Progress 09/15/03 to 09/14/06
Outputs
OUTPUTS: This report covers the duration of project from 9/15/03-9/14/06. 1) T. Bradley. During the course of this project we initiated characterization of several novel genes upregulated in salmon by osmotic stress associated with transfer from freshwater to seawater net pens. The two genes had a high degree of homology with proteins associated with osmotic stress or cold stress in mammals. Marked upregulation of these genes in fish undergoing smoltification and during osmotic stress suggest a role in osmotic adaption. 2) P.Cohen: We have investigated the role of gluconeogenesis, the glyoxylate bypass, and the TCA cycle in Salmonella enterica serovar Typhimurium virulence in mice. We found that neither gluconeogenesis nor the glyoxylate bypass is required for full virulence, but that a complete TCA cycle is. In addition, we found that for full virulence, malate must be removed from the TCA cycle and converted to pyruvate. The emerging picture is that serovar Typhimurium grows in an environment scarce in glycolytic sugars. 3) M. Gomez Chiarri: We have investigated the role of several candidate molecules in the immune responses of oysters. Infection with the protozoan parasite Perkinsus marinus resulted in changes of matrix metalloproteinases, histone H4 and nitric oxide in oyster plasma. In vitro treatment of P. marinus with nitric oxide resulted in a significant decrease of parasite viability. Inhibition of nitric oxide synthase in infected oysters resulted in higher levels of infection, indicating that nitric oxide is involved in clearing the parasite in vivo during infection. 4) A. Kausch: We have established robust gene transfer technologies for corn, rice and several species of grasses. These systems are important to the development and evaluation of transgenic traits and technologies. We have established a collaboration with Dr. S. Dellaporta's laboratory at Yale University and developed the constructs to evaluate a system for creating a transposon library in maize and rice. 5) T. Mather: We demonstrated the role of vesicle and plasma membrane-bound protein receptors (SNAREs) in regulating protein secretion in tick salivary gland cells. Our hypothesis is that similar exocytotic processes regulate pathogen transmission. We demonstrated the presence of SNARE proteins in the tick Amblyomma americanum, in Ixodes scapularis cell line IDE8, and in I. scapularis salivary glands. Using RNAi, we silenced SNARE proteins YKT6 and NSF to assess their role in tick feeding on vertebrate hosts. In vitro, salivary glands dissected from I. scapularis and incubated for 6 h with YKT6 and NSF dsRNA demonstrated 100% decrease in YKT6 and NSF transcript levels. Unfed live female ticks injected with NSF and YKT6 dsRNA showed complete silencing of respective gene transcripts. 6) D. Nelson: We have investigated the role of rpoS and luxS in regulating virulence and cell-cell signaling in Vibrio anguillarum. We have shown that rpoS regulates transcription of the extracellular metalloprotease empA, a virulence factor, but does not regulate luxS expression. Further, we could find no evidence that luxS directly regulates virulence in V. anguillarum. PARTICIPANTS: Postdocs: Dr.Subrata Chowdhury, Dr.Shahid Karim, Dr.Sudha Pichu, Dr. Merlin Tchawa-Yimga, Dr. Eric J. Gauger, Dr. Andrew M. Staroscik, Dr. Hamid Farahmand, Dr.Jacques Zarate. Principal investigators: Dr. T. Bradley, Dr. P. Cohen, Dr. A. Kausch, Dr. M. Gomez Chiarri, Dr. T. Mather, Dr. D. Nelson. TARGET AUDIENCES: The target audience includes: scientists, researchers, extension personnel, an array of agricultural interests, students, public health workers and consumers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1) Characterization of the genes and processes involved in adaptation of salmon to the marine environment will assist producers in enhancing survival and result in economic gain. 2) The nutrients and metabolic pathways that serovar Typhimutium requires for pathogenesis are unknown. Such knowledge should allow innovative approaches to preventing pathogenesis. 3) Control of protozoan and bacterial diseases threatening the eastern oyster Crassostrea virginica, requires a good understanding of the defense mechanisms of the oyster. Molecules characterized in this research may provide markers for breeding of disease resistance in oysters. 4) Understanding specific molecular pathways critical to pathogen multiplication in vacuoles and secretion into saliva may provide opportunities for developing novel therapeutic strategies to block tick-borne pathogen transmission. 5) The physiological state of pathogenic bacteria, like V. anguillarum does affect the expression of virulence factors such as the EmpA metalloprotease; however, we could find no evidence that cell-cell signaling plays a significant role in promoting pathogenesis in this bacterium.
