Identification of Virulence and Virulence Regulatory Factors in Escherichia coli using Dictyostelium discoideum as a Model Phagocyte

Abstract

Abstract Escherichia coli is normally found as part of the human gut microbiota, however there exist a number of diarrheagenic strains. Identification and disruption of the virulence factors that promote infection could lead to a reduction in infection period of these E. coli strains and potentially of other species with similar means of virulence. To identify E. coli strains with altered virulence, we used Dictyostelium discoideum, a social amoeba that phagocytizes bacteria for nutritional purposes. Using random mutagenesis, we created mutant stocks of E. coli, each. These stocks were subjected to several rounds of phagocytosis by D. discoideum, enriching the culture and selecting for mutants resistant to intracellular killing. Resistant mutants were isolated and we found that some of the resistant strains showed increased survival when compared to the wild-type strain. Once isolated, the full genomes of our resistant mutant strains were sequenced using Illumina’s next generation MiSeq technology. While analyzing the sequencing data from these mutant strains, we found several potential genes of interest, including genes related to flagella, oxidoreductases, and acid resistance. We have begun to characterize genes of interest through various qualitative and quantitative assays using individual gene knockout strains. These studies should allow for identification of novel factors associated with regulation of virulence in E. coli. In addition, given that the development of mechanisms to resist D. discoideum predation may have contributed to the selection and maintenance of bacterial virulence factors against mammalian hosts, these studies may provide insight on the evolution of host-pathogen interactions.