Genome-wide transcriptome analysis of Enterohemorrhagic Escherichia coli (EHEC) isolates for identification of fresh produces-specific virulence factors

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen causing serious threats to human. Recent studies have revealed that many outbreaks of EHEC were mediated by spoiled produce such as raw sprouts or lettuce. To investigate how the plant-derived EHEC cells adapt to fresh produces, EHEC #736 isolated from kimchi in Korea was cultivated with canola spouts (Brassica napus) and water dropwort (Oenanthe javanica). Planktonic bacteria in fresh produces were subjected to RNA sequencing to achieve bacterial mRNA profiles responding to contacts with canola sprouts and water dropwort. As a result, among a total of 4,882 annotated coding sequences, 6.06% and 5.12% of total genes were significantly up- or down-regulated when exposed to canola sprouts and water dropwort respectively after 1 hour. Similarly, 5.90% and 4.53% of total genes were significantly up- or down-regulated when exposed to canola sprouts and water dropwort respectively for 3 hours. Genes with different expression (p<0.05) could be grouped mainly into five categories: translation and ribosome biogenesis, cell motility, carbohydrate transport, energy conversion and secondary metabolites biosynthesis/transport. In detail, multiple nutrition transport systems for carbohydrate, amino acid and nitrate that are known to be prevalent in plants were activated in EHEC #736. This indicates the availability to EHEC #736 of a variety of factors released from fresh produces that may promote the growth of bacteria. Particularly, RNA-Seq revealed that iron uptake related genes including Fur (Ferric-uptake regulator) regulons are differentially expressed when the bacteria were exposed to plants. It was further demonstrated that EHEC #736 ΔentB mutant defective in siderophore biosynthesis had growth defects in water dropwort, and the defective phenotype was complemented by the addition of ferrous ion. This indicates that iron uptake is essential for survival of EHEC in plant. Considering the importance of iron acquisition for bacterial survival, iron disrupting mechanism, such as treating gallium, a transition metal, might prevent fresh produce from bacterial contamination.