Enteroendocrine cells (EEs) are interspersed between enterocytes and stem cells in the Drosophila intestinal epithelium. Like enterocytes, EEs express components of the immune deficiency (IMD) innate immune pathway, which activates transcription of genes encoding antimicrobial peptides. The discovery of large lipid droplets in intestines of IMD pathway mutants prompted us to investigate the role of the IMD pathway in the host metabolic response to its intestinal microbiota. Here we provide evidence that the short-chain fatty acid acetate is a microbial metabolic signal that activates signaling through the enteroendocrine IMD pathway in a PGRP-LC-dependent manner. This, in turn, increases transcription of the gene encoding the endocrine peptide Tachykinin (Tk), which is essential for timely larval development and optimal lipid metabolism and insulin signaling. Our findings suggest innate immune pathways not only provide the first line of defense against infection but also afford the intestinal microbiota control over host development and metabolism.

The Snf1 protein kinase of Saccharomyces cerevisiae has been shown to have a role in regulating haploid invasive growth in response to glucose depletion. Cells contain three forms of the Snf1 kinase, each with a different ␤-subunit isoform, either Gal83, Sip1, or Sip2. We present evidence that different Snf1 kinases play distinct roles in two aspects of invasive growth, namely, adherence to the agar substrate and filamentation. The Snf1-Gal83 form of the kinase is required for adherence, whereas either Snf1-Gal83 or Snf1-Sip2 is sufficient for filamentation. Genetic evidence indicates that Snf1-Gal83 affects adherence by antagonizing Nrg1-and Nrg2-mediated repression of the FLO11 flocculin and adhesin gene. In contrast, the mechanism(s) by which Snf1-Gal83 and Snf1-Sip2 affect filamentation is independent of FLO11. Thus, the Snf1 kinase regulates invasive growth by at least two distinct mechanisms.

The yeast Saccharomyces cerevisiae responds to environmental stress by rapidly altering the expression of large sets of genes. We report evidence that the transcriptional repressors Nrg1 and Nrg2 (Nrg1/Nrg2), which were previously implicated in glucose repression, regulate a set of stress-responsive genes. Genome-wide expression analysis identified 150 genes that were upregulated in nrg1⌬ nrg2⌬ double mutant cells, relative to wild-type cells, during growth in glucose. We found that many of these genes are regulated by glucose repression. Stress response elements (STREs) and STRE-like elements are overrepresented in the promoters of these genes, and a search of available expression data sets showed that many are regulated in response to a variety of environmental stress signals. In accord with these findings, mutation of NRG1 and NRG2 enhanced the resistance of cells to salt and oxidative stress and decreased tolerance to freezing. We present evidence that Nrg1/Nrg2 not only contribute to repression of target genes in the absence of stress but also limit induction in response to salt stress. We suggest that Nrg1/Nrg2 fine-tune the regulation of a set of stress-responsive genes.

