesis (sGAGs) in macrophages is required to efficiently take up MAB. Both enzymatic and genetic approaches found key sGAG biosynthesis regulators, UGDH, B3GLCT, B4GALT7 and B3GAT3, influence macrophage interactions with MAB and other pathogens like Mycobacterium tuberculosis. Using a recently developed alveolar macrophage model, we are now dissecting the role of sGAGs in lung specific macrophages. Future work will determine how sGAG pathways modulate bacterial replication, cytokine responses and disease progression following MAB infection with the goal of defining key mechanisms driving host-pathogen interactions in the lungs.