Ethanol- and PARP-Mediated Regulation of Ribosome-Associated Long Non-Coding RNA (lncRNA) in Pyramidal Neurons.

Although, by definition, long noncoding RNAs (lncRNAs) are not translated, they are sometimes associated with ribosomes. In fact, some estimates suggest the existence of more than 50 K lncRNA molecules that could encode for small peptides. We examined the effects of an ethanol and Poly-ADP Ribose Polymerase (PARP) inhibitor (ABT-888) on ribosome-bound lncRNAs. Mice were administered via intraperitoneal injection (i.p.) either normal saline (CTL) or ethanol (EtOH) twice a day for four consecutive days. On the fourth day, a sub-group of mice administered with ethanol also received ABT-888 (EtOH+ABT). Ribosome-bound lncRNAs in CaMKIIα-expressing pyramidal neurons were measured using the Translating Ribosome Affinity Purification (TRAP) technique. Our findings show that EtOH altered the attachment of 107 lncRNA transcripts, while EtOH+ABT altered 60 lncRNAs. Among these 60 lncRNAs, 49 were altered by both conditions, while EtOH+ABT uniquely altered the attachment of 11 lncRNA transcripts that EtOH alone did not affect. To validate these results, we selected eight lncRNAs (Mir124-2hg, 5430416N02Rik, Snhg17, Snhg12, Snhg1, Mir9-3hg, Gas5, and 1110038B12Rik) for qRT-PCR analysis. The current study demonstrates that ethanol-induced changes in lncRNA attachment to ribosomes can be mitigated by the addition of the PARP inhibitor ABT-888.

Effects of alcohol and PARP inhibition on RNA ribosomal engagement in cortical excitatory neurons.

We report on the effects of ethanol (EtOH) and Poly (ADP-ribose) polymerase (PARP) inhibition on RNA ribosomal engagement, as a proxy for protein translation, in prefrontal cortical (PFC) pyramidal neurons. We hypothesized that EtOH induces a shift in RNA ribosomal-engagement (RE) in PFC pyramidal neurons, and that many of these changes can be reversed using a PARP inhibitor. We utilized the translating ribosome affinity purification (TRAP) technique to isolate cell type-specific RNA. Transgenic mice with EGFP-tagged Rpl10a ribosomal protein expressed only in CaMKIIα-expressing pyramidal cells were administered EtOH or normal saline (CTL) i.p. twice a day, for four consecutive days. On the fourth day, a sub-group of mice that received EtOH in the previous three days received a combination of EtOH and the PARP inhibitor ABT-888 (EtOH + ABT-888). PFC tissue was processed to isolate both, CaMKIIα pyramidal cell-type specific ribosomal-engaged RNA (TRAP-RNA), as well as genomically expressed total-RNA from whole tissue, which were submitted for RNA-seq. We observed EtOH effects on RE transcripts in pyramidal cells and furthermore treatment with a PARP inhibitor "reversed" these effects. The PARP inhibitor ABT-888 reversed 82% of the EtOH-induced changes in RE (TRAP-RNA), and similarly 83% in the total-RNA transcripts. We identified Insulin Receptor Signaling as highly enriched in the ethanol-regulated and PARP-reverted RE pool and validated five participating genes from this pathway. To our knowledge, this is the first description of the effects of EtOH on excitatory neuron RE transcripts from total-RNA and provides insights into PARP-mediated regulation of EtOH effects.

Complete Genome Sequence of Arthrobacter Phage GantcherGoblin Exhibits Both Conservation with Subcluster AU6 Phages and Genetic Novelty.

GantcherGoblin is a lytic siphovirus that was isolated on Arthrobacter globiformis B-2979 from soil collected in Massachusetts. The 55,368-bp genome has a GC content of 50.1% and 91 predicted protein-coding genes. Based on gene content similarity to phages in the Actinobacteriophage Database, GantcherGoblin was assigned to phage subcluster AU6.

Complete Genome Sequence of Gordonia rubripertincta Bacteriophage Hexbug Suggests Potential for a New CT Subcluster.

Through the SEA-PHAGES program at Tufts University, a bacteriophage infecting Gordonia rubripertincta NRRL B-16540 was isolated and characterized. Hexbug is a lytic phage and is currently one of 44 phages belonging to cluster CT. The Hexbug genome shares >96% nucleotide identity with cluster CT phage Orla.

