Mutations in the IMD Pathway and Mustard Counter Vibrio cholerae Suppression of Intestinal Stem Cell Division in Drosophila

ABSTRACT Vibrio cholerae is an estuarine bacterium and an intestinal pathogen of humans that causes severe epidemic diarrhea. In the absence of adequate mammalian models in which to study the interaction of V. cholerae with the host intestinal innate immune system, we have implemented Drosophila melanogaster as a surrogate host. We previously showed that immune deficiency pathway loss-of-function and mustard gain-of-function mutants are less susceptible to V. cholerae infection. We find that although the overall burden of intestinal bacteria is not significantly different from that of control flies, intestinal stem cell (ISC) division is increased in these mutants. This led us to examine the effect of V. cholerae on ISC division. We report that V. cholerae infection and cholera toxin decrease ISC division. Because IMD pathway and Mustard mutants, which are resistant to V. cholerae, maintain higher levels of ISC division during V. cholerae infection, we hypothesize that suppression of ISC division is a virulence strategy of V. cholerae and that accelerated epithelial regeneration protects the host against V. cholerae. Extension of these findings to mammals awaits the development of an adequate experimental model

NELF Potentiates Gene Transcription in the Drosophila Embryo

A hallmark of genes that are subject to developmental regulation of transcriptional elongation is association of the negative elongation factor NELF with the paused RNA polymerase complex. Here we use a combination of biochemical and genetic experiments to investigate the in vivo function of NELF in the Drosophila embryo. NELF associates with different gene promoter regions in correlation with the association of RNA polymerase II (Pol II) and the initial activation of gene expression during the early stages of embryogenesis. Genetic experiments reveal that maternally provided NELF is required for the activation, rather than the repression of reporter genes that emulate the expression of key developmental control genes. Furthermore, the relative requirement for NELF is dictated by attributes of the flanking cis-regulatory information. We propose that NELF-associated paused Pol II complexes provide a platform for high fidelity integration of the combinatorial spatial and temporal information that is central to the regulation of gene expression during animal development

Infection decreases short chain phospholipids in the intestines of <i>V</i>. <i>cholerae</i>-infected flies.

<p><b>Lysophospholipids are more abundant in the intestines of Δ<i>gcvT</i> mutant infected flies.</b> (A) Schematic representation of the lipid species discussed in the text. Grey circles represent the polar headgroups, lines represent the fatty acid chains, and surrounding shaded shapes represent the relative space filled by the head group and fatty acid chains of each lipid species. The larger the area of the head group relative to the area filled by fatty acid chains, the greater the propensity to form a highly-curved structure such as a small lipid droplet. (B) Two mechanisms by which small lipid droplets may form a larger lipid droplet are illustrated. Enlargement, shown on the left, results when a large influx of triglycerides must be accommodated. Coalescence results when the supply of phospholipids is inadequate to coat the triglyceride core, and smaller lipid droplets join to minimize exposed surface area. (C-K) LC-MS/MS-based lipidomic analysis of the intestines of flies fed LB alone or inoculated with wild-type <i>V</i>. <i>cholerae</i> (WT) or a Δ<i>gcvT</i> mutant. (C) Triglycerides (TG), (D) Total phosphatidylcholine species with a total of 30 carbons or less in the two fatty acid chains (PC≤30), (E) Individual phosphatidylcholine species with the total number of fatty acid carbons indicated below. (F) Total phosphatidylethanolamine species with a total of 30 carbons or less in the two fatty acid chains (PE≤30) (G) Individual phosphatidylethanolamine species with the total number of fatty acid carbons indicated below. (H) Total lysophosphatidylcholine species. (I) Individual lysophosphatidylcholine species with the total number of fatty acid carbons indicated below. (J) Total lysophosphatidylethanolamine species. (K) Individual lysophosphatidylethanolamine species with the total number of fatty acid carbons indicated below. (L) Putative phospholipase cascade. (M-P) Lipidomic analysis of the intestines of flies infected with wild-type <i>V</i>. <i>cholerae</i> (WT) or a <i>Δacs1</i> mutant. (M) Total phosphatidylcholine species with fatty acid carbons less than or equal to 30 (PC≤30). (N) Total phosphatidylethanolamine species with fatty acid carbons less than or equal to 30 (PE≤30). (O) Total lysophosphatidylcholine species. (P) Total lysophosphatidylethanolamine species. For pooled data, the mean and SD are shown. Pairwise statistical significance was calculated using a student’s t-test (*p<0.05, **p<0.01, ***p<0.001).</p

