Competing dopamine neurons drive oviposition choice for ethanol in Drosophila

The neural circuits that mediate behavioral choice evaluate and integrate information from the environment with internal demands and then initiate a behavioral response. Even circuits that support simple decisions remain poorly understood. In Drosophila melanogaster, oviposition on a substrate containing ethanol enhances fitness; however, little is known about the neural mechanisms mediating this important choice behavior. Here, we characterize the neural modulation of this simple choice and show that distinct subsets of dopaminergic neurons compete to either enhance or inhibit egg-laying preference for ethanol-containing food. Moreover, activity in α′β′ neurons of the mushroom body and a subset of ellipsoid body ring neurons (R2) is required for this choice. We propose a model where competing dopaminergic systems modulate oviposition preference to adjust to changes in natural oviposition substrates.In nature, rotting fruit is the social hub for the fruit fly Drosophila melanogaster. Flies use fermenting fruit as a food source (1) and a site for oviposition (2). The choice of a suitable oviposition substrate is an ecologically important decision with a direct impact on species fitness. However, other than having a clear preference for fermenting fruit, how females choose oviposition sites in nature is largely unknown.One of the main metabolites of fermentation is ethanol, which is present in ripe fleshy fruits (3). Although ethanol concentrations in the fruit are rather low [≤5% (vol/vol)] (4), plumes containing ethanol vapor can act as long-distance signals to attract flies to rotting fruit (3, 5). When given the choice, female flies prefer to lay their eggs on media containing low concentrations of ethanol (up to 5%) (6), which leads to enhanced fitness of the developing larva and the adult fly.D. melanogaster’s resistance to ethanol toxicity may have evolved to allow inhabitation of ethanol-containing environments (7). For example, adult flies allowed to mate on ethanol-containing media improve mating success and fecundity (8). Although rearing larvae on food containing relatively high ethanol concentrations delays development and decreases survival (9–11), larvae reared on low concentrations of ethanol develop into heavier adults (7, 12). This weight increase may be a result of D. melanogaster larvae metabolizing ethanol and using it as a food source (12). Ingestion of ethanol during the larval stage has additional benefits, such as protection from natural parasites such as endoparasitoid wasps (13).Studies on the neural circuits underlying the oviposition program and choice of oviposition substrates have been initiated only recently in D. melanogaster (14, 15). Ethanol is a particularly intriguing stimulus for oviposition preference, because it has, depending on concentration, both beneficial and detrimental effects on developing larvae. In flies, the function of dopaminergic neurons has been implicated in responses to both rewarding and aversive stimuli (16–19), making it a candidate neuromodulator to signal the beneficial and detrimental effects of ethanol. Dopamine signaling has also been implicated in other innate behaviors required for survival, such as food consumption, sex, and social interaction (20–22). Although the circuitry for ethanol oviposition preference is unknown, both dopaminergic and mushroom body (MB) signaling is required for flies to remember ethanol as a reward (23).We report that females show concentration-dependent preference for oviposition on food containing ethanol, with highest preference for food substrates containing the most ecologically beneficial concentrations of ethanol. This simple behavior relies on functional dopamine neurons, with different subsets regulating oviposition site preference in opposite ways. Our data also suggest that dopaminergic innervation of higher-order brain regions, including specific subsets of the MB and ellipsoid body (EB) neurons modulate the decision to lay eggs on ethanol.     

Acute ethanol responses in Drosophila are sexually dimorphic

In mammalian and insect models of ethanol intoxication, low doses of ethanol stimulate locomotor activity whereas high doses induce sedation. Sex differences in acute ethanol responses, which occur in humans, have not been characterized in Drosophila. In this study, we find that male flies show increased ethanol hyperactivity and greater resistance to ethanol sedation compared with females. We show that the sex determination gene transformer (tra) acts in the developing nervous system, likely through regulation of fruitless (fru), to at least partially mediate the sexual dimorphism in ethanol sedation. Although pharmacokinetic differences may contribute to the increased sedation sensitivity of females, neuronal tra expression regulates ethanol sedation independently of ethanol pharmacokinetics. We also show that acute activation of fru-expressing neurons affects ethanol sedation, further supporting a role for fru in regulating this behavior. Thus, we have characterized previously undescribed sex differences in behavioral responses to ethanol, and implicated fru in mediating a subset of these differences.Alcohol is one of the most widely used and abused drugs in the world. The acute effects of ethanol are biphasic: at lower internal concentrations, ethanol acts as a stimulant, whereas, at higher concentrations, it acts as a depressant (1). The stimulant effects of ethanol manifest as elevated mood and energy level in humans and as increased locomotor activity in animal models, and are thought to reflect the reinforcing properties of ethanol (2, 3). In contrast, the depressant effects of ethanol manifest in humans as depressed mood, fatigue, and cognitive and motor impairment (2, 4); animal models similarly exhibit motor incoordination and ultimately sedation (1). Several studies have suggested that susceptibility to alcohol use disorders (AUDs) is correlated with increased sensitivity to the stimulant effects of ethanol and decreased sensitivity to its depressant effects (5, 6). Characterizing the mechanisms underlying acute ethanol responses may therefore provide insight into alcohol addiction.Men and women are differentially affected by acute and long-term ethanol exposure. Men exhibit increased alcohol consumption and a higher incidence of alcohol use disorders compared with women (7, 8). However, women are more susceptible to the negative physical consequences of heavy drinking, such as organ damage and risk of death, and exhibit a faster progression from first use to alcohol abuse and addiction (9, 10). Women are also more strongly affected by acute ethanol intoxication. Part of this effect is pharmacological, as the same ethanol dose (adjusted for body weight) induces a higher blood alcohol content (BAC) in women as a result of differences in body water content (11). However, even when BAC is equalized between the sexes, women exhibit greater ethanol-induced motor impairment and subjective feelings of intoxication than men (4). Thus, there are likely to be sex differences in how ethanol affects the nervous system, but these mechanisms have not yet been identified.The fruit fly Drosophila melanogaster is an established model for studying the genes underlying acute ethanol responses (12). As in humans and rodents, lower doses of ethanol stimulate locomotor activity in flies (13), whereas higher doses induce motor incoordination and sedation (14, 15). Several evolutionarily conserved genes and neuronal signaling pathways regulate ethanol responses in flies and mammals (12). Drosophila therefore offers powerful tools for dissecting the molecular and neural pathways regulating ethanol responses.Despite the number of studies examining acute ethanol responses in flies, sex differences in these behaviors have not been reported. In this study, we find clear sexual dimorphisms in Drosophila ethanol responses. We report that male flies show increased ethanol-induced hyperactivity and greater resistance to ethanol sedation compared with females. The sex difference in ethanol sedation is at least partially mediated by neuronal expression of transformer (tra), which regulates splicing of the neural sex determination gene fruitless (fru). In addition, acute activation of fru-expressing neurons enhances ethanol sedation sensitivity. Thus, we have identified sex differences in ethanol-induced behavior and linked a subset of these differences to fru.     

