Progress 10/01/06 to 09/30/09
Outputs
OUTPUTS: Our initial observations indicate that bacterial colonization of squid light organs restricts super-infection by other bacteria. This mirrors mutualistic and commensal bacterial associations with all animals that help provide a barrier to pathogenic infections, although few models allow their molecular dissection. One mutant bacteria lacking the GacA protein was unable to direct changes in its host, could not restrict super infection by other bacteria, and could not evade attachment by host macrophages. Based on DNA microarray data by the mutant, we had two primary targets for investigation for their role in host interaction: PatA, a gene involved in LPS modification, and RpoS2, a novel sigma factor found only in symbiotic strains that is linked to a biofilm regulon conserved across multiple Vibrio species. Generating clean mutants in these two genes proved extremely difficult. As necessitated by the project, we created a new method for the isolation of difficult bacterial mutants using Penicillin (streptozotocin) enrichment. Over the life of this project several poster presentation were made at scientific meetings, including one to American Society for Microbiology, and the annual Pow Wow Squid-Vibrio symposium. A manuscript on the RpoS2 mutant and penicillin enrichment method developed as part of this project is currently under preparation: Brian M. Schuster, Randi Desy, Lauren A. Perry, and Cheryl A. Whistler. Isolation and characterization of a novel RpoS mutant reveals its role in bioluminescence and symbiotic colonization. Lauren Perry, Brian Schuster, Cheryl Whistler, 2007. Annual meeting for the American Society for Microbiology, (Toronto). Poster presentation; You Can Teach an Old Dog New Tricks: Revitalizing Penicillin Enrichment for the Isolation of Mutants. Lauren Perry, Brian Schuster, Vaughn Cooper, Cheryl Whistler, 2008. Annual meeting for the American Society of Microbiology (Boston,MA). Poster presentation for; Experimental evolution of Vibrio fischeri to squid symbiosis. PARTICIPANTS: Graduate students Lauren Perry and Brian Schuster worked collaboratively to develop the enrichment protocol with the help of a technician Randi Desy. Undergraduates Jill Brellsford and Micelle Desrosiers conducted preliminary characterization of RpoS2 and PatA respectively. Lauren Perry successfully completed her thesis and graduated August 2009. Jill Brellsford completed a B.S. in Microbiology in May 2007, and is currently in graduate school for an Masters in Public Health at George Washington University. Michelle Desrosiers completed a B.S. in Biology in May 2007, and has applied to graduate school at the University of Colorado for Fall 2010. TARGET AUDIENCES: Our work on developing methods for mutagenesis have a broad target audience, and the work was received with enthusiasm when presented at the ASM annual meeting. The target audience for commensal interactions that lead to protection include scientists interested in symbiosis and disease. PROJECT MODIFICATIONS: Although our initial focus was on PatA, due to the difficulty in developing a mutant, we expanded our study to include RpoS2, as we had a polar mutant in hand and were able to generate a clean mutant this last year.
Impacts
Identification of the mechanism of these interactions could allow us to manipulate host-microbe interactions to promote animal health. This method will greatly enhance the ability of microbiologist working with any bacterial species with limited mutagenesis tools to more rapidly generate mutants for direct hypothesis testing. As part of this project, we generated two mutants with the method, one for amino acid auxotrophy as a control, the other a component of bacterial RNA polymerase that had proven difficult to generate (RpoS2/RpoSQ). Characterization of the RpoS2 mutant reveals it is enhanced in bioluminescence, but not altered in colonization or biofilm production as was predicted, and appears to trigger normal host development and restriction to superinfection. Although we attempted to make a PatA mutant to investigate the role of LPS in tissue remodelling, even with the new approach we were unable to generate the mutant, thus it is likely an essential gene and the mutation lethal. We have several alternative approaches we will employ for continued study of the gene, and are currently developing a larger project to investigate the macrophage interactions.