As compared with infection with wild-type <i>V</i>. <i>cholerae</i>, <i>Drosophila</i> infection with a <i>V</i>. <i>cholerae</i> Δ<i>gcvT</i> mutant results in an increase in oxidation of protein-associated methionine within the intestine.

<p>Western blot analysis of proteins in the intestines of <i>Drosophila</i> fed (A) conventional LB broth (LB) and fly food (Food) or (B) a wild-type <i>V</i>. <i>cholerae</i> (WT) and a Δ<i>gcvT</i> mutant. An antibody recognizing oxidized methionine (anti-MetO) was used. BSA-MetO protein was used as a positive control (CTL), and tubulin was used as a loading control (Tub). (C-E) Densitometry analysis of indicated bands and entire lanes (Total) for Western blots shown in (A) and (B). Relative density represents the density measurement for the individual band or lane normalized to the density measurement of the relevant tubulin loading control. Experimental replicates were performed with similar trends noted.</p

<i>Vibrio cholerae</i> ensures function of host proteins required for virulence through consumption of luminal methionine sulfoxide

<div><p><i>Vibrio cholerae</i> is a diarrheal pathogen that induces accumulation of lipid droplets in enterocytes, leading to lethal infection of the model host <i>Drosophila melanogaster</i>. Through untargeted lipidomics, we provide evidence that this process is the product of a host phospholipid degradation cascade that induces lipid droplet coalescence in enterocytes. This infection-induced cascade is inhibited by mutation of the <i>V</i>. <i>cholerae</i> glycine cleavage system due to intestinal accumulation of methionine sulfoxide (MetO), and both dietary supplementation with MetO and enterocyte knock-down of host methionine sulfoxide reductase A (MsrA) yield increased resistance to infection. MsrA converts both free and protein-associated MetO to methionine. These findings support a model in which dietary MetO competitively inhibits repair of host proteins by MsrA. Bacterial virulence strategies depend on functional host proteins. We propose a novel virulence paradigm in which an intestinal pathogen ensures the repair of host proteins essential for pathogenesis through consumption of dietary MetO.</p></div

Activation of Vibrio cholerae quorum sensing promotes survival of an arthropod host

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The <i>V</i>. <i>cholerae</i> glycine cleavage system promotes host metabolic disruption and suppression of intestinal stem cell division by a novel mechanism.

<p>(A) Components of the glycine cleavage system. (B) Chromosomal environments of <i>glyA2</i>, <i>gcvH</i>, <i>gcvP</i>, VC2638 and <i>glyA1</i>. (C) Survival curves of Oregon R flies fed LB broth inoculated with wild-type <i>V</i>. <i>cholerae</i> (WT), glycine cleavage system mutants or serine catabolism mutants. (D) <i>V</i>. <i>cholerae</i> colony-forming units (cfu) per fly after 48h of exposure to LB broth inoculated with the indicated <i>V</i>. <i>cholerae</i> strains. (E) Enumeration of PH3⁺ cells/fly intestine after 72h of exposure to LB broth alone or inoculated with the indicated <i>V</i>. <i>cholerae</i> strains. (F) Nile red staining of neutral lipids in the fat body and intestine of flies fed LB broth alone or inoculated with the indicated <i>V</i>. <i>cholerae</i> strains. (G) Quantification of cells containing lipid droplets in the midgut of flies fed the indicated <i>V</i>. <i>cholerae</i> strains. (H) Western blot analysis of phosphorylated AKT (pAKT) or total AKT levels in whole flies fed LB broth alone or inoculated with wild-type <i>V</i>. <i>cholerae</i> (WT) or a Δ<i>gcvT</i> mutant. (I) Acetate levels in the spent supernatants of wild-type <i>V</i>. <i>cholerae</i> (WT) or Δ<i>crbS</i> and Δ<i>gcvT</i> mutants cultured in LB. (J) Fluorescence ratios of flies fed LB supplemented with fluorescein either alone or inoculated with wild-type <i>V</i>. <i>cholerae</i> or a Δ<i>gcvT</i> mutant and harvested at the indicated time. (K) Survival curves of Oregon R flies fed LB broth inoculated with wild-type <i>V</i>. <i>cholerae</i> (WT), a Δ<i>crbS</i> mutant, a Δ<i>gcvT</i> mutant, or a combination of both. For pooled data, the mean and SD are shown. Pairwise statistical significance was calculated using a student’s t-test (*p<0.05, **p<0.01, ***p<0.001). For survival curves, statistical significance was calculated by log-rank analysis.</p