GIRK3 gates activation of the mesolimbic dopaminergic pathway by ethanol

G protein-gated inwardly rectifying potassium (GIRK) channels are critical regulators of neuronal excitability and can be directly activated by ethanol. Constitutive deletion of the GIRK3 subunit has minimal phenotypic consequences, except in response to drugs of abuse. Here we investigated how the GIRK3 subunit contributes to the cellular and behavioral effects of ethanol, as well as to voluntary ethanol consumption. We found that constitutive deletion of GIRK3 in knockout (KO) mice selectively increased ethanol binge-like drinking, without affecting ethanol metabolism, sensitivity to ethanol intoxication, or continuous-access drinking. Virally mediated expression of GIRK3 in the ventral tegmental area (VTA) reversed the phenotype of GIRK3 KO mice and further decreased the intake of their wild-type counterparts. In addition, GIRK3 KO mice showed a blunted response of the mesolimbic dopaminergic (DA) pathway to ethanol, as assessed by ethanol-induced excitation of VTA neurons and DA release in the nucleus accumbens. These findings support the notion that the subunit composition of VTA GIRK channels is a critical determinant of DA neuron sensitivity to drugs of abuse. Furthermore, our study reveals the behavioral impact of this cellular effect, whereby the level of GIRK3 expression in the VTA tunes ethanol intake under binge-type conditions: the more GIRK3, the less ethanol drinking.G protein-gated inwardly rectifying potassium (GIRK) channels mediate slow inhibitory postsynaptic potentials following activation of G_i/o-coupled receptors, thereby regulating membrane excitability in neuronal, cardiac, and endocrine cells. In neurons, GIRK channels exist as GIRK2 homotetramers or heterotetramers of GIRK1, GIRK2, and/or GIRK3 (reviewed in ref. 1). Despite overlapping distributions in the central nervous system, the three subunits exhibit cell type-specific patterns of expression within some brain regions (2–7). In particular, in the ventral tegmental area (VTA), dopaminergic (DA) neurons express only GIRK2 and GIRK3, whereas non-DA neurons also express GIRK1, a discrepancy that drives differential sensitivity of the two cell populations to G_i/o-coupled receptor (e.g., GABA_B receptor) activation (8–10).In addition to their activation by G_i/o-coupled receptors, GIRK channels also can be directly activated by ethanol (11–14). The behavioral significance of GIRK channel activation by ethanol (either directly or through G proteins) is poorly understood, however. GIRK2 knockout (KO) mice are less sensitive to ethanol’s rewarding and aversive effects, as measured in conditioned place preference and conditioned taste aversion tests (15). Ethanol-induced locomotor stimulation, anxiolytic-like effect, and withdrawal severity are also blunted in the absence of GIRK2 (16). Constitutive GIRK2 deletion produces numerous behavioral abnormalities, however, including increased seizure susceptibility, reduced anxiety-like behavior, hyperactivity, hyperalgesia, and enhanced operant response for food, making it difficult to interpret the effects of ethanol in GIRK2 KO mice (reviewed in ref. 17). In contrast, GIRK3 KO mice are indistinguishable from wild-type (WT) mice in many behavioral assays (e.g., locomotor activity, anxiety-like behavior, motor balance, response for food), suggesting that GIRK3 subunits contribute in a more subtle manner to the function of GIRK channels in vivo (18, 19); however, GIRK3 KO mice exhibit reduced response for cocaine self-administration and blunted hyperexcitability during withdrawal from sedative-hypnotic drugs, suggesting that GIRK3 plays an important role in the neuronal activity and plasticity evoked by drugs of abuse (20, 21).In the present study, we examined the contribution of GIRK3 to the cellular and behavioral effects of ethanol, as well as to ethanol consumption. We show that GIRK3 in VTA neurons is essential for the activation of the mesolimbic DA system by ethanol, and that it selectively modulates binge-like drinking without influencing behavioral manifestations of ethanol intoxication. Our findings support the notion that the subunit composition of VTA GIRK channels is a critical determinant of DA neuron sensitivity to drugs of abuse (9, 10, 22).     

From the Cover: Mitogen-activated protein kinase modulates ethanol inhibition of cell adhesion mediated by the L1 neural cell adhesion molecule