Publications
- No publications reported this period
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: One poster presentation at a national symposium, and one oral presentation was given at a regional meeting. ASM 2007 (Toronto; 1) You Can Teach an Old Dog New Tricks: Revitalizing Penicillin Enrichment for the Isolation of Mutants Lauren Perry, Brian Schuster, Cheryl Whistler 2) 20th Annual Squid Vibrio Symposium: Pow Wow 2008 (Hawaii; Experimental evolution of Vibrio fischeri H905 to squid Symbiosis Lauren Perry, Brian Schuster, Vaughn Cooper, Cheryl Whistler; ASM 2008; 3)(Boston)Experimental evolution of Vibrio fischeri to squid symbiosis Lauren Perry, Brian Schuster, Vaughn Cooper, Cheryl Whistler; 4) Boston Bacteriological 2008 Experimental evolution of Vibrio fischeri to squid symbiosis Lauren Perry (talk), Brian Schuster, Vaughn Cooper, Cheryl Whistler PARTICIPANTS: Cheryl Whistler, PI, conducted molecular and animal experiments; Ms. Lauren Perry, Graduate student, conducted animal colonization experiments, Mr. Brian Schuster, undergraduate student, assisted with animal experimetns; Jenny Mahoney, graduate student, conducted molecular genetic experiments and developed assays; Ms Randi Desy, technician, developed assays and ran the animal facility. TARGET AUDIENCES: Scientific community studying commensal interactions with host cells, especially those interested in probiotics, prebiotics, biocontrol, and innate immunity. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We established a role for several bacterial traits in colonization and tissue remodelling. We determined that our symbiotic bacterium regulates its ability to evade host immune cell recognition. The bacterial factors are LPS (called an LOS for lipooligosaccharide in bacteria like V. fischeri that do not have an elaborate LPS or polysaccharide), and more specifically remodelling of the higher molecular weight components of the LOS. Its not yet known if this is a change in the sugar or in the lipid A component, but it is visible in the colony morphology and also from LPS extractions run on polyacrylamide gens. GacA is required for this remodeling of the membrane and in its absence the symbiont does not trigger changes in the host through this normal signalling. Only symbionts with the higher molecular weight LOS trigger host tissue remodelling. Furthermore, host hemocytes actually attach to and engulf GacA mutants as if they no longer recognize this bacterium as a symbiont. We believe a gene called patA may play a role in the LOS remodelling.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: One poster presentation was also made at the American Society for Microbiology Meeting, in Toronto, Canada May 2007: "You can teach an old dog new tricks: revitalizing penecillin enrichment for the isolation of mutants", L. Perry, B. Schuster, C. Whistler
PARTICIPANTS: There was significant undergraduate and graduate training as part of this project. Lauren Perry, M.S. expected June 2008. Ms Perry worked towards her masters degree on several aspects of the project. Brian Schuster, B.S. expected June 2008. Mr. Schuster worked with Ms Perry on developing animal assays and genetic manipulation protocols for the study of microbial exclusion. Jennifer Mahoney, Ph.D. expected 2010. Ms Mahoney conducted genetic manipulations of strains and oversaw several undergraduates working on the project. Mariah Gavin, B.S. expected June 2009. Ms Gavin began making bacterial mutants this year for this project. Ms. Alicia Ballok, M.S. 2007. Ms. Ballok generated mutant bacteria for use in the project. This work was part of her degree training. Michelle Desrosiers, B.B. June 2007. Ms Desrosiers generated a mutant in LPS modification that is altered in its ability to trigger host tissue responses as part of normal commensal recognition.
TARGET AUDIENCES: The audiences are researchers and students in medical microbiology. Work with symbionts challenges the current paradigms of immunology and infection and we aim to broaden their understanding of the important role of beneficial microbes. Mechanisms of immune recognition which are critical in health are poorly understood, and this model system is ideal for investigating these important interactions. As we further develop the project, we expect to more broadly disseminate the information beyond medical researchers to the general public and industries interested in developing probiotics.
PROJECT MODIFICATIONS: Much of our time this year was used developing the model system. We now have the tools and assays in line for a detailed assessment of mechanism, focusing on innate immune recognition of bacteria and Bacterial cell wall LPS components involved in host signaling.
Impacts
A new method for genetic manipulation of bacteria was generated during the study. This method can be applied to any microbial organism, was disseminated publicly in a poster presentation, and a manuscript on the method is in preparation. The technique will be broadly useful for bacterial manipulation. This year we developed the experimental system for study. We have shared our bacterial strains and methods with colleagues at University of Connecticut and Cal Poly San Luis Obispo. In a recent study, we determined that the GacA mutant, which is incapable of normal host signaling, does not have normal innate immune accomodation; specifically, unlike wild-type symbionts which are not attacked by host macrophages, GacA mutants are attacked and engulfed by these immune cells. This indicates that our work on this regulon will elucidate how host immune systems learn to recognize their commensals. Disruption of this immune recognition and accomodation is the basis for many auto
immune diseases.
Publications
- C. A. Whistler, T. A. Koropatnick, A. Pollack, M. J. McFall-Ngai, and E. G. Ruby. 2007. The GacA global regulator of Vibrio fischeri is required for normal host tissue responses that limit subsequent bacterial colonization. Cellular Microbiology. 9:766-778
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