Inactivation of host methionine sulfoxide reductase in the setting of wild-type <i>V</i>. <i>cholerae</i> infection phenocopies infection of control flies with a <i>V</i>. <i>cholerae</i> Δ<i>gcvT</i> mutant.

<p>Transcription levels of (A) d<i>msrA</i> and (B) <i>dmsrB</i> in the intestines of flies infected with wild-type <i>V</i>. <i>cholerae</i> (WT) or a Δ<i>gcvT</i> mutant. (C) Transcriptional levels of <i>msrA</i> in control flies (yw) as well as fly lines carrying the two mutant alleles studied here, <i>msrA</i>^{<i>MI14018</i>} and <i>msrA</i> ^{<i>EY05753</i>}. (D) Fractional survival of control (yw) or d<i>msrA</i> mutant flies infected with wild-type <i>V</i>. <i>cholerae</i>. (E) Bacterial burden of control (yw) flies and <i>dmsrA</i> mutants after infection with <i>V</i>. <i>cholerae</i>. (F) Nile red staining of neutral lipids in the fat body and intestine of wild-type (yw) or d<i>msrA</i> fly mutant flies fed wild-type <i>V</i>. <i>cholerae</i>. (G) Quantification of midgut cells containing lipid droplets. (H) Western blot analysis of phosphorylated AKT(pAKT) or total AKT levels in whole control (yw) or d<i>msrA</i> mutant flies fed LB broth alone (LB) or inoculated with wild-type <i>V</i>. <i>cholerae</i> (Vc). (I) Enumeration of PH3⁺ cells in the intestines of control (yw) or d<i>msrA</i> mutant flies fed LB broth alone or inoculated with wild-type <i>V</i>. <i>cholerae</i> (WT) or a Δ<i>gcvT</i> mutant at 72h. (J) Levels of <i>msrA</i> transcription in control (Act-Gal4>) and Act-Gal4><i>msrA</i>^V48990-RNAi flies. (K) Fractional survival of control (yw), Da-Gal4>, or Da-Gal4><i>msrA</i> ^V48990-RNAi flies infected with wild-type <i>V</i>. <i>cholerae</i>. (L) Fractional survival of control (yw), NP1>, or NP1><i>msrA</i> ^V48990-RNAi flies fed with wild-type <i>V</i>. <i>cholerae</i>. (M-P) Lipidomics analysis of the intestines of <i>msrA</i> ^{<i>EY05753</i>} flies fed LB alone (LB) or inoculated with wild-type <i>V</i>. <i>cholerae</i> (Vc). (M) Total phosphatidylcholine species with fatty acid carbons less than or equal to 30 (PC≤30). (N) Total phosphatidylethanolamine species with fatty acid carbons less than or equal to 30 (PE≤30). (O) Total lysophosphatidylcholine species. (P) Total lysophosphatidylethanolamine species. For pooled data, the mean and SD are shown. Pairwise statistical significance was calculated using a student’s t-test (*p<0.05, **p<0.01, ***p<0.001). Log-rank analysis for survival curves (*p<0.05, **p<0.01, ***p<0.001).</p