There is a genetic contribution to fetal alcohol spectrum disorders (FASD), but the identification of candidate genes has been elusive. Ethanol may cause FASD in part by decreasing the adhesion of the developmentally critical L1 cell adhesion molecule through interactions with an alcohol binding pocket on the extracellular domain. Pharmacologic inhibition or genetic knockdown of ERK2 did not alter L1 adhesion, but markedly decreased ethanol inhibition of L1 adhesion in NIH/3T3 cells and NG108-15 cells. Likewise, leucine replacement of S1248, an ERK2 substrate on the L1 cytoplasmic domain, did not decrease L1 adhesion, but abolished ethanol inhibition of L1 adhesion. Stable transfection of NIH/3T3 cells with human L1 resulted in clonal cell lines in which L1 adhesion was consistently sensitive or insensitive to ethanol for more than a decade. ERK2 activity and S1248 phosphorylation were greater in ethanol-sensitive NIH/3T3 clonal cell lines than in their ethanol-insensitive counterparts. Ethanol-insensitive cells became ethanol sensitive after increasing ERK2 activity by transfection with a constitutively active MAP kinase kinase 1. Finally, embryos from two substrains of C57BL mice that differ in susceptibility to ethanol teratogenesis showed corresponding differences in MAPK activity. Our data suggest that ERK2 phosphorylation of S1248 modulates ethanol inhibition of L1 adhesion by inside-out signaling and that differential regulation of ERK2 signaling might contribute to genetic susceptibility to FASD. Moreover, identification of a specific locus that regulates ethanol sensitivity, but not L1 function, might facilitate the rational design of drugs that block ethanol neurotoxicity.Prenatal alcohol exposure causes fetal alcohol spectrum disorders (FASD) in up to 2–5% of school-age children and is the leading preventable cause of mental retardation in the Western world (1, 2). The prevalence and presentation of FASD are influenced by the quantity, frequency, and timing of drinking and are modified by a variety of environmental, nutritional, epigenetic, and genetic factors (3–7). The observation that there is greater concordance for fetal alcohol syndrome (FAS) in monozygotic twins than in dizygotic twins suggests that there are susceptibility genes for FASD (8); however, their identification remains elusive. The identification of molecular pathways that regulate sensitivity to ethanol teratogenesis would be helpful in the search for FASD susceptibility genes.One potentially important target of ethanol in the pathogenesis of FASD is the developmentally critical immunoglobulin neural cell adhesion molecule, L1. The homophilic binding of L1 molecules on adjacent cells mediates neuronal migration, axon guidance, and axon fasciculation (9)—developmental events that are disrupted in FASD (10-12). Mutations in the human L1 gene cause brain lesions and neurological abnormalities. Some of these mutations also disrupt L1 homophilic binding (13–16). We noted that brain lesions in children with FASD resemble those of children with mutations in the gene for L1 and demonstrated that concentrations of ethanol attained after just one or two drinks inhibit L1 adhesion in cerebellar granule neurons, neural cell lines, and NIH/3T3 fibroblasts (17-19). Importantly, drugs that block ethanol inhibition of L1 adhesion also prevent ethanol teratogenesis in mice (20–25).L1 adhesion is not universally inhibited by ethanol. For example, ethanol does not inhibit the adhesion of human L1 (hL1) when expressed in myeloma cells or Drosophila S2 cells (26, 27). Even clonal NIH/3T3 cell lines derived from a single transfection with hL1 have shown either an ethanol-sensitive or ethanol-insensitive phenotype over multiple passages and many years (19). These findings suggest that cell-specific factors regulate the sensitivity of L1 to ethanol. The characterization of these factors might prove valuable in identifying candidate genes that govern susceptibility to ethanol teratogenesis.L1 homophilic binding is mediated by its extracellular domain (ECD), which comprises six Ig and five fibronectin type III (Fn) repeats (9). Homophilic binding is potentiated by the folding of the L1-ECD into a horseshoe structure in which the Ig1 and Ig4 domains lie opposed to each other (28–30). Using photolabeling, we demonstrated the interaction of alcohols with a binding pocket at this functionally important Ig1–Ig4 domain interface (31). Mutation of a single alcohol binding residue, Glu-33 on Ig1, did not reduce L1 adhesion, but markedly altered the pharmacology of alcohol inhibition of L1 adhesion (31). These findings suggest that subtle changes in the conformation of an alcohol binding pocket can significantly alter alcohol inhibition of L1 adhesion.If ethanol inhibits L1 adhesion by interacting with an extracellular binding pocket, how, then, might intracellular events regulate these extracellular interactions? The L1 cytoplasmic domain (L1-CD) is highly conserved across species and contains numerous sites for phosphorylation by casein kinase II, p90 ribosomal S6 kinase, extracellular signal-regulated kinase 2 (ERK2) [a member of the mitogen-activated protein kinase (MAPK) family], pp60c-src, chicken embryonic kinase 5, and potentially other kinases (32–35). Phosphorylation of the L1-CD regulates the conformation and function of the extracellular domain (ECD) (36, 37), a phenomenon known as “inside-out” signaling and could conceivably modify the conformation of the alcohol binding pocket in the L1-ECD. Here, we show that L1 sensitivity to ethanol is regulated by phosphorylation of S1248, an ERK2 substrate on the L1-CD. Furthermore, differences in MAPK activity and S1248 phosphorylation determine the ethanol-sensitive or ethanol-insensitive phenotype of clonal L1-expressing NIH/3T3 cells. Finally, two substrains of C57BL mice that differ in susceptibility to ethanol teratogenesis show corresponding differences in MAPK activity.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Refining the pathovar paradigm via phylogenomics of the attaching and effacing Escherichia coli

