Construction of enterotoxinogenic Escherichia coli infection model using Caenorhabditis elegans as an alternative host

Background and objectives: Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveler's diarrhea in developed countries and infantile diarrhea in developing countries. Caenorhabditis elegans (C. elegans) has been used as an alternative model animal for studying pathogen interaction. ETEC was previously reported to shorten the survival time of C. elegans (Hoshino et al. 2008). However, the genetic background of the ETEC strain and host defense responses remain largely uncharacterized. This study aimed to uncover the molecular basis of host-ETEC interaction and establish a simple model system for ETEC infection using C. elegans. Methods: The clinical strain ETEC1 (O6:H16), used by Hoshino et al. (2008), was also examined in this study. The draft genome sequence of the strain was assembled using short reads obtained by Illumina Miseq. Existence of enterotoxin genes were verified by tBLASTn and VirulenceFinder 2.0. Colonization factors (CSs) were also verified as same as the toxin genes. Wild-type C. elegans N2 was used as the experimental animal. The synchronized L1 larvae were grown on mNGM medium (agar medium for nematode growth) with non-pathogenic E. coli OP50, a standard food, until they reached adulthood. Thereafter, they were divided into groups fed OP50 or ETEC1 for lifespan assay. Survival rates were calculated by the Kaplan-Meier method, and differences in survival rates between the two groups were compared by the log-rank test Results: Whole genome sequencing revealed that ETEC1 possessed heat-labile toxin LT1 and heat-stable toxin STh. Out of the major CSs, CS2 and CS3 exist on the ETEC1 genome. Worms fed ETEC1 showed significantly shorter survival time in comparison with those fed OP50, as shown in the previous study. We are currently validating differentially expressed genes (DEGs) using RNA-seq and real-time PCR to estimate host defense responses to ETEC1. Conclusions:We established an C. elegans -ETEC infection model. It could be valuable for understanding the molecular basis on host-ETEC interaction and screens of functional food materials for anti-infective effects.