Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Expression data of Vibrio fischeri in response to nitric oxide (NO)

ABSTRACT: The bioluminescent bacterium Vibrio fischeri initiates a specific, persistent symbiosis in the light organ of the squid Euprymna scolopes. During the early stages of colonization, V. fischeri is exposed to host-derived nitric oxide (NO). While NO can be both an antimicrobial component of innate immunity and a common signaling molecule of eukaryotes, its roles in beneficial host-microbe associations remain undescribed. V. fischeri encodes HnoX, a member of a family of bacterial NO-binding proteins of unknown function. We hypothesized that HnoX acts as a NO sensor that is involved in regulating symbiosis-related genes during initiation of symbiosis. With an aim to discover the genes whose regulations respond to NO signal, and in an HnoX-mediated fashion in particular, we carried out a whole-genome expression study on the wild-type and an insertional mutant of hnoX. The wild-type parent and an insertional mutant (hnoX-) of the hnoX gene were grown to early log phase in a minimal-salts medium. One half of each culture was treated with 80ÂµM of the NO-generator, DEA-NONOate, and the other half was left untreated as a control. After 30 min, cells from all the cultures were fixed with RNAprotect Bacteria Reagent. Total RNA was isolated, labeled and hybridized to the Custom Vibrio fischeri GeneChip Array (Affymetrix). Three independent experiments were performed on separate days for statistical analysis.

ORGANISM(S): Vibrio fischeri

SUBMITTER: Yanling Wang

PROVIDER: E-GEOD-15522 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The squid, Euprymna scolopes, and the luminescent bacterium, Vibrio fischeri, form a highly specific beneficial light organ symbiosis. Not only does the host have to select V. fischeri from the environment, but it must also prevent subsequent colonization by non-symbiotic microorganisms. Host macrophage-like hemocytes are believed to play a role in mediating the symbiosis with V. fischeri. Previous studies have shown that the colonization state of the light organ influences the host’s hemocyte response to the symbiont. To further understand the molecular mechanisms behind this process, two quantitative mass spectrometry-based proteomic techniques, isobaric tags for relative and absolute quantification (iTRAQ) and label-free spectral counting, were used to compare and quantify the adult hemocyte proteomes from colonized (sym) and uncolonized (antibiotic-treated/cured) squid. Overall, iTRAQ allowed for the quantification of 1,024 proteins with 2 or more peptides. Thirty-seven unique proteins were determined to be significantly different between sym and cured hemocytes (p-value < 0.05), with 20 more abundant proteins and 17 less abundant in sym hemocytes. The label-free approach resulted in 1,241 proteins that were identified in all replicates. Of 185 unique proteins present at significantly different amounts in sym hemocytes (as determined by spectral counting), 92 were more abundant and 93 were less abundant. Comparisons between iTRAQ and spectral counting revealed that 30 of the 37 proteins quantified by iTRAQ exhibited similar trends identified by the label-free method. Both proteomic techniques mutually identified 16 proteins that were significantly different between the two groups of hemocytes (p-value <0.05). The presence of V. fischeri in the host light organ influenced the abundance of proteins associated with the cytoskeleton, adhesion, lysosomes, proteolysis, and the innate immune response. These data provide evidence that colonization by V. fischeri alters the hemocyte proteome and reveals proteins that may be important for maintaining host-symbiont specificity.

Project description:The light-organ symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri offers the opportunity to decipher the hour-by-hour events that occur during the natural colonization of an animal's epithelial surface by its microbial partners. To determine the genetic basis of these events, a glass-slide microarray was used to characterize the light-organ transcriptome of juvenile squid in response to the initiation of symbiosis. Patterns of gene expression were compared between animals not exposed to the symbiont, exposed to the wild-type symbiont, or exposed to a mutant symbiont defective in either of two key characters of this association: bacterial luminescence or autoinducer (AI) production. Hundreds of genes were differentially regulated as a result of symbiosis initiation, and a hierarchy existed in the magnitude of the host's response to three symbiont features: bacterial presence > luminescence > AI production. Putative host receptors for bacterial surface molecules known to induce squid development are up-regulated by symbiont light production, suggesting that bioluminescence plays a key role in preparing the host for bacteria-induced development. Further, because the transcriptional response of tissues exposed to AI in the natural context (i.e., with the symbionts) differed from that to AI alone, the presence of the bacteria potentiates the role of quorum signals in symbiosis. Comparison of these microarray data with those from other symbioses, such as germ-free/conventionalized mice and zebrafish, revealed a set of shared genes that may represent a core set of ancient host responses conserved throughout animal evolution. Six experimental treatments of juvenile animals were performed for the microarray matrix: uncolonized (Apo); uncolonized, but supplemented with AI (Apo + AI); colonized by wild-type V. fischeri (wild type); colonized by a mutant defective in luciferase synthesis (luxA); colonized by a mutant defective in AI synthesis (luxI); and, colonized by the luxI mutant, but supplemented with AI (luxI + AI). At 18 h postinoculation, animals were anesthetized in 2% ethanol in HOSW, and the light organs were removed into RNAlater (Ambion Biosystems).

