Effector-mediated subversion of proteasome activator (PA)28αβ enhances lysosomal targeting of Legionella pneumophila within cytokine-activated macrophages

Abstract

AbstractLegionella pneumophila is a natural pathogen of protozoa that causes Legionnaires’ Disease pneumonia via replication within host macrophages using an arsenal of hundreds of translocated virulence factors termed effector proteins. Effectors are critical for intracellular replication but can also enhance pathogen clearance by mammalian hosts via effector-triggered immunity. The effector LegC4 confers a fitness disadvantage on L. pneumophila in mouse models of Legion-naires’ Disease and uniquely potentiates the antimicrobial activity of macrophages activated with either tumor necrosis factor (TNF) or interferon (IFN)-γ. Here, we investigated the mechanism by which LegC4 enhances macrophage restriction of L. pneumophila and found a central role for the induced proteasome activator (PA)28αβ. PA28αβ facilitates ubiquitin-independent pro-teasomal degradation of oxidant-damaged proteins to relieve oxidative stress and impaired proteasome activity results in compensatory upregulation of lysosomal degradation pathways. We found that LegC4 binds the conserved PA28α subunit and that the LegC4 restriction phenotype is abolished within PA28αβ-deficient macrophages. Moreover, LegC4 impaired the resolution of oxidative proteotoxic stress and enhanced cytokine-dependent phagolysosomal fusion with the Legionella-containing vacuole. PA28αβ has been traditionally associated with antigen presentation and adaptive immunity; however, our data support a model whereby suppression of PA28αβ by LegC4 culminates in the cell-autonomous lysosomal killing of L. pneumophila within activated macrophages. This work provides a solid foundation on which to evaluate induced proteasome regulators as mediators of innate immunity.Significance StatementPro-inflammatory cytokines induce antimicrobial host defense pathways within macrophages to control intracellular pathogens. We discovered that the Legionella pneumophila effector protein LegC4 potentiates pathogen clearance within cytokine-activated macrophages. Here, we investigated LegC4 function and found that host proteasome activator (PA)28αβ was required for LegC4 restriction within macrophages. PA28αβ is cytokine-induced and facilitates proteasomal degradation of oxidant-damaged proteins. We found that LegC4 binds PA28α and impairs the resolution of oxidative stress. Loss of proteasome activity causes compensatory upregulation of lysosomal degradation pathways and, concomitantly, LegC4 increases phagolysosomal fusion with Legionella-containing vacuoles. These findings support a model whereby pathogen subversion of host proteostasis machinery triggers lysosomal pathogen targeting within activated macrophages.