Transcript AI-2 - SPUR - University of Oregon

Quantifying the Effect of AutoInducer-2 on Helicobacter pylori Biofilm Formation
Katie Canul1, Jeneva Foster2, Christopher Wreden, PhD2, and Karen Guillemin, PhD2
2012 Summer Program for Undergraduate Research
1 California State University Monterey Bay, Seaside, CA
2, University of Oregon Institute of Molecular Biology, Eugene, OR
Methods cont’d.
Abstract
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.
Helicobacter pylori chemotaxis and dispersal in the stomach
(A) What signals mediate dispersal in the stomach?
Day 1-4
Culture H.
pylori cells on
blood-agar
plates
Day 10
Ethanol
extraction,
quantification
Day 5
Culture in Liquid
Media
AI-2 Production and Chemotaxis Mutants exhibit
slightly increased adherence to surfaces
Day 5-10
Day 10
Inoculate 12-well
trays with bacteria,
incubate
Ethanol
extraction,
quantification
Day 10
Day 10
Collect nonadherent
bacteria
Stain all cells
With Crystal
Violet Dye
Day 10
Collect
adherent
bacteria
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% CO2 at 37˚C and cultured on CHBA plates or in BB10.
(B) Does AutoInducer-2 play a role in dispersal?
WT
AI-2
?
∆tlpB
∆431
∆277
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.
chemotaxis
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.
Summary and Conclusions
Does AI-2 chemotaxis affect biofilm formation?
•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
431
LuxS
TlpB
AI-2
AI-2
Chemotaxis
LuxS Produces AI-2
277
Smaller Biofilms
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.
Three proteins required to transmit the signal
X
TlpB
X
277
X
431
X
LuxS
X
AI-2
Chemotaxis
Larger Biofilms
AI-2 Mutants Display Variable Biofilm Formation Phenotypes
•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.
Future Questions
•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?
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.
Acknowledgements
Methods
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.
Strain
AI-2 Production
AI-2 Chemotaxis
Predicted Surface
Adherence
WT
Normal
Normal
Normal
∆luxS
None
Normal
Increased
Excess
Normal
Decreased
Funding provided by NIH-1R25HD070817NICHD Summer Research Program at the University of Oregon.
∆tlpB
Normal
Deleted
Increased
References
∆277
Normal
Deleted
Increased
∆431
Normal
Deleted
Increased
∆277/431
Normal
Deleted
Increased
luxS
+
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.
A sincere thank you to the SPUR program, Peter O’Day, Adam Unger, and the interns of SPUR 2012.
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.
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): 24452455.