Since commensal bacteria residing in the GI tract are the source for most prokaryotic signals in the intestinal lumen, we hypothesized that the organization of commensal bacteria in the GI tract is a key determinant of pathogen colonization. In this work, we have adopted micropatterning techniques in order to manipulate and investigate the local microenvironment that EHEC encounters during colonization so that its role in EHEC infections can be determined. Conventional cell culture methods for studying EHEC attachment do not account for the effect of the GI tract microenvironment on EHEC colonization as it is difficult to localize bacteria in specific locations among eukaryotic cells. Using easy to operate pneumatic valves, we have fabricated several commensal bacterial “islands” ranging in diameter from 20 to 100 microns and surrounded them with eukaryotic cells so as to create an in-vivo like microenvironment. Microvalves were used to capture bacteria and localize them to specific regions where they colonize and form biofilms. The effect of commensal island size and bacterial attachment time was optimized as these two parameters are expected to directly influence the concentration of signaling molecules released into the microenvironment. The feasibility of localizing bacteria in specific regions was demonstrated using sequential introduction of dyes and bacteria expressing different fluorescent proteins. The ability to localize different bacterial species in different islands enables mimicking the in vivo GI tract heterogeneity. Following bacterial island formation, residual bacteria outside the island was lysed, followed by seeding of Hela cells to complete the co-culture system. Finally, EHEC was introduced into the micropatterned chamber and its colonization to the vicinity of specific bacterial islands studied. Our micropatterned co-culture of bacteria and epithelial cells is expected to enable investigation of signaling interactions between bacteria and host-cells that are crucial to infections.
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