The attaching and effacing Escherichia coli (AEEC) are characterized by the presence of a type III secretion system encoded by the locus of enterocyte effacement (LEE). Enterohemorrhagic E. coli (EHEC) are often identified as isolates that are LEE+ and carry the Shiga toxin (stx)-encoding phage, which are labeled Shiga toxin-producing E. coli; whereas enteropathogenic E. coli (EPEC) are LEE+ and often carry the EPEC adherence factor plasmid-encoded bundle-forming pilus (bfp) genes. All other LEE+/bfp−/stx− isolates have been historically designated atypical EPEC. These groups have been defined based on the presence or absence of a limited number of virulence factors, many of which are encoded on mobile elements. This study describes the comparative analysis of the genomes of 114 LEE+ E. coli isolates. Based on a whole-genome phylogeny and analysis of type III secretion system effectors, the AEEC are divided into five distinct genomic lineages. The LEE+/stx+/bfp− genomes were primarily divided into two genomic lineages, the O157/O55 EHEC1 and non-O157 EHEC2. The LEE+/bfp+/stx− AEEC isolates sequenced in this study separated into the EPEC1, EPEC2, and EPEC4 genomic lineages. A multiplex PCR assay for identification of each of these AEEC genomic lineages was developed. Of the 114 AEEC genomes analyzed, 31 LEE+ isolates were not in any of the known AEEC lineages and thus represent unclassified AEEC that in most cases are more similar to other E. coli pathovars than to text modification AEEC. Our findings demonstrate evolutionary relationships among diverse AEEC pathogens and the utility of phylogenomics for lineage-specific identification of AEEC clinical isolates.The attaching and effacing Escherichia coli (AEEC) are a significant, yet diverse group of pathogenic organisms that cause human disease (1). The AEEC pathogens include isolates defined by the presence of the locus of enterocyte effacement (LEE), encoding a type III secretion system (T3SS), responsible for the injection of effectors that result in the formation of attaching and effacing lesions (1–3). Within this group of pathogens are the subgroups or pathovars known as enterohemorrhagic E. coli (EHEC) and the enteropathogenic E. coli (EPEC). Both EHEC and EPEC have been associated with severe disease and high mortality rates (4). The EHEC are defined on the molecular level as LEE-positive, Shiga toxin-encoding E. coli based on the presence of the Shiga toxin genes (stxAB). As such, EHEC are a subset of Shiga toxin-producing E. coli (STEC) strains, which are defined solely by the presence of stxAB without regard to LEE status. Genome sequencing of AEEC pathogens has largely focused on the O157:H7 EHEC (5–9), which are a significant cause of severe gastrointestinal illness and hemolytic uremic syndrome (HUS) in the United States (10, 11). The O157:H7 EHEC [LEE+/stx+/bundle-forming pilus-negative (bfp−) AEEC] are hypothesized to have evolved from LEE+/stx−/bfp− O55:H7 by the stepwise acquisition of virulence factors (12–16). To date, only a few non-O157 EHEC/STEC genomes have been sequenced (17, 18). One noticeable exception is the rapid sequencing and analysis of the O104:H4 E. coli outbreak isolates from the 2011 European outbreak (19–21). Although the bacterium implicated in the outbreak contained more genomic similarity to enteroaggregative E. coli (EAEC) isolates than EHEC isolates, the presence of the Shiga toxin-encoding genes and the clinical presentation of HUS led to confusion related to how to accurately classify this organism (19–22). These studies demonstrated the utility of whole-genome sequencing in outbreak situations, as well as the requirement for proper reference genomes for comparison.The AEEC subgroup known as EPEC is a significant cause of persistent watery diarrhea among children worldwide (23). EPEC isolates belonging to a limited number of O:H serotypes (24) contain the LEE region and may contain the EPEC adherence factor (EAF) plasmid-encoding genes encoding the bundle-forming pilus (bfpA-bfpL) (1, 2, 25, 26). The LEE+/stx−/bfp+ AEEC isolates are classified as typical EPEC (tEPEC), whereas the LEE+/stx−/bfp− AEEC isolates are termed atypical EPEC (aEPEC). The LEE+/stx−/bfp− AEEC isolates have been characterized as highly heterogenous, and likely include isolates that were once stx+ EHEC or bfp+ tEPEC isolates, but have lost those features during culture or passage (27, 28). Indeed, stx− isolates cultured from HUS patients have been thought to have lost the Shiga toxin phage during the course of the infection or isolation (29, 30). Meanwhile, the loss of the EAF plasmid from tEPEC has been observed following the passage of EPEC through adults in clinical trials (31, 32). This level of heterogeneity is often observed when the lack of certain virulence factors or genomic features is used as an identifying characteristic, especially when mobile genetic elements such as bacteriophages or plasmids encode these factors (13–16). There is sparse information about the genomic distribution of EPEC, with only one genomic representative of each of the two major lineages of tEPEC from humans, one rabbit-adapted EPEC (E22), and one aEPEC representative (E110019) sequenced to date (33, 34). Although these isolates are excellent starting points for functional analysis, they do not provide enough information to properly describe the genomic diversity of this pathogenic group.In the current study, we demonstrate the diversity of AEEC pathogens using genome sequencing and comparative analysis of 114 AEEC isolates as well as a diverse collection of 24 reference commensal and pathogenic E. coli and Shigella (34). The 114 AEEC genomes include 101 genome sequences that are first analyzed in this study. Among the isolates sequenced were 35 AEEC isolates of the diarrheagenic E. coli (DEC) collection, which provide a link to established reference isolates used in the community (35). The remaining AEEC isolates sequenced in this study were selected to represent a diverse set of diarrheagenic LEE+ isolates that have a wide array of serotypes, geographic locations, and isolation dates. Phylogenomic comparisons demonstrate that the AEEC can be separated into at least five distinct lineages, each with five or more isolates. A whole-genome comparative approach identified regions that are overrepresented or exclusive in subgroupings of the AEEC isolates. Molecular assays targeting these novel regions were then developed to identify each of these phylogeny-based lineages. Additional bioinformatic analysis of type III secretion effector proteins and other virulence-associated genomic islands, demonstrated that some features are lineage restricted in a pattern that is consistent with the whole-genome phylogeny. Importantly, and reminiscent of the recent German outbreak caused by a Shiga toxin-containing O104:H4 EAEC isolate (19–22), our findings demonstrate that the stx-encoding phage is not restricted to specific AEEC lineages. Multiple examples of isolates that contain inconsistent virulence gene and phylogenetic markers were identified in the same phylogenomic lineage, demonstrating a greater genomic variation in these isolates than was previously appreciated. The detection of the lineage-specific markers should be used concurrently with virulence gene detection to assess not only the pathogenic potential, but also the potential evolutionary history of LEE-containing E. coli.     

