1. Katie Canul 1, Jeneva Foster 2, Christopher Wreden, PhD 2, and Karen Guillemin, PhD 2 1 California State University Monterey Bay, Seaside, CA 2, University of Oregon Institute of Molecular Biology, Eugene, OR Abstract Does AI-2 chemotaxis affect biofilm formation? Figure 5. Quantification of biofilm adherence to surface. Each bar represents the amount of bacterial cells adhering to the surface dived by the total amount of bacteria in the culture. 6 to 24 replicates from 1-4 independent experiments. AI-2 Production and Chemotaxis Mutants exhibit slightly increased adherence to surfaces References Acknowledgements AI-2 Mutants Display Variable Biofilm Formation Phenotypes Figure 3. Representative growth of biofilms used for adherence assays. Biofilms that developed along the walls of the microtiter tray and on the glass frit after staining with 1% crystal violet dye. 2012 Summer Program for Undergraduate Research Helicobacter pylori infections exist in nearly half of the world’s population and are associated with the formation of gastric ulcers and some types of gastric cancer. H. pylori adhere to surfaces and form biofilms in response to extracellular signals, which may play a role in dispersal in and colonization of the gastric mucosa. One such signal is the quorum-sensing small molecule AutoInducer-2 (AI-2), a product of the enzyme LuxS. The Guillemin lab has previously shown that AI-2 is sensed by the chemoreceptor TlpB to produce a chemotactic repellant response. Others have reported that strains of H. pylori that do not express LuxS have been shown to form larger biofilms. We hypothesize that H. pylori may form larger biofilms when the repellent AI-2 is not produced or sensed. H. pylori may form larger biofilms when the repellent AI-2 is not produced or sensed. This study investigated the influence of AI-2 in biofilm formation by quantifying biofilm growth in mutant H. pylori strains with altered or deleted portions of the signaling pathway involved in AI-2 synthesis and chemotaxis. We found that disrupting the chemotaxis pathway results in the formation of larger biofilms. From this work, we hope to gain a clearer understanding of the function of AI-2 sensing in H. pylori, which in turn will lead to better understanding of the ways that these bacteria disperse upon entering the stomach. Methods cont’d. Day 1-4 Culture H. pylori cells on blood-agar plates Day 5-10 Inoculate 12-well trays with bacteria, incubate Day 10 Stain all cells With Crystal Violet Dye Day 5 Culture in Liquid Media Day 10 Collect non- adherent bacteria Day 10 Ethanol extraction, quantification Table 1. Mutant strains of H. pylori used in biofilm experiments. Strains were generated from the G27 WT strain. Genes were either deleted from the genome or manipulated to disrupt protein functionality. Figure 2. Design of biofilm experiments. Each individual experiment consisted of 6 replicates of 1-4 mutant strains of bacteria grown under the same condition as WT bacteria. Cultures and biofilms were incubated in 5% CO 2 at 37˚C and cultured on CHBA plates or in BB10. LuxS X 431 TlpB 277 AI-2 Chemotaxis Smaller Biofilms Larger Biofilms AI-2 Chemotaxis X 431 TlpB 277 X X X Figure 4. Quantification of biofilm size. Each point represents the average number of cells adherent to the well surface and glass frit from six replicates during independent experiments. To minimize variation in growth rate between experiments, cell counts were normalized to the Wild Type control from the corresponding experiment. Summary and Conclusions Biofilm formation is quantifiable in H. pylori by measuring the amount of cells adherent to a surface and comparing to total cells in the culture Strains of H. pylori with genetic deletions of AI-2 production or chemotaxis machinery exhibit increased adherence to surfaces. AutoInducer-2 chemotaxis is involved in the formation of biofilms, which implies its involvement in dispersal and colonization of the stomach. Figure 2. Hypothesized Autoinducer-II Chemotaxis pathway. LuxS produces AI-2, which enters the environment and interacts with three proteins in the periplasm, leading to a chemotaxis response. If this pathway is genetically disrupted, we hypothesize the formation of larger biofilms will occur. Day 10 Collect adherent bacteria Day 10 Ethanol extraction, quantification This work would not have been possible without the generous support and encouragement of the Guillemin lab, particularly the chemotaxis subgroup: Jeneva Foster, Chris Wreden, Emily-Goers Sweeney, and Karen Guillemin. A sincere thank you to the SPUR program, Peter O’Day, Adam Unger, and the interns of SPUR 2012. Funding provided by NIH-1R25HD070817NICHD Summer Research Program at the University of Oregon. WT∆tlpB ∆277 ∆431 StrainAI-2 ProductionAI-2 Chemotaxis Predicted Surface Adherence WTNormal ∆luxSNoneNormalIncreased luxS + ExcessNormalDecreased ∆tlpBNormalDeletedIncreased ∆277NormalDeletedIncreased ∆431NormalDeletedIncreased ∆277/431NormalDeletedIncreased Quantifying the Effect of AutoInducer-2 on Helicobacter pylori Biofilm Formation AI-2 Helicobacter pylori chemotaxis and dispersal in the stomach What signals mediate dispersal in the stomach? Three proteins required to transmit the signal LuxS Produces AI-2 Cole et al. 2004. Characterization of monospecies biofilm formation in Helicobacter pylori. J. Bacteriology. 186(10):3124-3132. Monds RD and George O’Toole. 2008. The developmental model of microbial biofilms: ten years of a paradigm up for review. Cell Press. 17(2): 73-87. Rader BA et al. 2007. The quorum-sensing molecule autoinducer 2 regulates motility and flagellar morphogenesis in Helicobacter pylori. J. Bacteriology. 189(17):6109-6117. Rader BA et al. 2011. Helicobacter pylori perceives the quorum-sensing molecule AI-2 as a chemorepellent via the chemoreceptor TlpB. Microbiology. 157 (9): 2445- 2455. Does AutoInducer-2 play a role in dispersal? ? Figure 1. Biofilm formation and dispersal. (A) H. pylori biofilm formation via surface attachment to the gastric epithelium. (B) AI-2 is a self-produced molecule that produces a repellent response in H. pylori. (A)(B) chemotaxis AI-2 Do mutants that overexpress AI-2 make smaller biofilms? Does adding back AI-2 to recover biofilm phenotypes? Do mutant strains display colonization defects in vivo? Future Questions Methods