The yeast Snf1 kinase and its mammalian ortholog, AMP-activated protein kinase (AMPK), regulate responses to metabolic stress. Previous studies identified a glycogen-binding domain in the AMPK ␤1 subunit, and the sequence is conserved in the Snf1 kinase ␤ subunits Gal83 and Sip2. Here we use genetic analysis to assess the role of this domain in vivo. Alteration of Gal83 at residues that are important for glycogen binding of AMPK ␤1 abolished glycogen binding in vitro and caused diverse phenotypes in vivo. Various Snf1/Gal83-dependent processes were upregulated, including glycogen accumulation, expression of RNAs encoding glycogen synthase, haploid invasive growth, the transcriptional activator function of Sip4, and activation of the carbon source-responsive promoter element. Moreover, the glycogen-binding domain mutations conferred transcriptional regulatory phenotypes even in the absence of glycogen, as determined by analysis of a mutant strain lacking glycogen synthase. Thus, mutation of the glycogen-binding domain of Gal83 positively affects Snf1/Gal83 kinase function by a mechanism that is independent of glycogen binding.The Saccharomyces cerevisiae Snf1 kinase and mammalian AMP-activated protein kinase (AMPK) are highly conserved kinases with roles in metabolic stress responses (for review, see references 14 and 24). Both Snf1 and AMPK monitor nutritional status. AMPK is activated by increases in AMP during metabolic stress, by the hormones leptin and adiponectin (32, 51), and by drugs used in the treatment of type 2 diabetes (52). Snf1 is also activated by stresses, notably glucose limitation (22,29,48,50). Snf1 and AMPK regulate glucose and lipid metabolism both by controlling the activity of metabolic enzymes and by controlling transcription. Snf1 is required for the expression of glucose-repressed genes involved in respiration, gluconeogenesis, peroxisome biogenesis, and metabolism of alternate carbon sources (4, 12).Snf1 and AMPK are heterotrimeric kinases with multiple subunit isoforms. Snf1 kinase comprises the catalytic subunit Snf1, a ␤ subunit (Gal83, Sip1, or Sip2), and the regulatory subunit Snf4. Snf1 kinase containing the Gal83 ␤ subunit will be referred to here as Snf1/Gal83 kinase. For AMPK, the corresponding subunit isoforms are designated ␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3. Individual ␤ subunits of Snf1 and AMPK have distinct subcellular localizations (44, 47) and thereby presumably regulate access of the kinase to substrates. The Gal83 ␤ subunit is known to mediate interaction of the kinase with Sip4, a transcriptional activator of gluconeogenic genes that is dependent on Snf1 kinase for its function (27,38,42).Recent studies identified a glycogen-binding domain in the AMPK ␤1 subunit that is related to isoamylase domains found in glycogen and starch branching enzymes (21, 34). Mutation of conserved residues abolishes binding to glycogen in vitro (34). In mammalian cells, AMPK ␤1 colocalizes with glycogen phosphorylase (34) and glycogen synthase (21); with the caveat that AMPK ␤1 was overexpr...

SummaryWe previously demonstrated that Vibrio cholerae is able to colonize the intestine of the fly to produce a lethal infection. Here we present the results of a genetic screen undertaken to identify factors that alter susceptibility of the fly to intestinal V. cholerae infection. In this model of infection, the Eiger/ Wengen signalling pathway protects the fly against infection. Furthermore, mutations within the IMD signalling pathway increase resistance to intestinal V. cholerae infection and increase programmed cell death within the intestinal epithelium during infection. We propose that programmed cell death protects the intestinal epithelium against V. cholerae infection and therefore that the fly may serve as a useful model in which to study modulation of intestinal epithelial cell survival by commensal and pathogenic intestinal bacteria as well as the pathological processes leading to erosion of the intestinal epithelium and intestinal malignancy.

Here, we describe a Drosophila melanogaster transposon insertion mutant with tolerance to V. cholerae infection and markedly decreased transcription of diptericin as well as other genes regulated by the IMD innate immunity signaling pathway. We present genetic evidence that this insertion affects a locus previously implicated in pupal eclosion. This genetic locus, which we have named mustard (mtd), contains a LysM domain, often involved in carbohydrate recognition, and a TLDc domain of unknown function. Over twenty Mtd isoforms containing one or both of these conserved domains are predicted. We establish that the mutant phenotype represents a gain of function and can be replicated by increased expression of a short, nuclearly localized Mtd isoform comprised almost entirely of the TLDc domain. We show that this Mtd isoform does not block Relish cleavage or translocation into the nucleus. Lastly, we present evidence suggesting that the eclosion defect previously attributed to the Mtd locus may be the result of the unopposed action of the NF-κB homolog, Relish. Mtd homologs have been implicated in resistance to oxidative stress. However, this is the first evidence that Mtd or its homologs alter the output of an innate immunity signaling cascade from within the nucleus.