Publications
- Gomez-Chiarri, M. and Munoz, P. 2003 Differential gene expression in Eastern oysters, Crassostrea virginica, experimentally infected with the protozoan parasite Perkinsus marinus. Plant and Animal Genomics XI, San Diego, CA.
- Dorrington, T. 2005. Recombinant antimicrobials for feed based delivery in aquaculture. Diss. Abst. Int.,Pt.B - Sci.& Eng. 4548. Dorrington, T., Villamil, L. and Gomez-Chiarri, M. 2005. Gene Expression in Eastern Oysters at early time points after experimental infection with the parasite Perkinsus marinus. Plant and Animal Genomics XIV. San Diego.
- Gomez-Chiarri, M. 2007. Molecular responses of American oysters to infection with the protozoan parasite Perkinsus marinus: Lessons from the functional genomics and candidate gene approaches. International Workshop on Perkinsosis. Vigo, Spain.
- Pan F., Zarate J., Choudhury, A., Rupprecht, R. and Bradley, T.M. 2004. Osmotic stress of salmon stimulates upregulation of a cold inducible RNA binding protein (CIRP) similar to that of mammals and amphibians. Biochimie 86: 451-461.
- Starocik, A.M., S.M. Denkin, and D.R. Nelson. 2005. Regulation of the Vibrio anguillarum metalloprotease EmpA by post-translational modification. J. Bacteriol. 187:2257-2260.
- Tchawa Yimga, M., Leatham, M.P., Allen, J.H., Laux, D.C., Conway, T. and Cohen, P.S. 2006. The role of gluconeogenesis and the TCA cycle in the virulence of Salmonella enterica serovar Typhimurium in BALB/c Mice. Infect. Immun. 74:1130-1140.
- Villamil, L., Gomez-Leon, J., Dorrington, T. and Gomez-Chiarri, M. 2006. Activation of nitric oxide response in Crassostrea virginica and in Crassostrea gigas after experimental infection with the protozoan parasite Perkinsus marinus. International Society of Comparative and Developmental Immunology, Charleston,SC.
- Villamil, L., Gomez-Leon, J. and Gomez-Chiarri, M. 2007. Role of nitric oxide in the defenses of Crassostrea virginica to experimental infection with the protozoan parasite Perkinsus marinus. Developmental and Comparative Immunology 31(10):968-977.
- Zarate, J. 2007. The role of osmolyte transporters and heat shock proteins in adaptation of Atlantic salmon to selected stressors. Diss.Abstr.Internat., Part B 68/01.
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Progress 01/01/05 to 12/31/05
Outputs
OUTPUTS: This report covers the period from 1/1/05-12/31/05. 1) T. Bradley: Characterization of the cDNA of a novel osmotic stress protein isolated by our laboratory and designated salmon glycine rich protein was undertaken. The gene was found to be upregulated in Atlantic salmon in response to a range of osmotic stress conditions. The protein has a high degree of similarity to a cold stress induced protein in amphibians and mammals. A fusion protein with GFP was used to follow expression of the gene. 2) P. Cohen: Previously, we found that gluconeogenesis is not required for full Salmonella enterica serovar Typhimurium virulence in mice, but that a complete TCA cycle is required. During this period, we showed that the glyoxylate bypass is not required for full serovar Typhimurium virulence, but that for full virulence malate must be removed from the TCA cycle and converted to pyruvate. The emerging picture is that serovar Typhimurium grows in an environment scarce in glycolytic sugars in vivo. 3) M. Gomez Chiarri: As part of our research on the characterization of early responses of oysters to infection, we have shown that nitric oxide is upregulated in response to infection. Another molecule with antimicrobial activity, histone H4, is also upregulated in oysters in response to infection with bacterial and parasitic pathogens. 4) A. Kausch: The outset of this research project necessitated the establishment of robust and routine transgenic systems for corn, rice and grass species which is a prerequisite for conducting genetic modification in these species. We have successfully established plant transformation systems in these plant species using Agrobacterium-mediated transformation and selection with a several selectable marker genes. 