Mutation of the Enterohemorrhagic Escherichia coli Core LPS Biosynthesis Enzyme RfaD Confers Hypersusceptibility to Host Intestinal Innate Immunity In vivo

Abstract

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen causing severe diseases in humans worldwide. Currently, there is no specific treatment available for EHEC infection and the use of conventional antibiotics is contraindicated. Therefore, identification of potential therapeutic targets and development of effective measures to control and treat EHEC infection are needed. Lipopolysaccharides (LPS) are surface glycolipids found on the outer membrane of gram-negative bacteria, including EHEC, and LPS biosynthesis has long been considered as potential anti-bacterial target. Here, we demonstrated that the EHEC rfaD gene that functions in the biosynthesis of the LPS inner core is required for the intestinal colonization and pathogenesis of EHEC in vivo. Disruption of the EHEC rfaD confers attenuated toxicity in Caenorhabditis elegans and less bacterial colonization in the intestine of C. elegans and mouse. Moreover, rfaD is also involved in the control of susceptibility of EHEC to antimicrobial peptides and host intestinal immunity. It is worth noting that rfaD mutation did not interfere with the growth kinetics when compared to the wild-type EHEC cells. Taken together, we demonstrated that mutations of the EHEC rfaD confer hypersusceptibility to host intestinal innate immunity in vivo, and suggested that targeting the RfaD or the core LPS synthesis pathway may provide alternative therapeutic regimens for EHEC infection.

Keywords: Caenorhabditis elegans; RfaD/GmhD/WaaD; antimicrobial peptides (AMPs); enterohemorrhagic Escherichia coli (EHEC); intestinal innate immunity; lipopolysaccharide (LPS).

Figure 1

Lipopolysaccharide (LPS) structure and the core-lipid A biosynthetic pathway. (A) A graphic representation of the LPS structure of E. coli. LPS contains several conserved components: lipid A, inner core, outer core and O-antigen (Raetz and Whitfield, 2002). (B) Pathways for the biosynthesis of the LPS core lipid A (Raetz and Whitfield, 2002). The genes encode the catalytic enzymes for ADP-L-glycero-D-manno-heptose and core-lipid A biosynthesis are presented in rectangular boxes.

Figure 2

Disruptions of rfaD and the genes involved in EHEC core LPS biosynthesis confer attenuated toxicity. (A–D) Survival curves of N2 animals fed with E. coli strain OP50 (OP50), EHEC strain EDL933 (EDL933), and EDL933 with Tn5 transposon insertion in rfaD (EDL933 rfaD::Tn5), rfaD deletion (EDL933:ΔrfaD), rfaE deletion (EDL933:ΔrfaE) and rfaC deletion (EDL933:ΔrfaC), and EDL933:ΔrfaD with prfaD complementation (EDL933:ΔrfaD-prfaD) were examined. All survival experiments were conducted independently at least three times with approximately 100 animals each time. (A) Animals feeding on the EDL933 rfaD::Tn5 mutant plates (P < 0.001) and the EDL933:ΔrfaD plates (P < 0.001) lived significantly longer than animals feeding on the wild-type EDL933 plates. (B) N2 animals fed with EDL933:ΔrfaE (P < 0.001) lived significantly longer than animals fed with the wild-type EDL933, but were similar to those fed with EDL933:ΔrfaD (P = 0.100). (C) Animals feeding on EDL933:ΔrfaC plates (P < 0.001) lived significantly longer than animals feeding on the wild-type EDL933 plates, but were similar to those on EDL933:ΔrfaD (P = 0.938). (D) N2 animals fed with EDL933:ΔrfaD (P < 0.001) lived significantly longer than animals fed with EDL933 wild type. The survival curve of N2 animals fed with EDL933:ΔrfaD-prfaD was similar to that of wild-type EDL933 (P = 0.128). (E) Growth curves of wild-type EDL933 and rfaD mutants in LB broth at 37°C. Both the growth kinetic curves of EDL933:ΔrfaD and EDL933:ΔrfaD-prfaD, were similar to wild-type EDL933. (F) The LPS samples of OP50, EDL933, EDL933 rfaD::Tn5, EDL933:ΔrfaD, and EDL933:ΔrfaD-prfaD was examined by silver staining. O-antigens are indicated by the black arrow.

Figure 3

Mutation in rfaD reduces EDL933 colonization in C. elegans. Images of wild-type N2 nematodes fed with GFP-labeled OP50 (A), EDL933 (B), EDL933: ΔrfaD (C) or EDL933:ΔrfaD-prfaD (D) for 1 days at 20°C and then chased with non GFP-labeled OP50 for 3 days at 20°C, respectively. Animals previously exposed on GFP-labeled EDL933 and EDL933:ΔrfaD-prfaD plates showed significant GFP signals in their intestines. Animals fed with wild-type EDL933 and EDL933:ΔrfaD-prfaD exhibited unhealthy apperances with smaller and paler body compared to animals fed with OP50 and EDL933:ΔrfaD. Representative images are shown, and the scale bar represents 100 μm. (E) The number of bacteria colonized in C. elegans was determined by the colony forming units (CFU) assay. Values represent the means of three independent assays, and error bars indicate the standard deviations. P-values denote the results of statistical analysis. The total numbers of animals tested in each group are indicated by n.

