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

<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

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

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

<i>Vibrio cholerae</i> augments virulence by modulation of host intestinal methionine sulfoxide reductase.

<p>(A) Wild-type <i>V</i>. <i>cholerae</i> (WT) catabolizes dietary methionine sulfoxide (MetO) in the intestinal lumen, leaving host enterocyte methionine sulfoxide reductase (MsrA) free to repair proteins that have been inactivated by methionine oxidation. (B) In a <i>V</i>. <i>cholerae ΔgcvT</i> mutant infection, dietary MetO is not consumed by <i>V</i>. <i>cholerae</i> but rather taken up by host enterocytes. Dietary MetO competitively inhibits reduction of protein-associated MetO by MsrA within enterocytes.</p

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

Decreased consumption of methionine sulfoxide (MetO) by the <i>V</i>. <i>cholerae</i> Δ<i>gcvT</i> mutant attenuates virulence by promoting intestinal lipid mobilization and insulin signaling.

<p>(A) Survival curves of flies fed LB broth inoculated with wild-type <i>V</i>. <i>cholerae</i> (WT) supplemented with 50 mM methionine (Met) or 25 mM and 50 mM methionine sulfoxide (MetO). (B) Bacterial burden of flies measured in colony forming units (cfu) after ingestion of LB broth supplemented with 25mM and 50mM methionine sulfoxide (MetO) and inoculated with wild-type <i>V</i>. <i>cholerae</i> (WT). (C) Enumeration of PH3⁺ cells in the intestines of flies fed PBS or PBS supplemented with 50 mM methionine (Met) and 50 mM methionine sulfoxide (MetO) for 72h. (D) Nile red staining of neutral lipids in the fat body and intestine of flies fed LB broth or LB broth inoculated with wild-type <i>V</i>. <i>cholerae</i> (WT) supplemented with methionine (Met) or methionine sulfoxide (MetO). (E) Quantification of midgut cells containing lipid droplets. (F) 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) supplemented with methionine (Met) or methionine sulfoxide (MetO). 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

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

Mutation of the <i>V</i>. <i>cholerae</i> glycine cleavage system alters the extracellular environment in LB broth, <i>Drosophila</i> intestine, and the rabbit intestine.

<p>LC-MS/MS based metabolomic analysis of wild-type <i>V</i>. <i>cholerae</i> (WT) or a Δ<i>gcvT</i> mutant in (A and B) LB culture supernatants, (C) infected <i>Drosophila</i> intestines, and (D) cecal fluid of infected infant rabbits. Only metabolites that were significantly different under the two conditions are shown (p<0.05). (E) Levels of MetO and methionine (Met) in LB broth alone, in the intestines of flies fed LB broth alone (LB), LB broth inoculated with wild-type <i>V</i>. <i>cholerae</i> (WT) or LB broth inoculated with a <i>V</i>. <i>cholerae</i> Δ<i>gcvT</i> mutant, and in the cecal fluid of infant rabbits inoculated with wild-type <i>V</i>. <i>cholerae</i> or a <i>V</i>. <i>cholerae</i> Δ<i>gcvT</i> mutant. (F) Compounds identified in cecal fluid metabolomics are shown in green along with their relationship to the methionine cycle. For pooled data, the mean and SD are shown.</p