Recombinant transfer in the basic genome of Escherichia coli

An approximation to the ∼4-Mbp basic genome shared by 32 strains of Escherichia coli representing six evolutionary groups has been derived and analyzed computationally. A multiple alignment of the 32 complete genome sequences was filtered to remove mobile elements and identify the most reliable ∼90% of the aligned length of each of the resulting 496 basic-genome pairs. Patterns of single base-pair mutations (SNPs) in aligned pairs distinguish clonally inherited regions from regions where either genome has acquired DNA fragments from diverged genomes by homologous recombination since their last common ancestor. Such recombinant transfer is pervasive across the basic genome, mostly between genomes in the same evolutionary group, and generates many unique mosaic patterns. The six least-diverged genome pairs have one or two recombinant transfers of length ∼40–115 kbp (and few if any other transfers), each containing one or more gene clusters known to confer strong selective advantage in some environments. Moderately diverged genome pairs (0.4–1% SNPs) show mosaic patterns of interspersed clonal and recombinant regions of varying lengths throughout the basic genome, whereas more highly diverged pairs within an evolutionary group or pairs between evolutionary groups having >1.3% SNPs have few clonal matches longer than a few kilobase pairs. Many recombinant transfers appear to incorporate fragments of the entering DNA produced by restriction systems of the recipient cell. A simple computational model can closely fit the data. Most recombinant transfers seem likely to be due to generalized transduction by coevolving populations of phages, which could efficiently distribute variability throughout bacterial genomes.The increasing availability of complete genome sequences of many different bacterial and archaeal species, as well as metagenomic sequencing of mixed populations from natural environments, has stimulated theoretical and computational approaches to understand mechanisms of speciation and how prokaryotic species should be defined (1–8). Much genome analysis and comparison has been at the level of gene content, identifying core genomes (the set of genes found in most or all genomes in a group) and the continually expanding pan-genome. Population genomics of Escherichia coli has been particularly well studied because of its long history in laboratory research and because many pathogenic strains have been isolated and completely sequenced (9–14). Proposed models of how related groups or species form and evolve include isolation by ecological niche (7–9, 11, 15), decreased homologous recombination as divergence between isolated populations increases (2–4, 8, 14, 16), and coevolving phage and bacterial populations (6).E. coli genomes are highly variable, containing an array of phage-related mobile elements integrated at many different sites (17), random insertions of multiple transposable elements (18), and idiosyncratic genome rearrangements that include inversions, translocations, duplications, and deletions. Although E. coli grows by binary cell division, genetic exchange by homologous recombination has come to be recognized as a significant factor in adaptation and genome evolution (9, 10, 19). Of particular interest has been the relative contribution to genome variability of random mutations (single base-pair differences referred to as SNPs) and replacement of genome regions by homologous recombination with fragments imported from other genomes (here referred to as recombinant transfers or transferred regions). Estimates of the rate, extent, and average lengths of recombinant transfers in the core genome vary widely, as do methods for detecting transferred regions and assessing their impact on phylogenetic relationships (12–14, 20, 21).In a previous comparison of complete genome sequences of the K-12 reference strain MG1655 and the reconstructed genome of the B strain of Delbrück and Luria referred to here as B-DL, we observed that SNPs are not randomly distributed among 3,620 perfectly matched pairs of coding sequences but rather have two distinct regimes: sharply decreasing numbers of genes having 0, 1, 2, or 3 SNPs, and an abrupt transition to a much broader exponential distribution in which decreasing numbers of genes contain increasing numbers of SNPs from 4 to 102 SNPs per gene (22). Genes in the two regimes of the distribution are interspersed in clusters of variable lengths throughout what we referred to as the basic genome, namely, the ∼4 Mbp shared by the two genomes after eliminating mobile elements. We speculated that genes having 0 to 3 SNPs may primarily have been inherited clonally from the last common ancestor, whereas genes comprising the exponential tail may primarily have been acquired by horizontal transfer from diverged members of the population.The current study was undertaken to extend these observations to a diverse set of 32 completely sequenced E. coli genomes and to analyze how SNP distributions in the basic genome change as a function of evolutionary divergence between the 496 pairs of strains in this set. We have taken a simpler approach than those of Touchon et al. (13), Didelot et al. (14), and McNally et al. (21), who previously analyzed multiple alignments of complete genomes of E. coli strains. The appreciably larger basic genome derived here is not restricted to protein-coding sequences and retains positional information.     

Rsu1 regulates ethanol consumption in Drosophila and humans

Alcohol abuse is highly prevalent, but little is understood about the molecular causes. Here, we report that Ras suppressor 1 (Rsu1) affects ethanol consumption in flies and humans. Drosophila lacking Rsu1 show reduced sensitivity to ethanol-induced sedation. We show that Rsu1 is required in the adult nervous system for normal sensitivity and that it acts downstream of the integrin cell adhesion molecule and upstream of the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase to regulate the actin cytoskeleton. In an ethanol preference assay, global loss of Rsu1 causes high naïve preference. In contrast, flies lacking Rsu1 only in the mushroom bodies of the brain show normal naïve preference but then fail to acquire ethanol preference like normal flies. Rsu1 is, thus, required in distinct neurons to modulate naïve and acquired ethanol preference. In humans, we find that polymorphisms in RSU1 are associated with brain activation in the ventral striatum during reward anticipation in adolescents and alcohol consumption in both adolescents and adults. Together, these data suggest a conserved role for integrin/Rsu1/Rac1/actin signaling in modulating reward-related phenotypes, including ethanol consumption, across phyla.Alcohol consumption has a worldwide prevalence of 42% (1), and alcohol is the third most serious risk factor for health loss worldwide (2). The genetic contribution to the development of alcohol use disorders (AUDs) has been estimated at 40–60% based on family, adoption, and twin studies (3, 4). Although several studies in humans and model organisms have described genes and molecular pathways involved in alcohol responses (5, 6), our molecular understanding of how AUDs develop is still incomplete.The vinegar fly, Drosophila melanogaster, is a genetically tractable organism used to model addiction-relevant, ethanol-induced behaviors (7, 8). When exposed to ethanol vapor, flies display biphasic behaviors similar to those elicited in humans. Low ethanol doses induce a state of disinhibition and increased locomotor activity, whereas higher doses lead to loss of postural control and sedation (9, 10). Flies also display addiction-like behaviors similar to mammals. In an ethanol consumption and preference assay (11), for example, flies gradually acquire alcohol preference and will overcome an aversive stimulus to consume alcohol (12).In addition to the similarities that mammals and flies display in their behavioral responses to ethanol, numerous genes and signaling pathways affect alcohol-induced behaviors across organisms. In vitro and in vivo studies in Drosophila and mammals have revealed a link between alcohol and the actin cytoskeleton (13). When cultured primary mouse neurons are exposed to ethanol, there is a gradual decay in filamentous actin that correlates with decreased NMDA receptor current (14). Mice with a genetic KO of the actin-capping protein epidermal growth factor receptor kinase substrate 8 (EPS8), which displays reduced decay of both filamentous actin and NMDA receptor current in the presence of acute ethanol, show increased alcohol preference (14). Flies with mutations in the arouser gene, encoding an EPS8 homolog, also show an ethanol sensitivity phenotype (15).A major regulator of actin cytoskeleton dynamics is the Rho family of small GTPases, including Rho, Rac, and Cdc42, and mutations in these genes affect alcohol-induced behaviors (13). Adult loss of Ras-related C3 botulinum toxin substrate 1 (Rac1) activity, for example, leads to enhanced sensitivity to alcohol-induced sedation, whereas loss of the Rac1 down-regulator RhoGAP18B causes reduced sensitivity (16). Although these studies have shown that Rho family GTPases play a role in alcohol responses, the upstream signaling pathways modulating their effects on actin cytoskeletal dynamics are not understood.Here, we describe the identification and characterization of mutations in the icarus (ics) gene encoding Ras suppressor 1 (Rsu1), which exhibits reduced sensitivity to ethanol-induced sedation. Our experiments reveal that ics mediates normal behavioral responses to ethanol in the adult nervous system by regulating actin dynamics downstream of integrin and upstream of the Rac1 GTPase. Although WT flies gradually acquire ethanol consumption preference over several days, flies completely lacking Rsu1 show heightened naïve preference that does not increase further over the time of the assay. Conversely, flies lacking Rsu1 only in the mushroom bodies (MBs) show no naïve preference and also, fail to acquire preference over time, suggesting that distinct neural circuits mediate naïve and acquired ethanol preference. In humans, RSU1 was associated with frequency of lifetime drinking in an adolescent sample and alcohol dependence in an independent adult replication sample. In adolescents, RSU1 was also associated with altered functional MRI activation in the ventral striatum (VS) during reward anticipation. Our findings, thus, highlight Rsu1 and the integrin/Rsu1/Rac1 signaling pathway as important modulators of reward-related phenotypes, including ethanol consumption across phyla.     

