A genome-wide screen in macrophages defines host genes regulating the uptake ofMycobacterium abscessus

Abstract

ABSTRACTThe 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 B4galt7resulted 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.IMPORTANCEPathogen interactions with immune cells like macrophages contribute to pathogenesis, yet the mechanisms underlying these interactions remain largely undefined. For emerging respiratory pathogens, likeMycobacterium abscessus, understanding these host-pathogen interactions is important to fully understand disease progression. Given thatM. abscessusis broadly recalcitrant to antibiotic treatments, new therapeutic approaches are needed. Here, we leveraged a genome-wide knockout library in murine macrophages to globally define host genes required forM. abscessusuptake. We identified new macrophage uptake regulators duringM. abscessusinfection, including a subset of integrins and the glycosaminoglycan synthesis (sGAG) pathway. While ionic characteristics of sGAGs are known to drive pathogen-cell interactions, we discovered a previously unrecognized requirement for sGAGs to maintain robust surface expression of key uptake receptors. Thus, we developed a flexible forward-genetic pipeline to define important interactions duringM. abscessusinfection and more broadly identified a new mechanism by which sGAGs control pathogen uptake.