SHC-3: a previously unidentified C. elegans Shc family member functions in the insulin-like signaling pathway to enhance survival during L1 arrest.

Shc proteins function in many different signaling pathways where they mediate phosphorylation-dependent protein-protein interactions. These proteins are characterized by the presence of two phosphotyrosine-binding domains, an N-terminal PTB and a C-terminal SH2. We describe a previously unrecognized C. elegans Shc gene, shc-3 and characterize its role in stress response. Both shc-3 and shc-1 are required for long-term survival in L1 arrest and survival in heat stress, however, they do not act redundantly but rather play distinct roles in these processes. Loss of shc-3 did not further decrease survival of daf-16 mutants in L1 arrest, suggesting that like SHC-1, SHC-3 functions in the Insulin-like signaling pathway. In the absence of SHC-3, DAF-16 nuclear entry and exit are slowed, suggesting that SHC-3 is required for rapid changes in DAF-16 signaling.

Lessons from assembling a microbial natural product and pre-fractionated extract library in an academic laboratory.

Microbial natural products are specialized metabolites that are sources of many bioactive compounds including antibiotics, antifungals, antiparasitics, anticancer agents, and probes of biology. The assembly of libraries of producers of natural products has traditionally been the province of the pharmaceutical industry. This sector has gathered significant historical collections of bacteria and fungi to identify new drug leads with outstanding outcomes-upwards of 60% of drug scaffolds originate from such libraries. Despite this success, the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds in the drug industry. The advent of advanced mass spectrometry tools, along with rapid whole genome sequencing and in silico identification of biosynthetic gene clusters that encode the machinery necessary for the synthesis of specialized metabolites, offers the opportunity to revisit microbial natural product libraries with renewed vigor. Assembling a suitable library of microbes and extracts for screening requires the investment of resources and the development of methods that have customarily been the proprietary purview of large pharmaceutical companies. Here, we report a perspective on our efforts to assemble a library of natural product-producing microbes and the establishment of methods to extract and fractionate bioactive compounds using resources available to most academic labs. We validate the library and approach through a series of screens for antimicrobial and cytotoxic agents. This work serves as a blueprint for establishing libraries of microbial natural product producers and bioactive extract fractions suitable for screens of bioactive compounds. ONE-SENTENCE SUMMARY: Natural products are key to discovery of novel antimicrobial agents: Here, we describe our experience and lessons learned in constructing a microbial natural product and pre-fractionated extract library.

Simple model systems reveal conserved mechanisms of Alzheimer's disease and related tauopathies.

The lack of effective therapies that slow the progression of Alzheimer's disease (AD) and related tauopathies highlights the need for a more comprehensive understanding of the fundamental cellular mechanisms underlying these diseases. Model organisms, including yeast, worms, and flies, provide simple systems with which to investigate the mechanisms of disease. The evolutionary conservation of cellular pathways regulating proteostasis and stress response in these organisms facilitates the study of genetic factors that contribute to, or protect against, neurodegeneration. Here, we review genetic modifiers of neurodegeneration and related cellular pathways identified in the budding yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, and the fruit fly Drosophila melanogaster, focusing on models of AD and related tauopathies. We further address the potential of simple model systems to better understand the fundamental mechanisms that lead to AD and other neurodegenerative disorders.

Microbiomes: How a gut bacterium promotes healthier living in a nematode.

A new study finds that a bacterium from the microbiome of the nematode Pristionchus pacificus can promote rapid growth, increased body size, and increased fecundity by inducing neuronal expression of TGF-ß ligands. This is an intriguing example of how the microbiota induces systemic effects in the host by stimulating neuroendocrine signaling.

Cuticle Collagen Expression Is Regulated in Response to Environmental Stimuli by the GATA Transcription Factor ELT-3 in Caenorhabditis elegans.

The nematode Caenorhabditis elegans is protected from the environment by the cuticle, an extracellular collagen-based matrix that encloses the animal. Over 170 cuticular collagens are predicted in the C. elegans genome, but the role of each individual collagen is unclear. Stage-specific specialization of the cuticle explains the need for some collagens; however, the large number of collagens suggests that specialization of the cuticle may also occur in response to other environmental triggers. Missense mutations in many collagen genes can disrupt cuticle morphology, producing a helically twisted body causing the animal to move in a stereotypical pattern described as rolling. We find that environmental factors, including diet, early developmental arrest, and population density can differentially influence the penetrance of rolling in these mutants. These effects are in part due to changes in collagen gene expression that are mediated by the GATA family transcription factor ELT-3 We propose a model by which ELT-3 regulates collagen gene expression in response to environmental stimuli to promote the assembly of a cuticle specialized to a given environment.

