A pathoadaptive deletion in an enteroaggregative Escherichia coli outbreak strain enhances virulence in a Caenorhabditis elegans model

Abstract

Enteroaggregative Escherichia coli (EAEC) strains are important diarrheal pathogens. EAEC strains are defined by their characteristic stacked-brick pattern of adherence to epithelial cells but show heterogeneous virulence and have different combinations of adhesin and toxin genes. Pathoadaptive deletions in the lysine decarboxylase (cad) genes have been noted among hypervirulent E. coli subtypes of Shigella and enterohemorrhagic E. coli. To test the hypothesis that cad deletions might account for heterogeneity in EAEC virulence, we developed a Caenorhabditis elegans pathogenesis model. Well-characterized EAEC strains were shown to colonize and kill C. elegans, and differences in virulence could be measured quantitatively. Of 49 EAEC strains screened for lysine decarboxylase activity, 3 tested negative. Most notable is isolate 101-1, which was recovered in Japan, from the largest documented EAEC outbreak. EAEC strain 101-1 was unable to decarboxylate lysine in vitro due to deletions in cadA and cadC, which, respectively, encode lysine decarboxylase and a transcriptional activator of the cadAB genes. Strain 101-1 was significantly more lethal to C. elegans than control strain OP50. Lethality was attenuated when the lysine decarboxylase defect was complemented from a multicopy plasmid and in single copy. In addition, restoring lysine decarboxylase function produced derivatives of 101-1 deficient in aggregative adherence to cultured human epithelial cells. Lysine decarboxylase inactivation is pathoadapative in an important EAEC outbreak strain, and deletion of cad genes could produce hypervirulent EAEC lineages in the future. These results suggest that loss, as well as gain, of genetic material can account for heterogeneous virulence among EAEC strains.

FIG. 1.

Schematic of the yjdC-yjdL intergenic region in E. coli K-12 MG1655 and EAEC strain 101-1. This region of the chromosome contains the cadABC genes in MG1655. Strain 101-1 is identical to MG1655 at this locus with the exception of a 5′ 30-nucleotide deletion in cadA and a 558-nucleotide deletion in cadC. Genes harboring inactivating deletions are shaded.

FIG. 2.

Survival of C. elegans fed with EAEC strains. (A) Kaplan-Meier plots for worms fed on OP50, P. aeruginosa PA14, and five EAEC strains. (B) Median time to death for C. elegans fed with P. aeruginosa or one of six EAEC strains. Each experiment used 40 to 60 worms for each strain, and survival data were compared statistically by the Kaplan-Meier method (7) using the PEPI version 4.0 SURVIVAL program. Survival of all EAEC strains was significantly less than that of OP50 (P < 0.05), which had a median time to death of >400 h.

FIG. 3.

Survival of C. elegans fed with wild-type EAEC outbreak strain 101-1, 101-1 in the presence of exogenous cadaverine, and 101-1 complemented with pCADA. Strain OP50 was used as a negative control in the presence and absence of cadaverine. The pCADA-bearing derivative was significantly attenuated compared to the wild-type 101-1 strain (P = 0.001). However, the comparison of strain 101-1 fed in the presence and absence of cadaverine and OP50 fed in the presence and absence of cadaverine did not show a significant difference in survival.

FIG. 4.

Bacterial adherence to HEp-2 cell monolayers after 3-h infections. (A) E. coli OP50. (B) EAEC strain 101-1. (C) Strain 101-1 in the presence of 300 μM cadaverine. (D) Strain 101-1 complemented with plasmid pCADA and induced with IPTG. (E) Adherent bacteria quantified by viable counting. The differences between 101-1 and 101-1(pCADA) were significant at P < 0.00001.

FIG. 5.

Adherence of 101-1 and its cad cis-complement to cultured HEp-2 cells. (A) Adherence quantified by viable counting. Adherence of INK1300 and adherence of 101-1(pCADA) are not significantly different from one another, but both adhere to a lesser degree than wild-type strain 101-1 (P < 0.00001). (B to G) Bacterial adherence to cultured HEp-2 cells after 3-h infections. (B) Prototypical EAEC strain 042. (C) Outbreak strain 101-1. (D) 101-1(pCADA). (E) INK1300. (F) OP50. (G) Uninfected HEp-2 cells.

FIG. 6.

(A) C. elegans survival rates of wild-type EAEC outbreak strain 101-1, 101-1 complemented with pCADA, and INK1300, the derivative of 101-1 with the cad operon integrated into the chromosome. Strain OP50 was used as a negative control. Both strain INK1300 and 101-1 bearing pCADA were significantly attenuated compared to the wild-type 101-1 strain (P = 0.001). The observed differences between strains fed on INK1300 and 101-1(pCADA) did not show statistical significance. (B to G) Visualization of bacterial strains OP50, 101-1, and INK1300 expressing dsRed from pHKT4 inside infected C. elegans. (B) Fluorescence of bacteria within a worm that has used 101-1(pHTK4) as a food source. Note intense colonization of the pharynx and intact bacteria present inside the worm anterior to the grinder (marked with an arrow in panels B, D, and F). Autofluorescence of the lumen is visualized in green. (D) Fluorescence of a worm that has used INK1300(pHTK4) as a food source. As with 101-1, the pharynx is colonized but there are fewer bacteria posterior to the grinder. (F) Fluorescence of a worm that has used OP50 as a food source, showing very little pharyngeal colonization and no detectable red fluorescence distal to the grinder. (C, E, and G) Light field views of worms in panels B, D, and F, respectively. Photomicrographs were taken 3 days postseeding with a fluorescence microscope at ×100 magnification.