Distinct quaternary structures of the AAA+ Lon protease control substrate degradation

Lon is an ATPase associated with cellular activities (AAA+) protease that controls cell division in response to stress and also degrades misfolded and damaged proteins. Subunits of Lon are known to assemble into ring-shaped homohexamers that enclose an internal degradation chamber. Here, we demonstrate that hexamers of Escherichia coli Lon also interact to form a dodecamer at physiological protein concentrations. Electron microscopy of this dodecamer reveals a prolate structure with the protease chambers at the distal ends and a matrix of N domains forming an equatorial hexamer–hexamer interface, with portals of ∼45 Å providing access to the enzyme lumen. Compared with hexamers, Lon dodecamers are much less active in degrading large substrates but equally active in degrading small substrates. Our results support a unique gating mechanism that allows the repertoire of Lon substrates to be tuned by its assembly state.Protein quality control is vital under stress conditions that promote protein unfolding and aggregation. Escherichia coli Lon degrades many unfolded proteins (1–3) and also degrades folded proteins, including SulA (supressor of Lon protein) and the inclusion-body binding proteins A and B (IbpA and B) (4–6). In E. coli and many other bacteria, Lon is up-regulated under numerous stress conditions (7–10). In mitochondria, Lon helps combat oxidative stress (11–14), and human mitochondrial Lon was recently identified as a potential antilymphoma target (15). It is widely believed that a major role of Lon in all organisms is to degrade misfolded proteins (2, 10, 16).Lon subunits consist of an N domain, a central ATPase associated with cellular activities (AAA+) ATPase module, and a C-terminal peptidase domain. Although early reports suggested that Lon might be a tetramer (17), it is now clear that six subunits of the E. coli enzyme assemble into a hexamer with an internal degradation chamber accessible via an axial pore in the AAA+ ring (18, 19). Lon substrates are recognized, unfolded if necessary by ATP-dependent reactions mediated by the AAA+ ring, and then translocated through the pore and into the peptidase chamber for degradation (20).In many families of ATP-dependent proteases, the AAA+ unfolding/translocation ring and the self-compartmentalized peptidase are encoded by distinct polypeptides, which assemble into independent oligomers before interacting to form the functional protease (21, 22). For example, the ClpXP protease consists of AAA+ ClpX hexamers, which dock with the self-compartmentalized ClpP peptidase. This interaction suppresses the ATPase rate of ClpX and enhances the peptidase activity of ClpP (22). Lon activity cannot be controlled in this way because the ATPase and protease domains are always physically attached. Little is currently known about how Lon activity is regulated, although mutational studies show that the AAA+ and peptidase domains influence each other’s activities (23–25). In some cases, the function of the two domains also appears to be linked via allosteric communication mediated by substrate binding (26, 27).Here, we demonstrate that Lon forms dodecamers that equilibrate with hexamers at physiological concentrations. A structure determined by EM at low resolution reveals a unique protease architecture with the degradation chambers of each hexamer at opposite ends of a prolate ellipsoid. Near the equator of this structure, the arrangement of N domains creates portals, which could serve as entry sites for protein substrates. Formation of the dodecamer suppresses proteolysis of large but not small protein substrates, suggesting that the dodecamer uses a gating mechanism that allows the repertoire of Lon substrates to be tuned by its state of assembly.     

