The intestinal tract is colonized by hundreds of non-pathogenic bacterial species, including those belonging to the genus Escherichia, that are important in maintaining normal gastrointestinal (GI) tract function. Colonization of the GI tract by pathogenic bacteria such as Escherichia coli O157:H7 (EHEC) proceeds in three distinct steps: (i) migration of the pathogen to intestinal epithelial cell surface, (ii) colonization, and (iii) injection of virulence factors. While the driving forces underlying EHEC infections are not fully understood, it is becoming increasingly clear that soluble signal-mediated interactions are important in EHEC infections. Recent work from our lab has shown that phenotypes relevant in EHEC infections - chemotaxis, colonization, and attachment – are differentially regulated by eukaryotic hormones in the GI tract (e.g., norepinephrine) and bacterial quorum sensing signals (e.g., indole) [1, 2]. These results underscore the importance of GI tract signals in EHEC infections.

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.

REFERENCES

1. Bansal T, Englert DL, Lee J, Hegde M, Wood TK and Jayaraman A. Infection Immunity. 75: 4597 (2007).

2. Bansal T, Jesudhasan P, Pillai S, Wood TK, and Jayaraman A. Applied Microbiology & Biotechnology. 73: 4100 (2008).