Differential H3K9me2 heterochromatin levels and concordant mRNA expression in postmortem brain tissue of individuals with schizophrenia, bipolar, and controls.

The existence of repressive and durable chromatin assemblies along gene promoters or networks, especially in the brain, is of theoretical and therapeutic relevance in a subset of individuals diagnosed with schizophrenia who experience a chronic, persistent, and treatment-resistant trajectory. We used chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) to generate an epigenomic map that includes differential sites occupied by di-methylated lysine 9 of histone 3 (H3K9me2), a repressive modification that is yet unexplored in human postmortem brain tissue. We have discovered over 150 significantly differential promoter sites in the postmortem prefrontal cortex tissue of individuals diagnosed with schizophrenia (n = 15) when compared to controls (n = 15). Potentially dysregulated gene categories include postsynaptic proteins, processing enzymes (for proproteins, lipids, and oxidative stress), cadherin family genes, the complement system, and peptide hormones. Ten genes with significantly increased or decreased H3K9me2 promoter occupation were selected through statistical analysis, function, or previous GWAS association, and Quantitative RT-PCR (qRT-PCR) was performed on an extended sample of postmortem brain tissue, adding an additional 17 controls, 7 individuals with schizophrenia, and 19 individuals with bipolar samples (n = 32 control, 22 schizophrenia, 19 bipolar). This approach revealed that mRNA expression levels correlated with chromatin modification levels in eight of 10 selected genes, and mRNA expression in the total sample could be predicted by the occupancy of H3K9me2. Utilization of this method and replication in a larger sample open a pathway to durable and restrictive epigenomic assemblies whose accumulation across the lifespan of individuals diagnosed with schizophrenia may explain treatment resistance, and advance therapeutic options.

Rapid evolutionary turnover of mobile genetic elements drives bacterial resistance to phages.

Although it is generally accepted that phages drive bacterial evolution, how these dynamics play out in the wild remains poorly understood. We found that susceptibility to viral killing in marine Vibrio is mediated by large and highly diverse mobile genetic elements. These phage defense elements display exceedingly fast evolutionary turnover, resulting in differential phage susceptibility among clonal bacterial strains while phage receptors remain invariant. Protection is cumulative, and a single bacterial genome can harbor 6 to 12 defense elements, accounting for more than 90% of the flexible genome among close relatives. The rapid turnover of these elements decouples phage resistance from other genomic features. Thus, resistance to phages in the wild follows evolutionary trajectories alternative to those predicted from laboratory-based evolutionary experiments.

RRSP and RID Effector Domains Dominate the Virulence Impact of Vibrio vulnificus MARTX Toxin.

BACKGROUND: The bacterial pathogen Vibrio vulnificus causes severe septic foodborne infections. The multifunctional autoprocessing repeats-in-toxins (MARTX) toxin is an important secreted virulence factor. The effector domain region is essential for lethal intestinal infection in mice, but the contribution of each of the 5 effector domains to infection has not been investigated. METHODS: V. vulnificus mutants with varying effector domain content were inoculated intragastrically to mice, and the time to death was monitored to establish the contribution of each effector domain to overall virulence. Each strain was also tested for bacterial dissemination from the intestine to internal organs and for inhibition of phagocytosis. RESULTS: The effector domain region was required for V. vulnificus to inhibit phagocytosis by J774 macrophages, but no single effector domain was required. No single MARTX effector domain was necessary for bacterial dissemination. Nonetheless, overall survival of infected mice differed with respect to the infecting V. vulnificus strain. Removal of rid or rrsp significantly reduced the virulence potential of V. vulnificus, while deletion of duf1 or abh accelerated the time to death. CONCLUSION: Rho GTPases inactivation domain and Ras/Rap1-specific endopeptidase each exert greater effects on virulence than other MARTX domains, suggesting that modulation of the Rho/Ras family of GTPases is a critical function of the toxin during intestinal infection.

Efficacy of Ceftriaxone, Cefepime, Doxycycline, Ciprofloxacin, and Combination Therapy for Vibrio vulnificus Foodborne Septicemia.