Nutrient Sensing and Response Drive Developmental Progression in Caenorhabditis elegans.

In response to nutrient limitation, many animals, including Caenorhabditis elegans, slow or arrest their development. This process requires mechanisms that sense essential nutrients and induce appropriate responses. When faced with nutrient limitation, C. elegans can induce both short and long-term survival strategies, including larval arrest, decreased developmental rate, and dauer formation. To select the most advantageous strategy, information from many different sensors must be integrated into signaling pathways, including target of rapamycin (TOR) and insulin, that regulate developmental progression. Here, how nutrient information is sensed and integrated into developmental decisions that determine developmental rate and progression in C. elegans is reviewed.

Biology is the root of variability: cautionary tales in Caenorhabditis elegans biology.

Reproducibility is critical for the standardization, interpretation, and progression of research. However, many factors increase variability and reduce reproducibility. In Caenorhabditis elegans research, there are many possible causes of variability that may explain why experimental outcomes sometimes differ between laboratories and between experiments. Factors contributing to experimental variability include the genetic background of both C. elegans and its bacterial diet, differences in media composition, intergenerational and transgenerational effects that may be carried over for generations, and the use of chemicals or reagents that may have unexpected consequences. This review summarizes sources of variability in C. elegans research and serves to identify laboratory practices that could influence reproducibility.

The Caenorhabditis elegans Oxidative Stress Response Requires the NHR-49 Transcription Factor.

The overproduction of reactive oxygen species (ROS) in cells can lead to the development of diseases associated with aging. We have previously shown that C. elegans BRAP-2 (Brca1 associated binding protein 2) regulates phase II detoxification genes such as gst-4, by increasing SKN-1 activity. Previously, a transcription factor (TF) RNAi screen was conducted to identify potential activators that are required to induce gst-4 expression in brap-2(ok1492) mutants. The lipid metabolism regulator NHR-49/HNF4 was among 18 TFs identified. Here, we show that knockdown of nhr-49 suppresses the activation of gst-4 caused by brap-2 inactivation and that gain-of-function alleles of nhr-49 promote gst-4 expression. We also demonstrate that nhr-49 and its cofactor mdt-15 are required to express phase II detoxification enzymes upon exposure to chemicals that induce oxidative stress. Furthermore, we show that NHR-49 and MDT-15 enhance expression of skn-1a/c These findings identify a novel role for NHR-49 in ROS detoxification by regulating expression of SKN-1C and phase II detoxification genes.

Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity.

Type IV pili are expressed by a wide range of prokaryotes, including the opportunistic pathogen Pseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The pilus is composed mainly of major pilin subunits while the low abundance minor pilins FimU-PilVWXE and the putative adhesin PilY1 prime pilus assembly and are proposed to form the pilus tip. The minor pilins and PilY1 are encoded in an operon that is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 was proposed to be a mechanosensory component that-in conjunction with minor pilins-triggers up-regulation of acute virulence phenotypes upon surface attachment. Here, we investigated the link between the minor pilins/PilY1 and virulence. pilW, pilX, and pilY1 mutants had reduced virulence towards Caenorhabditis elegans relative to wild type or a major pilin mutant, implying a role in pathogenicity that is independent of pilus assembly. We hypothesized that loss of specific minor pilins relieves feedback inhibition on FimS-AlgR, increasing transcription of the AlgR regulon and delaying C. elegans killing. Reporter assays confirmed that FimS-AlgR were required for increased expression of the minor pilin operon upon loss of select minor pilins. Overexpression of AlgR or its hyperactivation via a phosphomimetic mutation reduced virulence, and the virulence defects of pilW, pilX, and pilY1 mutants required FimS-AlgR expression and activation. We propose that PilY1 and the minor pilins inhibit their own expression, and that loss of these proteins leads to FimS-mediated activation of AlgR that suppresses expression of acute-phase virulence factors and delays killing. This mechanism could contribute to adaptation of P. aeruginosa in chronic lung infections, as mutations in the minor pilin operon result in the loss of piliation and increased expression of AlgR-dependent virulence factors-such as alginate-that are characteristic of such infections.