5) T. Mather: Previously, we demonstrated the role of vesicle and plasma membrane-bound protein receptors (SNAREs) in regulating protein secretion in tick salivary gland cells. Our working hypothesis is that similar exocytotic processes regulate pathogen transmission by ticks. During this period, we demonstrated the presence of SNARE proteins in the tick Amblyomma americanum, in Ixodes scapularis cell line IDE8 and in I. scapularis salivary glands. SNARE proteins NSF, YKT6 and Syntaxin were observed by confocal microscopy in unfed and partially fed I. scapularis cells. Using RNAi, we silenced YKT6 and NSF to assess their role in tick feeding on vertebrate hosts. In vitro, salivary glands dissected from partially fed I. scapularis and incubated for 6 h with YKT6 and NSF dsRNA demonstrated about a100% decrease in individual YKT6 and NSF transcript levels. Unfed live female ticks injected in vivo with NSF and YKT6 dsRNA showed complete silencing of respective gene transcripts, as well as tick feeding inhibition. 6) D. Nelson: Characterization of the luxS gene in Vibrio anguillarum was continued. An rpoS null mutant of V. anguillarum (M03) was used to show that there is both rpoS-dependent and rpoS-independent production of AI-2 in V. anguillarum. RT-qPCR was used to demonstrate that luxS transcription is not under the control of rpoS. V. anguillarum vanP (luxP homologue) transcription is regulated by rpoS. PARTICIPANTS: PI's: Dr. Jeffrey Seemann, Dr. Terence Bradley, Dr. Paul Cohen, Dr. Marta Gomez-Chiarri, Dr. David Nelson, Dr. Thomas Mather and Dr. Albert Kausch. Post-docs: Dr.Subrata Chowdhury, Dr. Merlin Tchawa-Yimga, Dr. Andrew M. Staroscik and Dr. Hamid Farahmand. Funding allowed for the hire of one postdoctoral fellow for each of the projects. Postdocs received significant training opportunities as part of the projects. TARGET AUDIENCES: General public, educators, public health specialists, researchers and aquaculturists. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
In addition to bringing new expertise and equipment to the institution the projects had the following impacts: 1) Information on the novel genes involved in parr-smolt transformation will assist growers in enhancing successful transfer of smolts to seawater. 2) The nutrients and metabolic pathways that serovar Typhimutium requires for pathogenesis are unknown. Such knowledge should allow innovative approaches to preventing pathogenesis. 3) Control of diseases threatening the eastern oyster, Crassostrea virginica, industry requires greater understanding of the defense mechanisms of the oyster. A better characterization of the early responses of oysters to infection may provide markers for breeding of disease resistance. 4) Ability to conduct routine plant transformation allows the investigation of a large number of trait genes and the development of transgenic technologies, such as gene confinement and transposon mutagenesis. 5) Understanding specific molecular pathways critical to pathogen multiplication in vacuoles and secretion into saliva may provide opportunities for developing novel therapeutic strategies to block tick-borne pathogen transmission. 6) Despite being thought of as high density, stationary phase signal, AI-2 production is not dependent upon RpoS, the stationary phase alternative sigma factor. Further, transcription of luxS, which encodes the AI-2 synthase, does not require RpoS.
Publications
- Luo, H., Hu, Q., Nelson, K., Longo, C. and Kausch, A.P. 2003. Controlling transgene escape in genetically modified grasses. Plant Biology, Annual Meetings of the American Society of Plant Biologists.
- Karim, S., Miller, N. J., Valenzuela, J. G., Sauer, J. R. and Mather, T. N. 2005. RNAi-mediated gene silencing to assess the role of synaptobrevin and cystatin in tick blood feeding, Biochemical and Biophysical Research Communications 334, 1336-1342.
- Bradley, T.M., Zarate, J. and Farahmand, H. 2005. Transporters and other genes involved in adaptation of salmon to the marine environment. Sixth International Smoltification Workshop, Tono, Japan.
- Dorrington, T., Villamil, L. and Gomez-Chiarri, M. 2005. Gene expression in eastern oysters at early time points after experimental infection with the parasite Perkinsus marinus. Plant and Animal Genomics XIII. San Diego, January.