Project description:The bioluminescent bacterium Vibrio fischeri forms a mutually beneficial symbiosis with the Hawaiian bobtail squid, Euprymna scolopes, in which the bacteria, housed inside a specialized light organ, produce light used by the squid in its nocturnal activities. Upon hatching, E. scolopes juveniles acquire V. fischeri from the seawater through a complex process that requires, among other factors, chemotaxis by the bacteria along a gradient of N-acetylated sugars into the crypts of the light organ, the niche in which the bacteria reside. Once inside the light organ, V. fischeri transitions into a symbiotic, sessile state in which the quorum-signaling regulator LitR induces luminescence. In this work we show that expression of litR and luminescence are repressed by a homolog of the V. cholerae virulence factor TcpP, which we have named HbtR. Further, we demonstrate that LitR represses genes involved in motility and chemotaxis into the light organ and activates genes required for exopolysaccharide production. Importance: TcpP homologs are widespread throughout the Vibrio genus; however, the only protein in this family described thus far is a V. cholerae virulence regulator. Here we show that HbtR, the TcpP homolog in V. fischeri, has both a biological role and regulatory pathway completely unlike that in V. cholerae. Through its repression of the quorum-signaling regulator LitR, HbtR affects the expression of genes important for colonization of the E. scolopes light organ. While LitR becomes activated within the crypts, and upregulates luminescence and exopolysaccharide genes and downregulates chemotaxis and motility genes, it appears that HbtR, upon expulsion of V. fischeri cells into seawater, reverses this process to aid the switch from a symbiotic to a planktonic state. The possible importance of HbtR to the survival of V. fischeri outside of its animal host may have broader implications for the ways in which bacteria transition between often vastly different environmental niches.

Project description:Quorum sensing (QS) is a cell density regulated communication system that bacteria use to coordinate activities, including biofilm formation, involved in colonization and pathogenesis. We have previously shown that inactivation of the QS master regulator LitR attenuates the Vibrio (Allivibrio) salmonicida strain LFI1238 in a fish model. In this work we show that LFI1238 as well as a panel of naturally occurring V. salmonicidia strains are poor biofilm producers. Inactivation of litR strongly enhances medium and temperature dependent adhesion, rugose colony morphology and biofilm formation. Chemical treatment and scanning electron microscopy of the biofilm identified an extracellular matrix consisting mainly of protein filaments and polysaccharides. Further, microarray analysis of planktonic and biofilm cells identified a number of genes regulated by LitR, and among these were homologues of the Vibrio fischeri symbiosis polysaccharide (syp) genes. Disruption of syp alleviated the different phenotypes regulated by LitR in V. salmonicida. Hence, LitR is a repressor of syp expression that is necessary for rugose colony morphology, adhesion and biofilm formation, three phenotypes of the DlitR mutant that are expressed at temperatures below 12M-BM-:C. The DlitR mutant mimics low cell density behavior suggesting that these phenotypes are important during the initial steps of colonization. Although the syp operon in V. salmonicida shows identical gene synteny to the one in the squid symbiont V. fischeri, its regulation is probably more related to vibrio polysaccharide (vps) expression in the human pathogenic Vibrio cholera which is controlled by the LitR homologue HapR. V. salmonicida wild type strain LFI1238 (control) and the isogenic DlitR mutant were grown as statical biofilm in SWT medium, at 4M-BM-0C and harvested after 72 hours. Biological replicates for each sample: 4 wild type, 4 DlitR mutant (including one dye swap), independently grown and harvested. One replicate per array.

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Expression data of Vibrio fischeri in response to nitric oxide (NO)

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