Foodborne Vibrio vulnificus infections are associated with higher rates of sepsis and mortality than wound infections; however, antibiotic efficacy studies have not been performed in foodborne infection models. The efficacies of ceftriaxone, cefepime, doxycycline, ciprofloxacin, and combination therapy were assessed in V. vulnificus intestinal infection in mice in order to model foodborne infections. In accordance with prior studies of cefotaxime, cefepime was synergistic with doxycycline and ciprofloxacin in vitro; combination therapy significantly decreased bacterial growth, by ≥2 log10 units, from that with antibiotic monotherapy (P < 0.01). In vivo, survival rates in the ceftriaxone (50%), doxycycline (79%), and ciprofloxacin (80%) groups were significantly higher than those in the control group (0%) (P < 0.0001). Survival was significantly higher with ceftriaxone-doxycycline (91%) or ceftriaxone-ciprofloxacin (100%) therapy than with ceftriaxone (50%) (P ≤ 0.05). Survival with cefepime-doxycycline (96%) or cefepime-ciprofloxacin (90%) therapy was significantly higher than that with cefepime alone (20%) (P < 0.001). There was no difference in survival between the combination therapy groups. Thus, we conclude that combination therapy was the most effective treatment for foodborne V. vulnificus septicemia. In a septic patient with a recent ingestion of raw seafood, cefepime in combination with doxycycline or ciprofloxacin should be initiated for coverage of resistant Gram-negative organisms and V. vulnificus pending a microbiological diagnosis. Once a diagnosis of foodborne V. vulnificus septicemia is established, treatment can safely transition to ceftriaxone in combination with doxycycline or ciprofloxacin.

Variable Virulence of Biotype 3 Vibrio vulnificus due to MARTX Toxin Effector Domain Composition.

Vibrio vulnificus is an environmental organism that causes septic human infections characterized by high morbidity and mortality. The annual incidence and global distribution of this pathogen are increasing as ocean waters warm. Clinical strains exhibit variations in the primary virulence toxin, suggesting a potential for the emergence of new strains with altered virulence properties. A clonal outbreak of tilapia-associated wound infections in Israel serves as a natural experiment for the sudden emergence of a new V. vulnificus strain. The effector domain content of the multifunctional autoprocessing RTX (MARTX) toxin of the outbreak-associated biotype 3 (BT3) strains was previously shown to harbor a modification generated by recombination. The modification introduced an actin-induced adenylate cyclase effector domain (ExoY) and an effector domain that disrupts the Golgi organelle (DmX). Here, we report that the exchange of these effector domains for a putative progenitor biotype 1 toxin arrangement produces a toxin that slows the lysis kinetics of targeted epithelial cells but increases cellular rounding phenotypes in response to bacteria. In addition, replacing the biotype 3 toxin variant with the putative progenitor biotype 1 variant renders the resulting strain significantly more virulent in mice. This suggests that the exchange of MARTX effector domains during the emergence of BT3 generated a toxin with reduced toxin potency, resulting in decreased virulence of this outbreak-associated strain. We posit that selection for reduced virulence may serve as a route for this lethal infectious agent to enter the human food chain by allowing it to persist in natural hosts. IMPORTANCEVibrio vulnificus is a serious infection linked to climate change. The virulence capacity of these bacteria can vary by gene exchange, resulting in new variants of the primary virulence toxin. In this study, we tested whether the emergence of an epidemic strain of V. vulnificus with a novel toxin variant correlated with a change in virulence. We found that restoring the biotype 3 toxin variant to the putative progenitor-type toxin resulted in dramatically increased virulence, revealing that the emergence of the biotype 3 strain could be linked to virulence reduction. This reduced virulence, previously found also in the biotype 1 strain, suggests that reduced virulence may stimulate outbreaks, as strains have greater capacity to enter the human food chain through reduced impact to environmental hosts.

Surface hypothermia predicts murine mortality in the intragastric Vibrio vulnificus infection model.

