Legionella Lem26 functions as an ATG8-activated effector that inhibits host autophagy

TL;DR

The Legionella protein Lem26 inhibits host autophagy by interacting with autophagosomal membranes and modifying key autophagy proteins.

Contribution

Lem26 is identified as a novel Legionella effector that inhibits autophagy through ATG8-dependent activation and ADP-ribosylation.

Findings

Lem26 blocks autophagy in yeast and mammalian cells by inhibiting core autophagy protein recruitment.

Lem26's ADP-ribosyltransferase activity is activated by binding to autophagosomal membranes containing ATG8.

Modification of autophagy proteins by Lem26 arrests the autophagy pathway.

Abstract

The intracellular pathogen Legionella pneumophila has evolved multiple effector proteins delivered into host cells by the Dot/Icm Type IVb secretion system that prevents recognition of the vacuole in which it resides by the host autophagy pathway. The number of effectors involved in this process remains unclear. Thus, we conducted a screen in Saccharomyces cerevisiae to identify Legionella effectors that were sufficient to block autophagy. This screen identified the Legionella protein Lem26 as an effector capable of autophagy inhibition. Lem26 production inhibited the recruitment of core autophagy proteins to autophagic targets and prevented the proteolytic processing of autophagy substrates in both yeast and mammalian systems. The Lem26 protein encodes an ADP-ribosyltransferase (ART) domain that was found to be essential for anti-autophagy activity. In vitro studies showed that purified Lem26 was inactive in solution, but the addition of pre-autophagosomal membranes obtained from fractionated mammalian cell lysates stimulated Lem26 ART activity. The addition of synthetic membranes containing lipid-conjugated ATG8 proteins was sufficient to stimulate Lem26 activity in vitro. An ATG8-interacting motif identified in Lem26 was critical for the activation of Lem26. These data establish that Lem26 is a Legionella effector that is recruited and activated upon interaction with autophagic membranes, and this promotes the posttranslational modification of proteins on the autophagic membrane to arrest the autophagy pathway.

Bacterial pathogens have evolved intricate mechanisms to specifically avoid detection by the host autophagy pathway, which is a cell-autonomous innate immune pathway conserved in all eukaryotic organisms. The intracellular pathogen Legionella pneumophila has co-evolved with evolutionarily diverse protozoan hosts for over 100 million years. Thus, these bacteria have devised multiple strategies for evading host autophagy. In this study, we analyzed roughly 300 different Legionella effector proteins for their ability to disrupt autophagy in yeast. The Legionella effector protein Lem26 was found to specifically block autophagy in both yeast and mammalian cells. Biochemical studies revealed that this protein is tightly regulated and is activated upon binding to autophagosomal membranes, which stimulates Lem26 ADP-ribosyltransferase activity and results in the modification of critical autophagy proteins colocalized to these membranes. Thus, Lem26 has evolved the capacity to disrupt host autophagy by proximity labeling of host determinants on autophagosomal membranes, which represents a unique strategy for autophagy inhibition.

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