- Hu, Q., Nelson, K., Kausch, A.P. and Luo, H. 2001. FLP-mediated site-specific DNA recombination in grass cells. Plant Biology, Quadrennial Joint Annual Meetings of the American Society of Plant Biologists and the Canadian Society of Plant Physiologists.
- Tchawa Yimga, M., Leatham, M., Laux, D., and Cohen, P.S. 2005. A complete TCA cycle is required for full virulence of Salmonella enterica serovar Typhimurium in BALB/c mice. General Meeting of the American Society for Microbiology, Atlanta, GA.
- Villamil L., Dorrington T., Gomez-Leon, J. and Gomez-Chiarri, M. 2005. Activation of nitric oxide response in Crassostrea virginica and in Crassostrea gigas after an experimental infection with Perkinsus marinus. National Shellfisheries Association, Philadelphia, April.
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Progress 01/01/04 to 12/31/04
Outputs
Characterization of Genes Upregulated by Hyperosmotic Stress of Salmon. PI: Terence M. Bradley. Characterization of organization of the Shop21 gene has been completed. Two alleles of the gene have been identified. The first allele (Shop21.1) is comprised of 4 exons and 3 introns. Intron 3 is located immediately upstream of the putative peroxisomal targeting sequence, disrupting the CDS. The second allele (Shop21.2) is identical to Shop21.1 with the exception that it lacks intron 3 and possesses a contiguous CDS. The Role of the TCA Cycle and Gluconeogenesis in Salmonella typhimurium Pathogenesis. PI: P. Cohen. This year, Dr. Tchawa Yimga showed that a full TCA cycle is required for full S. typhimurium expression in BALB/c mice, which suggests that S. typhimurium growth during the initial stages of infection in the intestine depends on low concentrations of sugars. Identifying the sugars and finding ways to further limit their availability to S. typhimurium in animals
should improve animal health and thereby limit infection in humans. Molecular Responses of Oysters to Parasitic Infection. PI: M. Gomez-Chiarri. We have isolated and sequenced several genes coding for MMPs in two species of oysters with known differences in their responses to parasitic infection and developed the tools to study the patterns of expression of these genes in oysters in response to infection. Contributions to the Development of a Transposon Mutagenesis Approach to Functional Genomics in Maize. PI: A. Kausch. We have established routine maize transformation with both hybrid and inbred lines, using constructs that will provide proof of principle for the development of a functional genomics approach in corn utilizing transposon mutagenesis. Dr. Peyui Zeng was recruited for this project and during has recovered numerous maize transgenic T0 lines using these constructs and others for related projects. These lines are currently in the greenhouse awaiting further molecular
analysis RNA-Mediated Gene Silencing in Ticks PI: T. Mather. Double-stranded RNA interference (RNAi) is being used to silence expression of several membrane associated proteins involved in exocytosis (SNAREs). In vitro incubation of tick salivary glands with dsRNA complimenting 3 SNARE genes demonstrated ~100% decrease in transcript levels. Unfed ticks injected with dsRNA to one of these SNARES showed about 90% silencing when ticks were partially fed. Silencing this gene resulted in significantly decreased repletion weight (mean=42 mg) when compared to control ticks (mean=192 mg). Cloning and Characterization of the Alternative Sigma-Factor, Sigmas, in Vibrio anguillarum. PI: David R. Nelson. The rpoS gene encodes the alternative sigma factor, RpoS, required for RNA polymerase recognition and binding to promoter sequences involved in stationary phase and starvation responses of many bacteria. The V. anguillarum rpoS gene has been cloned and sequenced. An rpoS mutant of V. anguillarum
was constructed and is unable to express the empA protease gene, but does express hemolysins.
Impacts
The funds provided for this project have enabled the hire of postdoctoral fellows to conduct research on topics of interest to USDA. The postdoctoral fellows have provided additional expertise and research capacity in the area of agriculture related molecular biology and biotechnology. Of significance, the funds have allowed the principal investigators to purchase equipment critical to the establishment of a core genomics facility at URI.
Publications
- Munoz P, Vance K, Gomez-Chiarri M (2003) Protease activity in the plasma of American oysters, Crassostrea virginica, experimentally infected with the protozoan parasite Perkinsus marinus. J Parasitol. 89:941-951.
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