The Nrg1 and Nrg2 repressors of Saccharomyces cerevisiae have highly similar zinc fingers and closely related functions in the regulation of glucose-repressed genes. We show that NRG1 and NRG2 are differently regulated in response to carbon source at both the RNA and protein levels. Expression of NRG1 RNA is glucose repressed, whereas NRG2 RNA levels are nearly constant. Nrg1 protein levels are elevated in response to glucose limitation or growth in nonfermentable carbon sources, whereas Nrg2 levels are diminished. Chromatin immunoprecipitation assays showed that Nrg1 and Nrg2 bind DNA both in the presence and absence of glucose. In mutant cells lacking the corepressor Ssn6(Cyc8)-Tup1, promoter-bound Nrg1, but not Nrg2, functions as an activator in a reporter assay, providing evidence that the two Nrg proteins have distinct properties. We suggest that the differences in expression and function of these two repressors, in combination with their similar DNA-binding domains, contribute to the complex regulation of the large set of glucose-repressed genes.The Nrg1 and Nrg2 proteins of S. cerevisiae are similar C 2 H 2 zinc finger proteins that function as transcriptional repressors. Considerable evidence indicates that these two proteins have broad roles in regulation of glucose-repressed genes. Nrg1 was first identified by its role in glucose repression of the STA1 (glucoamylase) gene (22). Nrg2 was identified by its two-hybrid interaction with Snf1 protein kinase, a component of a major glucose signaling pathway, and both Nrg proteins were shown to interact physically with Snf1 (30). The Nrg proteins contribute to repression of multiple glucose-repressed genes, including the DOG2, SUC2, GAL, STA2, and FLO11 genes (10,30,33). Nrg1 and Nrg2 also function as negative regulators of haploid invasive growth and initiation of biofilm formation, which are cellular responses to glucose limitation (3,10,25).The Nrg repressors have also been implicated in response to other environmental conditions. Both are negative regulators of diploid pseudohyphal growth (10), which occurs in response to nitrogen limitation; this function may be related to that of the Nrg1 ortholog of Candida albicans, which represses filamentous growth and expression of hypha-specific genes (2, 20). NRG1 is repressed by the alkaline pH response regulator Rim101, and Nrg1 represses alkaline pH-induced genes (11). Finally, levels of NRG2 RNA are induced by zinc limitation (18) and alkaline pH (12).Previous evidence indicated that Nrg1 and Nrg2 have closely related functions with respect to glucose regulation (10,29,30), consistent with the strong similarity of their DNA-binding domains (84% identity). However, we considered the possibility that these two proteins have distinct functions in glucose repression for two reasons. First, the Nrg proteins are less similar outside their DNA-binding domains (27% identity) and, hence, may interact differently with regulatory proteins or other transcription factors. Second, NRG1 and NRG2 RNAs are regulated differentl...

Protein kinase CK2 is highly conserved in eukaryotes and plays roles in many different cellular processes. CK2 is a tetramer comprising two catalytic and two regulatory subunits. Most organisms have two major isoforms of the catalytic subunit, and evidence suggests strongly overlapping function. In the yeast Saccharomyces cerevisiae, CK2 is essential for viability, and either catalytic subunit isoform, Cka1 or Cka2, suffices, but previous genetic evidence suggests that the isoforms have some distinct roles. In this work, we present evidence that the transcriptional repressor Nrg1, which regulates various stress-responsive genes, is a downstream target of CK2 containing the Cka1 isoform. We found that Nrg1 is phosphorylated in response to stress and that its phosphorylation was defective in cka1Delta, but not cka2Delta, mutants. Thus, the Cka1 catalytic subunit isoform is specifically required for phosphorylation of Nrg1 in vivo. The CK2 regulatory subunits were also required, indicating that the CK2 holoenzyme is involved. Both yeast and recombinant human CK2 phosphorylated recombinant Nrg1 in vitro. This identification of a protein whose phosphorylation requires a specific CK2 catalytic subunit isoform supports the view that the two isoforms exhibit functional specificity in vivo.