Generation of reactive oxygen species by lethal attacks from competing microbes

Whether antibiotics induce the production of reactive oxygen species (ROS) that contribute to cell death is an important yet controversial topic. Here, we report that lethal attacks from bacterial and viral species also result in ROS production in target cells. Using soxS as an ROS reporter, we found soxS was highly induced in Escherichia coli exposed to various forms of attacks mediated by the type VI secretion system (T6SS), P1vir phage, and polymyxin B. Using a fluorescence ROS probe, we found enhanced ROS levels correlate with induced soxS in E. coli expressing a toxic T6SS antibacterial effector and in E. coli treated with P1vir phage or polymyxin B. We conclude that both contact-dependent and contact-independent interactions with aggressive competing bacterial species and viruses can induce production of ROS in E. coli target cells.Microbial species exist predominantly in complex communities in the natural environment and animal hosts. To survive in a multispecies environment, bacteria have developed various strategies to compete with other species. For example, some bacteria can exert long-range inhibitory effects by secreting diffusible molecules, such as antibiotics, bacteriocins, and H₂O₂ (1), whereas others require direct cell-to-cell contact to kill nearby organisms (2, 3). One such contact-dependent inhibitory system is the type VI secretion system (T6SS), a protein translocating nanomachine expressed by many Gram-negative bacterial pathogens that can kill both bacterial and eukaryotic cells (3–5). Structurally analogous to an inverted bacteriophage tail, the T6SS delivers effectors into target cells by using a contractile sheath to propel an inner tube out of the producer cell and into nearby target cells. The inner tube (composed of Hcp protein) is thought to carry toxic effector proteins within its lumen or on its tip, which is decorated with VgrG and PAAR proteins (4, 6, 7). Given that some cells can detect T6SS attack but not suffer any measurable loss in viability (8, 9), it would seem that cell killing is likely due to the toxicity of effectors rather than membrane disruptions caused by insertion of the spear-like VgrG/PAAR/Hcp tube complex. T6SS-dependent effectors can attack a number of essential cellular targets, including the cell wall (10, 11), membranes (11, 12), and nucleic acids (13), and thus can mimic the actions of antibiotics and bacteriocins. As a model prey or target organism, Escherichia coli can be killed by the T6SS activities of a number of bacteria including Vibrio cholerae (14), Pseudomonas aeruginosa (10, 15), and Acinetobacter baylyi ADP1 (7).Collins and coworkers (16–18) have reported that antibiotic treatment of E. coli elicits the production of reactive oxygen species (ROS) resulting from a series of events involving perturbation of the central metabolic pathway, NADPH depletion, and the Fenton reaction. ROS can cause lethal damage to DNA, lipid, and proteins (19, 20) and thus can contribute to cell death in combination with the deleterious effects of antibiotics on their primary targets. The idea that antibiotics kill bacterial cells, in part, through the action of ROS has been supported by a number of follow-up studies (18, 21–23) but has also been challenged by others as a result of observations contradictory to a model where ROS is the sole mediator of antibiotic lethality (24–26). These observations include the fact that antibiotics kill under anaerobic conditions, oxidation of the hydroxyphenyl fluorescein fluorescence dye used to measure ROS levels is nonspecific, and the extracellular level of H₂O₂ is not elevated by antibiotic treatment (24, 26). To address these concerns, Dwyer et al. (27) used a panel of ROS-detection fluorescence dyes, a defined growth medium under stringent anaerobic conditions, and an in vivo H₂O₂ enzymatic assay to study the effects of antibiotics on cells. The results further support that antibiotics induce ROS generation, which contributes to the efficacy of antibiotics in addition to their primary lethal actions (18, 27, 28).     

Degenerate target sites mediate rapid primed CRISPR adaptation

Prokaryotes encode adaptive immune systems, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated), to provide resistance against mobile invaders, such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus (CRISPR), the formation of guide RNAs from this locus, and the degradation of cognate invader DNA (protospacer). Invaders can escape type I-E CRISPR-Cas immunity in Escherichia coli K12 by making point mutations in the seed region of the protospacer or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive-feedback process termed “priming.” Here, by using a randomized protospacer and PAM library and high-throughput plasmid loss assays, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6-nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of using degenerate target regions, with up to 13 mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position, and is nucleotide dependent. Our findings imply that even outdated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the coevolutionary arms race with their invaders.Bacteria and Archaea are regularly exposed to bacteriophages and other mobile genetic elements, such as plasmids. To control the competing effects of horizontal gene transfer, a spectrum of resistance strategies have evolved in prokaryotes (1). One of the most widespread and well-characterized are the CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated) systems, which provide bacterial “adaptive immunity” (1–8). Simply, CRISPR-Cas functions in three major steps. First, in a process termed “adaptation,” short sequences are derived from the invading element and incorporated into a CRISPR array (9). CRISPR arrays are composed of short repeats that are separated by the foreign-derived sequences, termed “spacers.” Second, CRISPRs are transcribed into a pre-CRISPR RNA (pre-crRNA), which is then processed into short crRNAs, which encompass portions of the repeats and most—or all—of the spacer. Finally, as part of a Cas ribonucleoprotein complex, the crRNAs guide a sequence-specific targeting of complementary nucleic acids (for recent reviews, see refs. 1–7).CRISPR-Cas systems are divided into three major types (I–III) and further categorized into subtypes (e.g., I-A to I-F) (10). The mechanisms of both crRNA generation and interference differ between the types and there are even significant differences between closely related subtypes. However, Cas1 and Cas2 are the only two Cas proteins completely conserved across all CRISPR-Cas systems and they are crucial for adaptation in Escherichia coli (10–12). The acquisition of new spacers is the most poorly understood stage in CRISPR-Cas immunity, mainly hindered by the paucity of robust laboratory assays to monitor this process (reviewed in ref. 9). Streptococcus thermophilus is highly proficient at spacer acquisition and provided much of the early insight into adaptation, showing that new spacers are typically acquired at one end of the CRISPR array from either phages (13–15) or plasmids (16). Recently, spacer acquisition has been detected in a variety of other systems (11, 12, 17–20). Adjacent to the expanding end of the array is the leader region, which harbors the promoter for pre-crRNA expression and sequences important for spacer acquisition (12, 21). Recent studies in E. coli in the type I-E system have shown that spacer acquisition can occur from phages and plasmids either when the Cas1 and Cas2 proteins are overexpressed or if the native cas genes are up-regulated, because of deletion of hns (11, 12, 20–22). The DNA targets (termed “protospacers”) of newly acquired spacers are consistently flanked by protospacer-adjacent motifs (PAMs), with the E. coli type I-E consensus 5′-protospacer-CTT-3′. PAMs were originally identified computationally (23) and were shown to play a role in interference in an early study (14). The importance of PAMs in the recognition and selection of precursor-spacers (prespacers) during adaptation was demonstrated unequivocally using assays that were independent of interference (12, 21). The simple overexpression of Cas1 and Cas2, in the absence of other cas genes, demonstrated these are the only Cas proteins essential for adaptation and are likely to recognize PAMs (12).Adaptation consists of two related stages, termed “naïve” and “primed” (9). Naïve adaptation occurs when a bacterium harboring a CRISPR-Cas system is infected by a new foreign element that it has not previously encountered. Although the acquisition of a new spacer can result in effective protection from the element, point mutations within the protospacer or PAM allow the element to escape CRISPR-Cas targeting (14, 24, 25). This aspect had been viewed as a weakness of CRISPR-Cas interference, but recent studies show that a positive feedback loop—called priming—occurs, which enables one or more new spacers to be acquired (11, 20, 22). Specifically, single mutations within either the PAM or the seed region of the protospacer, although inactive for interference, promote the rapid acquisition of new spacers from the same target (11). Priming is proposed to allow an effective response against viral or plasmid escapees through the incorporation of new spacers. Unlike naïve adaptation, priming is more complex, and in type I-E systems requires Cas1, Cas2, crRNA, the targeting complex termed Cascade [CRISPR-associated complex for antiviral defence, composed of Cse1, Cse2, Cas7, Cas5, and Cas6e (26–28)] and the Cas3 nuclease/helicase (11). Interestingly, the vast majority of spacers acquired through priming are derived from the same DNA strand as the original priming spacer (11, 20, 22). In addition, priming in E. coli was abolished by two mutations in the protospacer and PAM regions (11).In this study, we generated a mutagenic variant library of a protospacer and PAM region and used both individual high-throughput plasmid-loss assays and next-generation sequencing to determine the limits of both direct interference and indirect interference through priming. Our results demonstrate that direct interference tolerates mutations mostly at very specific positions in the protospacer, whereas priming tolerates extensive mutation of the PAM and protospacer regions. The results have wide evolutionary consequences for primed acquisition and could explain the retention of multiple “older” spacers in CRISPR arrays.     

