The methodologies used in this paper were not new, but the findings were; combining molecular microbiological and physiological approaches allowed us to discover the effect of this bacterial protein on the host target tissue, the intestinal epithelium. Since the publication of this paper, our labs and others’ labs have continued to build on the published findings. In addition, others have utilized our approach of combining molecular microbiology and cellular physiology to identify the roles of additional bacterial proteins in pathogenesis.

The paper described the role of the EspF protein in disruption of intestinal barrier function. In order to function effectively, the space between cells of the intestinal epithelium must be protected. This role is served by tight junctions, complex multi-protein structures that form a barrier across the space between cells. Earlier we found that the bacteria secreted the protein EspF out of their cells, but that mutant bacteria that were unable to make the protein because of a deletion of the gene were indistinguishable from wild-type (unmutated) bacteria in all of the assays we were able to perform at the time. Hence we did not know what the protein did. In this manuscript we reported that the bacteria not only secreted the protein, but were able to inject EspF into host cells. Furthermore, we showed that, whereas infection of layers of intestinal epithelial cells with the wild-type bacteria caused a breakdown of the tight junctions between the cells and a leakage of molecules across the cell layer, infection with the mutant bacteria unable to make EspF caused no such effect. Finally, we showed that re-introduction of the EspF gene into the mutant bacteria restored the ability of the bacteria to disrupt these junctions. The more EspF protein we directed the bacteria to make, the more disruption to barrier function ensued. Thus, we had discovered that this protein that the bacteria inject into cells was responsible for disrupting the barrier between the cells. This loss of barrier function could contribute to the diarrhea caused by the bacteria when they infect people.

Our laboratories had been independently studying EPEC for many years. We had earlier described many of the genes that enable these strains of E. coli to adhere to and damage cells and this led to the discovery of the EspF gene, the sequence of which suggested that the protein would interact with host cellular proteins. The EspF gene is found in both EPEC and EHEC strains. However, when we were unable to determine a phenotype associated with mutating the EspF gene, we turned to our collaborator, Gail Hecht at the University of Illinois, to test the hypothesis that the EspF protein might be involved in intestinal barrier function. She had earlier reported that EPEC and EHEC strains lead to a breakdown of tight junctions between cells when they infect layers of intestinal epithelial cells in culture.