When microbes contaminate the macrophage cytoplasm, leukocytes undergo a proinflammatory death that is initiated by nucleotide-binding-domain-, leucine-rich-repeat-containing proteins (NLR proteins) that bind and activate caspase-1. We report that these inflammasome components also regulate autophagy, a vesicular pathway to eliminate cytosolic debris. In response to infection with flagellate Legionella pneumophila, C57BL/6J mouse macrophages equipped with caspase-1 and the NLR proteins NAIP5 and NLRC4 stimulated autophagosome turnover. A second trigger of inflammasome assembly, K+ efflux, also rapidly activated autophagy in macrophages that produced caspase-1. Autophagy protects infected macrophages from pyroptosis, since caspase-1-dependent cell death occurred more frequently when autophagy was dampened pharmacologically by either 3-methyladenine or an inhibitor of the Atg4 protease. Accordingly, in addition to coordinating pyroptosis, both (pro-) caspase-1 protein and NLR components of inflammasomes equip macrophages to recruit autophagy, a disposal pathway that raises the threshold of contaminants necessary to trigger proinflammatory leukocyte death.IMPORTANCE An exciting development in the innate-immunity field is the recognition that macrophages enlist autophagy to protect their cytoplasm from infection. Nutrient deprivation has long been known to induce autophagy; how infection triggers this disposal pathway is an active area of research. Autophagy is encountered by many of the intracellular pathogens that are known to trigger pyroptosis, an inflammatory cell death initiated when nucleotide-binding-domain-, leucine-rich-repeat-containing proteins (NLR proteins) activate caspase-1 within inflammasome complexes. Therefore, we tested the hypothesis that NLR proteins and caspase-1 also coordinate autophagy as a barrier to cytosolic infection. By exploiting classical bacterial and mouse genetics and kinetic assays of autophagy, we demonstrate for the first time that, when confronted with cytosolic contamination, primary mouse macrophages rely not only on the NLR proteins NAIP5 and NLRC4 but also on (pro-)caspase-1 protein to mount a rapid autophagic response that wards off proinflammatory cell death.

During phagocytosis, the phosphoinositide content of the activated membrane decreases sharply, as does the associated surface charge, which attracts polycationic proteins. The cytosolic leaflet of the plasma membrane is enriched in phosphatidylserine (PS); however, a lack of suitable probes has precluded investigation of the fate of this phospholipid during phagocytosis. We used a recently developed fluorescent biosensor to monitor the distribution and dynamics of PS during phagosome formation and maturation. Unlike the polyphosphoinositides, PS persists on phagosomes after sealing even when other plasmalemmal components have been depleted. High PS levels are maintained through fusion with endosomes and lysosomes and suffice to attract cationic proteins like c-Src to maturing phagosomes. Phagocytic vacuoles containing the pathogens Legionella pneumophila and Chlamydia trachomatis, which divert maturation away from the endolysosomal pathway, are devoid of PS, have little surface charge, and fail to recruit c-Src. These findings highlight a function for PS in phagosome maturation and microbial killing.

The Tol proteins are involved in the outer membrane stability of gram-negative bacteria. The C-terminal domain of TolA was mutagenized to identify residues important for its functions. The isolation of suppressor mutants of tolA mutations in the tolB gene confirmed an interaction between TolAIII and the N-terminal domain of TolB.

Autophagy is a conserved membrane traffic pathway that equips eukaryotic cells to capture cytoplasmic components within a double-membrane vacuole, or autophagosome, for delivery to lysosomes. Although best known as a mechanism to survive starvation, autophagy is now recognized to combat infection by a variety of microbes.

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