Transformation of the Anticancer Drug Doxorubicin in the Human Gut Microbiome.

Bacteria living in the human gut are implicated in the etiology of several diseases. Moreover, dozens of drugs are metabolized by elements of the gut microbiome, which may have further implications for human health. Here, we screened a collection of gut isolates for their ability to inactivate the widely used antineoplastic drug doxorubicin and identified a strain of Raoultella planticola as a potent inactivator under anaerobic conditions. We demonstrate that R. planticola deglycosylates doxorubicin to metabolites 7-deoxydoxorubicinol and 7-deoxydoxorubicinolone via a reductive deglycosylation mechanism. We further show that doxorubicin is degraded anaerobically by Klebsiella pneumoniae and Escherichia coli BW25113 and present evidence that this phenotype is dependent on molybdopterin-dependent enzyme(s). Deglycosylation of doxorubicin by R. planticola under anaerobic conditions is found to reduce toxicity to the model species Caenorhabditis elegans, providing a model to begin understanding the role of doxorubicin metabolism by microbes in the human gut. Understanding the in vivo metabolism of important therapeutics like doxorubicin by the gut microbiome has the potential to guide clinical dosing to maximize therapeutic benefit while limiting undesirable side effects.

The Oxidative Stress Response in Caenorhabditis elegans Requires the GATA Transcription Factor ELT-3 and SKN-1/Nrf2.

Cellular damage caused by reactive oxygen species is believed to be a major contributor to age-associated diseases. Previously, we characterized the Caenorhabditis elegans Brap2 ortholog (BRAP-2) and found that it is required to prevent larval arrest in response to elevated levels of oxidative stress. Here, we report that C. elegans brap-2 mutants display increased expression of SKN-1-dependent, phase II detoxification enzymes that is dependent on PMK-1 (a p38 MAPK C. elegans ortholog). An RNA-interference screen was conducted using a transcription factor library to identify genes required for increased expression of the SKN-1 target gst-4 in brap-2 mutants. We identified ELT-3, a member of the GATA transcription factor family, as a positive regulator of gst-4p::gfp expression. We found that ELT-3 interacts with SKN-1 to activate gst-4 transcription in vitro and that elt-3 is required for enhanced gst-4 expression in the brap-2(ok1492) mutant in vivo Furthermore, nematodes overexpressing SKN-1 required ELT-3 for life-span extension. Taken together, these results suggest a model where BRAP-2 acts as negative regulator of SKN-1 through inhibition of p38 MAPK activity, and that the GATA transcription factor ELT-3 is required along with SKN-1 for the phase II detoxification response in C. elegans.

Packing on the Pounds in Response to Bacterial Growth Conditions.

Reporting in Nature Cell Biology, Lin and Wang (2017) show that bacterial methyl metabolism impacts host mitochondrial dynamics and lipid storage in C. elegans. The authors propose a model whereby bacterial metabolic products regulate a nuclear hormone receptor that promotes lipid accumulation through expression of a secreted Hedgehog-like protein.

Integrative Physiology: At the Crossroads of Nutrition, Microbiota, Animal Physiology, and Human Health.

Nutrition is paramount in shaping all aspects of animal biology. In addition, the influence of the intestinal microbiota on physiology is now widely recognized. Given that diet also shapes the intestinal microbiota, this raises the question of how the nutritional environment and microbial assemblages together influence animal physiology. This research field constitutes a new frontier in the field of organismal biology that needs to be addressed. Here we review recent studies using animal models and humans and propose an integrative framework within which to define the study of the diet-physiology-microbiota systems and ultimately link it to human health. Nutritional Geometry sits centrally in the proposed framework and offers means to define diet compositions that are optimal for individuals and populations.

Transcription Factor Activity Mapping of a Tissue-Specific in vivo Gene Regulatory Network.

A wealth of physical interaction data between transcription factors (TFs) and DNA has been generated, but these interactions often do not have apparent regulatory consequences. Thus, equating physical interaction data with gene regulatory networks (GRNs) is problematic. Here, we comprehensively assay TF activity, rather than binding, to construct a network of gene regulatory interactions in the C. elegans intestine. By manually observing the in vivo tissue-specific knockdown of 921 TFs on a panel of 19 fluorescent transcriptional reporters, we identified a GRN of 411 interactions between 19 promoters and 177 TFs. This GRN shows only modest overlap with physical interactions, indicating that many regulatory interactions are indirect. We applied nested effects modeling to uncover information flow between TFs in the intestine that converges on a small set of physical TF-promoter interactions. We found numerous cell nonautonomous regulatory interactions, illustrating tissue-to-tissue communication. Altogether, our study illuminates the complexity of gene regulation in the context of a living animal.

