Cell-selective proteomics reveal novel effectors secreted by an obligate intracellular bacterial pathogen.

Pathogenic bacteria secrete protein effectors to hijack host machinery and remodel their infectious niche. Rickettsia spp. are obligate intracellular bacteria that can cause life-threatening disease, but their absolute dependence on the host cell environment has impeded discovery of rickettsial effectors and their host targets. We implemented bioorthogonal non-canonical amino acid tagging (BONCAT) during R. parkeri infection to selectively label, isolate, and identify secreted effectors. As the first use of BONCAT in an obligate intracellular bacterium, our screen more than doubles the number of experimentally validated effectors for R. parkeri. The novel secreted rickettsial factors (Srfs) we identified include Rickettsia-specific proteins of unknown function that localize to the host cytoplasm, mitochondria, and ER. We further show that one such effector, SrfD, interacts with the host Sec61 translocon. Altogether, our work uncovers a diverse set of previously uncharacterized rickettsial effectors and lays the foundation for a deeper exploration of the host-pathogen interface.

The Third Coiled Coil Domain of Atg11 Is Required for Shaping Mitophagy Initiation Sites.

Selective autophagy is the capture of specific cytosolic contents in double-membrane vesicles that subsequently fuse with the vacuole or lysosome, thereby delivering cargo for degradation. Selective autophagy receptors (SARs) mark the cargo for degradation and, in yeast, recruit Atg11, the scaffolding protein for selective autophagy initiation. The mitochondrial protein Atg32 is the yeast SAR that mediates mitophagy, the selective autophagic capture of mitochondria. Atg11-Atg32 interactions concentrate Atg32 into puncta that are thought to represent sites of mitophagy initiation. However, it is unclear how Atg11 concentrates Atg32 to generate mitophagy initiation sites. We show here that the coiled coil 3 (CC3) domain of Atg11 is required for concentrating Atg32 into puncta. We determined the structure of the majority of the CC3, demonstrating that the CC3 forms a parallel homodimer whose dimer interface is formed by a small number of hydrophobic residues. We further show that the CC3 interface is not required for Atg11 dimerization but is required for shaping Atg32 into functional mitophagy initiation sites and for delivery of mitochondria to the vacuole. Our findings suggest that Atg11 self-interactions help concentrate SARs as a necessary precondition for cargo capture.