Cell Wall Lipids Promoting Host Angiogenesis During Mycobacterial Infection

Abstract

Mycobacterial infection leads to the formation of characteristic immune cell aggregates called granulomas. In humans and animal models, tuberculous granuloma formation is accompanied by dramatic remodeling of host vasculature which ultimately benefits the infecting mycobacteria, suggesting the bacteria may actively drive this host process. First, we sought to identify bacterial factors that promote granuloma vascularization. Using Mycobacterium marinum transposon mutants in a zebrafish infection model, we revealed the enzyme Proximal Cyclopropane Synthase of alpha-Mycolates (PcaA) as an important bacterial determinant of host angiogenesis. We found that PcaA-modified trehalose dimycolate, an abundant glycolipid in the mycobacterial cell wall, drives activation of host VEGF signaling and subsequent granuloma vascularization. To facilitate our continuing investigation of granuloma dynamics, we next sought to expand and improve upon the transgenic tools for studying macrophages in the zebrafish model. I describe two such tools: i) the macrophage-specific zebrafish mfap4 promoter, which allows long-term in vivo visualization and manipulation of macrophages during mycobacterial infection, and ii) the first zebrafish transgenic line with constitutive, ubiquitous Cas9 expression, as well as a transgene design capable of generating sgRNAs using macrophage-specific promoters. These tools allow CRISPR/Cas9 gene editing in vivo in the zebrafish in a macrophage-restricted manner.