BACKGROUND: The Gram-negative bacterium Vibrio vulnificus can cause severe disease in humans who consume undercooked, contaminated seafood. To study food-borne V. vulnificus disease in the laboratory, mouse virulence studies predominantly use death as the primary experimental endpoint because behaviorally based moribund status does not consistently predict lethality. This study assessed ventral surface temperature (VST) and its association with mouse survival during V. vulnificus virulence studies as an efficacious, humane alternative. METHODS: VST of mice intragastrically inoculated with V. vulnificus was measured every 2-h for 24 h and data for minimal VST analyzed for prediction of lethal outcome. RESULTS: In contrast to the relatively stable VST of mock-infected control animals, mice infected with V. vulnificus exhibited hypothermia with minima occurring 8 to 12 h post-inoculation. The minimum VST of mice that proceeded to death was significantly lower than that of surviving mice. VST ≤ 23.5 °C was predictive of subsequent death with a sensitivity of 68% and specificity of 95%. CONCLUSIONS: Use of VST ≤ 23.5 °C as an experimental endpoint during V. vulnificus infection has potential to reduce suffering of nearly 70% of mice for a mean of 10 h per mouse, without compromising experimental efficacy. Temperature cutoff of 23.5 °C exhibited 93% positive and 77% negative predictive value. For future V. vulnificus virulence studies requiring only binary comparison (e.g., LD50 assays), we find that VST can be applied as a humane endpoint. However, use of VST is not recommended when detailed survival kinetics are desired.

The Effector Domain Region of the Vibrio vulnificus MARTX Toxin Confers Biphasic Epithelial Barrier Disruption and Is Essential for Systemic Spread from the Intestine.

Vibrio vulnificus causes highly lethal bacterial infections in which the Multifunctional Autoprocessing Repeats-in-Toxins (MARTX) toxin product of the rtxA1 gene is a key virulence factor. MARTX toxins are secreted proteins up to 5208 amino acids in size. Conserved MARTX N- and C-terminal repeat regions work in concert to form pores in eukaryotic cell membranes, through which the toxin's central region of modular effector domains is translocated. Upon inositol hexakisphosphate-induced activation of the of the MARTX cysteine protease domain (CPD) in the eukaryotic cytosol, effector domains are released from the holotoxin by autoproteolytic activity. We previously reported that the native MARTX toxin effector domain repertoire is dispensable for epithelial cellular necrosis in vitro, but essential for cell rounding and apoptosis prior to necrotic cell death. Here we use an intragastric mouse model to demonstrate that the effector domain region is required for bacterial virulence during intragastric infection. The MARTX effector domain region is essential for bacterial dissemination from the intestine, but dissemination occurs in the absence of overt intestinal tissue pathology. We employ an in vitro model of V. vulnificus interaction with polarized colonic epithelial cells to show that the MARTX effector domain region induces rapid intestinal barrier dysfunction and increased paracellular permeability prior to onset of cell lysis. Together, these results negate the inherent assumption that observations of necrosis in vitro directly predict bacterial virulence, and indicate a paradigm shift in our conceptual understanding of MARTX toxin function during intestinal infection. Results implicate the MARTX effector domain region in mediating early bacterial dissemination from the intestine to distal organs-a key step in V. vulnificus foodborne pathogenesis-even before onset of overt intestinal pathology.

MARTX toxins as effector delivery platforms.

Bacteria frequently manipulate their host environment via delivery of microbial 'effector' proteins to the cytosol of eukaryotic cells. In the case of the multifunctional autoprocessing repeats-in-toxins (MARTX) toxin, this phenomenon is accomplished by a single, >3500 amino acid polypeptide that carries information for secretion, translocation, autoprocessing and effector activity. MARTX toxins are secreted from bacteria by dedicated Type I secretion systems. The released MARTX toxins form pores in target eukaryotic cell membranes for the delivery of up to five cytopathic effectors, each of which disrupts a key cellular process. Targeted cellular processes include modulation or modification of small GTPases, manipulation of host cell signaling and disruption of cytoskeletal integrity. More recently, MARTX toxins have been shown to be capable of heterologous protein translocation. Found across multiple bacterial species and genera--frequently in pathogens lacking Type 3 or Type 4 secretion systems--MARTX toxins in multiple cases function as virulence factors. Innovative research at the intersection of toxin biology and bacterial genetics continues to elucidate the intricacies of the toxin as well as the cytotoxic mechanisms of its diverse effector collection.

Distinct roles of the repeat-containing regions and effector domains of the Vibrio vulnificus multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin.