Pharmacological recruitment of aldehyde dehydrogenase 3A1 (ALDH3A1) to assist ALDH2 in acetaldehyde and ethanol metabolism in vivo

Correcting a genetic mutation that leads to a loss of function has been a challenge. One such mutation is in aldehyde dehydrogenase 2 (ALDH2), denoted ALDH2*2. This mutation is present in ∼0.6 billion East Asians and results in accumulation of toxic acetaldehyde after consumption of ethanol. To temporarily increase metabolism of acetaldehyde in vivo, we describe an approach in which a pharmacologic agent recruited another ALDH to metabolize acetaldehyde. We focused on ALDH3A1, which is enriched in the upper aerodigestive track, and identified Alda-89 as a small molecule that enables ALDH3A1 to metabolize acetaldehyde. When given together with the ALDH2-specific activator, Alda-1, Alda-89 reduced acetaldehyde-induced behavioral impairment by causing a rapid reduction in blood ethanol and acetaldehyde levels after acute ethanol intoxication in both wild-type and ALDH2-deficient, ALDH2*1/*2, heterozygotic knock-in mice. The use of a pharmacologic agent to recruit an enzyme to metabolize a substrate that it usually does not metabolize may represent a novel means to temporarily increase elimination of toxic agents in vivo.The aldehyde dehydrogenase (ALDH) superfamily comprises 19 enzymes that catalyze the oxidation and detoxification of a wide spectrum of short and long aliphatic and aromatic aldehydes (1, 2). Acetaldehyde is a product of ethanol metabolism, which is consumed by >80% of humans. In addition to the behavioral impairment risk, the ethanol metabolite, acetaldehyde, is a proven group 1 carcinogen (3). Above and beyond the health risk in the general population, ∼40% of East Asians [∼560 million or ∼8% of the world’s population (4, 5)] carry a point mutation in the ALDH2 gene that leads to a severe enzyme deficiency and accumulation of toxic acetaldehyde (6). After consuming two units of alcoholic beverage, blood acetaldehyde levels reach 60 μM and remain elevated for several hours in heterozygotic carriers of this mutation, whereas within 30 min, acetaldehyde levels are not detected in carriers of the wild-type enzyme (7). The inactivating glutamate 487 to lysine mutation (E487K) (8), denoted ALDH2*2 (vs. ALDH2*1 for the wild-type allele) (9), is dominant; heterozygotic ALDH2*1/*2 individuals have only 17–30% of wild-type activity (10, 11). ALDH2 deficiency is associated with severe facial flushing, longer behavioral impairment (intoxication), longer-lasting headache, nausea, and palpitations from moderate ethanol consumption compared with individuals with normal ALDH2*1/*1 (4).Despite the unpleasant reaction to acetaldehyde accumulation, 17–27% of individuals with ALDH2*1/*2 (heterozygotes) are heavy drinkers (4, 12, 13). These heterozygotic heavy drinkers (consuming >18 alcoholic drinks/week) have greater than 80-fold increased risk for squamous cell carcinomas in the upper aerodigestive track (UADT; i.e., oral cavity and pharynx, larynx, and esophagus) compared with a ∼fourfold increase in wild-type ALDH2*1/*1 heavy drinkers (4, 13–16). Further, an elevated risk of hepatocarcinoma and its recurrence occurs among hepatitis C-infected patients with the ALDH2*2 mutation (17). Acetaldehyde levels are particularly high in the saliva after ethanol ingestion (18), leading to a significant increase in acetaldehyde-DNA adduct levels in ALDH2*1/*2 heterozygotes, even after moderate ethanol consumption (19). Because acetaldehyde is a carcinogen, and the duration and extent of exposure influences its toxicity, increasing the rate of acetaldehyde elimination, especially in ALDH2*1/*2 heterozygotes, may reduce important health risks. We therefore set out to identify a pharmacologic tool to “recruit” another member of the ALDH family to enhance the elimination of acetaldehyde. We focused on ALDH3A1 because it is highly expressed in the epithelial cell layer of the UADT, stomach, liver, and kidney (20–22). ALDH3A1 metabolizes aromatic, aliphatic medium chain aldehydes and α,β-hydroxyalkenal aldehydes, but not acetaldehyde under basal condition (21, 23). The challenge therefore was to find a pharmacologic means to enable ALDH3A1 to assist in the elimination of acetaldehyde.Our laboratory has identified a group of small molecules, Aldas (aldehyde dehydrogenase activators), that increase the catalytic activity of ALDH2 (24). One of these molecules, Alda-1, interacts with the substrate-binding site of ALDH2 and accelerates acetaldehyde metabolism to carboxylic acid by about twofold (24, 25), probably by increasing productive interactions of the substrate with the catalytic Cys302 and reducing the K_m for the NAD⁺ coenzyme (25). We reasoned that another small molecule may increase productive interaction of acetaldehyde with Cys243 in the catalytic site of ALDH3A1, and thus temporarily recruit this enzyme to assist the mutant ALDH2 in eliminating acetaldehyde.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.