Figure 4

Mutations in rfaD reduce EHEC-induced intestinal microvillar actin rearrangement in C. elegans. (A) The confocal images showed the mCherry-tagged ACT-5 (intestinal microvillar actin) of GK454 transgenic animals fed with OP50, EDL933, EDL933 rfaD::Tn5 (rfaD::Tn5), EDL933:ΔrfaD (ΔrfaD) or EDL933:ΔrfaD-prfaD (ΔrfaD-prfaD) for 4 days respectively. Ectopic localization of the mCherry::ACT-5 signals from the apical membrane to the cytoplasm of the intestinal cells were significantly increased in the EDL933 treated group. Upper panels show mCherry images; lower panels are the merge images of the DIC and mCherry signals. Representative images are shown, and scale bar represents 10 μm. (B) Quantifications of animals with the EHEC-induced ectopic mCherry::ACT-5 signal were determined. Values represent the means of three independent assays, and error bars indicate the standard deviations. P-values denote the results of statistical analysis.

Figure 5

Disruption of rfaD reduces EDL933 colonization in the intestines of mice. (A) Represent images of mice inoculated with bioluminescence-labeled wild-type EHEC EDL933 (EDL933), EDL933:ΔrfaD (ΔrfaD), and EDL933:ΔrfaD-prfaD (ΔrfaD-prfaD) 1 h post infection. (B) Quantification of bioluminescence intensity of mice infected with EDL933, EDL933:ΔrfaD and EDL933:ΔrfaD-prfaD 1 h post infection. (C) Represent images of mice inoculated with bioluminescence-labeled wild-type EHEC EDL933 (EDL933), EDL933:ΔrfaD (ΔrfaD), and EDL933:ΔrfaD-prfaD (ΔrfaD-prfaD) 2 days post infection. (D) Quantification of bioluminescence intensity of mice infected with EDL933, EDL933:ΔrfaD and EDL933:ΔrfaD-prfaD 2 days post infection. (E) Represent images of intestinal tissues of mice infected with bioluminescence-labeled wild-type EHEC EDL933 (EDL933), EDL933:ΔrfaD (ΔrfaD), and EDL933:ΔrfaD-prfaD (ΔrfaD-prfaD). (F) Quantification of bioluminescence signals of intestinal tissues of bioluminescent EHEC infected mice 2 days post infection. The color scale represents the radiance (p/sec/cm²/sr). Representative images are shown. All experiments were conducted independently three times with 3 animals each time, and error bars indicate the standard deviations.

Figure 6

Deletion of EHEC rfaD increases its susceptibility to human serum killing and the antimicrobial peptide LL-37. The fold changes of bacterial cell numbers of wild-type EDL933, EDL933 rfaD::Tn5, EDL933:ΔrfaD and EDL933:ΔrfaD-prfaD after incubation in 10% normal human serum (NHS) (A) or 10% heat-inactivated normal human serum (HNHS) (B) at 37°C for 60 min were measured. Experiments were conducted independently at least three times, and error bars indicate the standard deviations. (A) The rfaD transposon mutant (rfaD::Tn5, P = 0.001) and rfaD deletion mutant (ΔrfaD, P = 0.001) showed significant decreased survival ratio compared to that of wild-type EDL933 (EDL933) in 10% NHS. The rfaD complement bacteria (ΔrfaD-prfaD) showed comparable survival ratio compared to that of EDL933 (P = 0.101) in 10% NHS. (B) The rfaD transposon mutant (rfaD::Tn5, P = 0.031) and rfaD deletion mutant (ΔrfaD, P = 0.034) showed significantly decreased survival ratio compared to that of wild-type EDL933 (EDL933) in 10% HNHS. The rfaD complement bacteria (ΔrfaD-prfaD) showed comparable survival ratio compared to that of EDL933 (P = 0.854) in 10% HNHS. n.s. indicates no statistical significance. (C) The OD₅₉₅ values of wild-type EDL933 (EDL933), EDL933 rfaD::Tn5 (rfaD::Tn5), EDL933:ΔrfaD (ΔrfaD), and EDL933:ΔrfaD-prfaD (ΔrfaD-prfaD) cultured with different dose of LL-37 at 37°C for 16 h were monitored. (D) The growth curves of EDL933, rfaD::Tn5, ΔrfaD and ΔrfaD-prfaD in the presence of 12.5 μg/ml LL-37 at 37°C.

Figure 7

The rfaD mutation increases the susceptibility of EHEC to host intestinal innate immunity. Images of DA597 pharynx defect C. elegans fed with (A) GFP-labeled OP50 (OP50-GFP), (B) EDL933 (EDL933-GFP), (C) EDL933:ΔrfaD (ΔrfaD-GFP), or (D) EDL933:ΔrfaD-prfaD (ΔrfaD-prfaD-GFP) at 20°C for 1 day. (E) Percentage of GFP-labeled bacteria (A–D) fed DA597 animals with GFP signals in intestine was calculated. Images of DA597 animals fed with (F) GFP-labeled OP50, (G) EDL933, (H) EDL933:ΔrfaD, or (I) EDL933:ΔrfaD-prfaD at 20°C for 1 days and chased on normal non-GFP OP50 plates for 3 days. (J) Percentage of GFP-labeled bacteria (F–I) fed DA597 animals with GFP signals in intestine was calculated. All experiments were conducted independently at least three times, and error bars indicate the standard deviations. The scale bar represents 100 μm.