RNA Interference in Caenorhabditis elegans.

RNAi has become an essential tool in C. elegans research. This unit describes procedures for RNAi in C. elegans by microinjecting with dsRNA, feeding with bacteria expressing dsRNA, and soaking in dsRNA solution, as well as high-throughput methods for RNAi-based screens.

Transcriptional regulation of Caenorhabditis elegans FOXO/DAF-16 modulates lifespan.

BACKGROUND: Insulin/IGF-1 signaling plays a central role in longevity across phylogeny. In C. elegans, the forkhead box O (FOXO) transcription factor, DAF-16, is the primary target of insulin/IGF-1 signaling, and multiple isoforms of DAF-16 (a, b, and d/f) modulate lifespan, metabolism, dauer formation, and stress resistance. Thus far, across phylogeny modulation of mammalian FOXOs and DAF-16 have focused on post-translational regulation with little focus on transcriptional regulation. In C. elegans, we have previously shown that DAF-16d/f cooperates with DAF-16a to promote longevity. In this study, we generated transgenic strains expressing near-endogenous levels of either daf-16a or daf-16d/f, and examined temporal expression of the isoforms to further define how these isoforms contribute to lifespan regulation. RESULTS: Here, we show that DAF-16a is sensitive both to changes in gene dosage and to alterations in the level of insulin/IGF-1 signaling. Interestingly, we find that as worms age, the intestinal expression of daf-16d/f but not daf-16a is dramatically upregulated at the level of transcription. Preventing this transcriptional upregulation shortens lifespan, indicating that transcriptional regulation of daf-16d/f promotes longevity. In an RNAi screen of transcriptional regulators, we identify elt-2 (GATA transcription factor) and swsn-1 (core subunit of SWI/SNF complex) as key modulators of daf-16d/f gene expression. ELT-2 and another GATA factor, ELT-4, promote longevity via both DAF-16a and DAF-16d/f while the components of SWI/SNF complex promote longevity specifically via DAF-16d/f. CONCLUSIONS: Our findings indicate that transcriptional control of C. elegans FOXO/daf-16 is an essential regulatory event. Considering the conservation of FOXO across species, our findings identify a new layer of FOXO regulation as a potential determinant of mammalian longevity and age-related diseases such as cancer and diabetes.

Interspecies systems biology uncovers metabolites affecting C. elegans gene expression and life history traits.

Diet greatly influences gene expression and physiology. In mammals, elucidating the effects and mechanisms of individual nutrients is challenging due to the complexity of both the animal and its diet. Here, we used an interspecies systems biology approach with Caenorhabditis elegans and two of its bacterial diets, Escherichia coli and Comamonas aquatica, to identify metabolites that affect the animal's gene expression and physiology. We identify vitamin B12 as the major dilutable metabolite provided by Comamonas aq. that regulates gene expression, accelerates development, and reduces fertility but does not affect lifespan. We find that vitamin B12 has a dual role in the animal: it affects development and fertility via the methionine/S-Adenosylmethionine (SAM) cycle and breaks down the short-chain fatty acid propionic acid, preventing its toxic buildup. Our interspecies systems biology approach provides a paradigm for understanding complex interactions between diet and physiology.

Diet-induced developmental acceleration independent of TOR and insulin in C. elegans.

Dietary composition has major effects on physiology. Here, we show that developmental rate, reproduction, and lifespan are altered in C. elegans fed Comamonas DA1877 relative to those fed a standard E. coli OP50 diet. We identify a set of genes that change in expression in response to this diet and use the promoter of one of these (acdh-1) as a dietary sensor. Remarkably, the effects on transcription and development occur even when Comamonas DA1877 is diluted with another diet, suggesting that Comamonas DA1877 generates a signal that is sensed by the nematode. Surprisingly, the developmental effect is independent from TOR and insulin signaling. Rather, Comamonas DA1877 affects cyclic gene expression during molting, likely through the nuclear hormone receptor NHR-23. Altogether, our findings indicate that different bacteria elicit various responses via distinct mechanisms, which has implications for diseases such as obesity and the interactions between the human microbiome and intestinal cells.