Macrophage vacuolar ATPase (v-ATPase) function controls Aspergillus fumigatus germination and hyphal growth independent of spore killing

Abstract

Tissue-resident macrophages efficiently internalize Aspergillus fumigatus spores, forming a critical first line of defense against infection. However, the mechanisms that these cells use to control spores in vivo remain incompletely defined. Here, we used the live imaging capabilities of the larval zebrafish host model to assess the role of the v-ATPase complex in macrophage-mediated defense against A. fumigatus in a whole vertebrate animal. For the first time we are able to visualize co-localization of A. fumigatus spores with the key v-ATPase subunit Atp6v1h in macrophages inside of an infected animal. As macrophages only have a low ability to kill spores, this co-localization occurs as early as 1-day post-injection and persists for multiple days. Surprisingly, macrophage spore killing is not further reduced by targeting of atp6v1h with CRISPR/Cas9. Instead, v-ATPase deficiency profoundly impacts macrophage-mediated control of spore swelling, decoupling the macrophage functions of spore killing and inhibition of germination. We also identify a role for the v-ATPase complex in macrophage control of extracellular hyphal growth. These effects on macrophage function drive significantly decreased host survival in larvae lacking a functional v-ATPase. We also report broad effects of v-ATPase deficiency on macrophage numbers, apoptosis in the hematopoietic tissue, and potential neutrophil functions, reflecting the importance of this complex in host antifungal immunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/664761v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@d494f2org.highwire.dtl.DTLVardef@7ecc81org.highwire.dtl.DTLVardef@a45584org.highwire.dtl.DTLVardef@ea5cd3_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIv-ATPase function in macrophages restricts Aspergillus germination and hyphal growth C_LIO_LISpore killing by macrophages is independent of v-ATPase function C_LIO_LILoss of macrophage v-ATPase mimics the effects of macrophage deficiency in vivo C_LI