Discovery and engineering of the antibody response against a prominent skin commensal.

The ubiquitous skin colonist Staphylococcus epidermidis elicits a CD8 + T cell response pre-emptively, in the absence of an infection 1 . However, the scope and purpose of this anti-commensal immune program are not well defined, limiting our ability to harness it therapeutically. Here, we show that this colonist also induces a potent, durable, and specific antibody response that is conserved in humans and non-human primates. A series of S. epidermidis cell-wall mutants revealed that the cell surface protein Aap is a predominant target. By colonizing mice with a strain of S. epidermidis in which the parallel ß-helix domain of Aap is replaced by tetanus toxin fragment C, we elicit a potent neutralizing antibody response that protects mice against a lethal challenge. A similar strain of S. epidermidis expressing an Aap-SpyCatcher chimera can be conjugated with recombinant immunogens; the resulting labeled commensal elicits high titers of antibody under conditions of physiologic colonization, including a robust IgA response in the nasal mucosa. Thus, immunity to a common skin colonist involves a coordinated T and B cell response, the latter of which can be redirected against pathogens as a novel form of topical vaccination.

Sestrin mediates detection of and adaptation to low-leucine diets in Drosophila.

Mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth and metabolism in response to multiple nutrients, including the essential amino acid leucine1. Recent work in cultured mammalian cells established the Sestrins as leucine-binding proteins that inhibit mTORC1 signalling during leucine deprivation2,3, but their role in the organismal response to dietary leucine remains elusive. Here we find that Sestrin-null flies (Sesn-/-) fail to inhibit mTORC1 or activate autophagy after acute leucine starvation and have impaired development and a shortened lifespan on a low-leucine diet. Knock-in flies expressing a leucine-binding-deficient Sestrin mutant (SesnL431E) have reduced, leucine-insensitive mTORC1 activity. Notably, we find that flies can discriminate between food with or without leucine, and preferentially feed and lay progeny on leucine-containing food. This preference depends on Sestrin and its capacity to bind leucine. Leucine regulates mTORC1 activity in glial cells, and knockdown of Sesn in these cells reduces the ability of flies to detect leucine-free food. Thus, nutrient sensing by mTORC1 is necessary for flies not only to adapt to, but also to detect, a diet deficient in an essential nutrient.

Structure of the nutrient-sensing hub GATOR2.

Mechanistic target of rapamycin complex 1 (mTORC1) controls growth by regulating anabolic and catabolic processes in response to environmental cues, including nutrients1,2. Amino acids signal to mTORC1 through the Rag GTPases, which are regulated by several protein complexes, including GATOR1 and GATOR2. GATOR2, which has five components (WDR24, MIOS, WDR59, SEH1L and SEC13), is required for amino acids to activate mTORC1 and interacts with the leucine and arginine sensors SESN2 and CASTOR1, respectively3-5. Despite this central role in nutrient sensing, GATOR2 remains mysterious as its subunit stoichiometry, biochemical function and structure are unknown. Here we used cryo-electron microscopy to determine the three-dimensional structure of the human GATOR2 complex. We found that GATOR2 adopts a large (1.1 MDa), two-fold symmetric, cage-like architecture, supported by an octagonal scaffold and decorated with eight pairs of WD40 ß-propellers. The scaffold contains two WDR24, four MIOS and two WDR59 subunits circularized via two distinct types of junction involving non-catalytic RING domains and α-solenoids. Integration of SEH1L and SEC13 into the scaffold through ß-propeller blade donation stabilizes the GATOR2 complex and reveals an evolutionary relationship to the nuclear pore and membrane-coating complexes6. The scaffold orients the WD40 ß-propeller dimers, which mediate interactions with SESN2, CASTOR1 and GATOR1. Our work reveals the structure of an essential component of the nutrient-sensing machinery and provides a foundation for understanding the function of GATOR2 within the mTORC1 pathway.

Multiplexed in vivo homology-directed repair and tumor barcoding enables parallel quantification of Kras variant oncogenicity.

Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. However, determining the functional significance of specific alterations remains a major bottleneck in our understanding of the genetic determinants of cancer. Here, we present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice. Using this approach, we introduce a barcoded library of non-synonymous mutations into hotspot codons 12 and 13 of Kras in adult somatic cells to initiate tumors in the lung, pancreas, and muscle. High-throughput sequencing of barcoded Kras HDR alleles from bulk lung and pancreas reveals surprising diversity in Kras variant oncogenicity. Rapid, cost-effective, and quantitative approaches to simultaneously investigate the function of precise genomic alterations in vivo will help uncover novel biological and clinically actionable insights into carcinogenesis.