UNLABELLED: Vibrio vulnificus is a seafood-borne pathogen that destroys the intestinal epithelium, leading to rapid bacterial dissemination and death. The most important virulence factor is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin comprised of effector domains in the center region flanked by long repeat-containing regions which are well conserved among MARTX toxins and predicted to translocate effector domains. Here, we examined the role of the repeat-containing regions using a modified V. vulnificus MARTX (MARTX(Vv)) toxin generated by replacing all the internal effector domains with ß-lactamase (Bla). Bla activity was detected in secretions from the bacterium and also in the cytosol of intoxicated epithelial cells. The modified MARTX(Vv) toxin without effector domains retained its necrotic activity but lost its cell-rounding activity. Further, deletion of the carboxyl-terminal repeat-containing region blocked toxin secretion from the bacterium. Deletion of the amino-terminal repeat-containing region had no effect on secretion but completely abolished translocation and necrosis. Neither secretion nor translocation was affected by enzymatically inactivating the cysteine protease domain of the toxin. These data demonstrate that the amino-terminal and carboxyl-terminal repeat-containing regions of the MARTXVv toxin are necessary and sufficient for the delivery of effector domains and epithelial cell lysis in vitro but that effector domains are required for other cytopathic functions. Furthermore, Ca(2+)-dependent secretion of the modified MARTX(Vv) toxin suggests that nonclassical RTX-like repeats found in the carboxyl-terminal repeat-containing region are functionally similar to classical RTX repeats found in other RTX proteins. IMPORTANCE: Up to 95% of deaths from seafood-borne infections in the United States are due solely to one pathogen, V. vulnificus. Among its various virulence factors, the MARTX(Vv) toxin has been characterized as a critical exotoxin for successful pathogenesis of V. vulnificus in mouse infection models. Similarly to MARTX toxins of other pathogens, MARTX(Vv) toxin is comprised of repeat-containing regions, central effector domains, and an autoprocessing cysteine protease domain. Yet how each of these regions contributes to essential activities of the toxins has not been fully identified for any of MARTX toxins. Using modified MARTX(Vv) toxin fused with ß-lactamase as a reporter enzyme, the portion(s) responsible for toxin secretion from bacteria, effector domain translocation into host cells, rapid host cell rounding, and necrotic host cell death was identified. The results are relevant for understanding how MARTX(Vv) toxin serves as both a necrotic pore-forming toxin and an effector delivery platform.

Impact of diet-induced obesity on intestinal stem cells: hyperproliferation but impaired intrinsic function that requires insulin/IGF1.

Nutrient intake regulates intestinal epithelial mass and crypt proliferation. Recent findings in model organisms and rodents indicate nutrient restriction impacts intestinal stem cells (ISC). Little is known about the impact of diet-induced obesity (DIO), a model of excess nutrient intake on ISC. We used a Sox9-EGFP reporter mouse to test the hypothesis that an adaptive response to DIO or associated hyperinsulinemia involves expansion and hyperproliferation of ISC. The Sox9-EGFP reporter mouse allows study and isolation of ISC, progenitors, and differentiated lineages based on different Sox9-EGFP expression levels. Sox9-EGFP mice were fed a high-fat diet for 20 weeks to induce DIO and compared with littermates fed low-fat rodent chow. Histology, fluorescence activated cell sorting, and mRNA analyses measured impact of DIO on jejunal crypt-villus morphometry, numbers, and proliferation of different Sox9-EGFP cell populations and gene expression. An in vitro culture assay directly assessed functional capacity of isolated ISC. DIO mice exhibited significant increases in body weight, plasma glucose, insulin, and insulin-like growth factor 1 (IGF1) levels and intestinal Igf1 mRNA. DIO mice had increased villus height and crypt density but decreased intestinal length and decreased numbers of Paneth and goblet cells. In vivo, DIO resulted in a selective expansion of Sox9-EGFP(Low) ISC and percentage of ISC in S-phase. ISC expansion significantly correlated with plasma insulin levels. In vitro, isolated ISC from DIO mice formed fewer enteroids in standard 3D Matrigel culture compared to controls, indicating impaired ISC function. This decreased enteroid formation in isolated ISC from DIO mice was rescued by exogenous insulin, IGF1, or both. We conclude that DIO induces specific increases in ISC and ISC hyperproliferation in vivo. However, isolated ISC from DIO mice have impaired intrinsic survival and growth in vitro that can be rescued by exogenous insulin or IGF1.