Development of Dictyostelium discoideum as a model to identify evolutionarily-conserved virulence factors in Escherichia coli

Abstract

Escherichia coli are a typical component of mammalian gut microbiota; however, a number of pathogenic strains remain a health concern for developing countries. Identification and disruption of virulence factors that promote infection could lead to reduction in infection. To identify novel bacterial virulence factors, we are using the soil amoeba, Dictyostelium discoideum, which preys upon bacteria, including E. coli, in the soil, as a model for mammalian phagocytic cells. Using random mutagenesis, we have created mutant stocks of MG1655 E. coli. These stocks were fed to D. discoideum, enriching the culture and selecting for mutants that were resistant to killing upon phagocytosis. After several rounds of selection, resistant mutant strains were isolated, and we found that some strains showed up to 100-fold increase in survival over a 2 h period. Once isolated, the genomes of the resistant mutant strains were sequenced and the sequences compared to that of the initial WT strain using Illumina’s MiSeq technology. While analyzing the sequences from these mutant strains, we found mutations in several potential genes of interest and are working to characterize their roles in the cell. We are testing E. coli strains reconstituted with specific mutations to identify particular mutations that are associated with phagocytosis-resistant phenotypes. These studies are aimed at identifying particular bacterial genes that may be involved in regulation of virulence. 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.