IgG Binds Escherichia coli Serine Protease EspP and Protects Mice From E. coli O157:H7 Infection

Abstract

Shiga toxin-producing Escherichia coli O157:H7 is a virulent strain causing severe gastrointestinal infection, hemolytic uremic syndrome and death. To date there are no specific therapies to reduce progression of disease. Here we investigated the effect of pooled immunoglobulins (IgG) on the course of disease in a mouse model of intragastric E. coli O157:H7 inoculation. Intraperitoneal administration of murine IgG on day 3, or both on day 3 and 6, post-inoculation improved survival and decreased intestinal and renal pathology. When given on both day 3 and 6 post-inoculation IgG treatment also improved kidney function in infected mice. Murine and human commercially available IgG preparations bound to proteins in culture filtrates from E. coli O157:H7. Bound proteins were extracted from membranes and peptide sequences were identified by mass spectrometry. The findings showed that murine and human IgG bound to E. coli extracellular serine protease P (EspP) in the culture filtrate, via the IgG Fc domain. These results were confirmed using purified recombinant EspP and comparing culture filtrates from the wild-type E. coli O157:H7 strain to a deletion mutant lacking espP. Culture filtrates from wild-type E. coli O157:H7 exhibited enzymatic activity, specifically associated with the presence of EspP and demonstrated as pepsin cleavage, which was reduced in the presence of murine and human IgG. EspP is a virulence factor previously shown to promote colonic cell injury and the uptake of Shiga toxin by intestinal cells. The results presented here suggest that IgG binds to EspP, blocks its enzymatic activity, and protects the host from E. coli O157:H7 infection, even when given post-inoculation.

Keywords: Escherichia coli O157:H7; EspP; Shiga toxin; hemolytic uremic syndrome; immunoglobulin G; mouse.

Figure 1

Murine IgG given on day 3 or day 3 and 6 protected mice from E. coli O157:H7-induced disease. (A) Survival in mice infected with E. coli O157:H7 (EHEC) and treated with IgG on day 3 and 6 (n=10, blue line), day 3 (n=5, red line), day 6 (n=5, green line), untreated (n=9, purple line) and uninfected controls treated with IgG (n=3, yellow line) and without IgG (n=8, black line). (B) Weight changes in infected and uninfected mice, starting 1 day before inoculation, at the start of fasting, until day 14 post-inoculation, when the experiment ended. (C) Bacterial colony forming units in feces of EHEC infected mice on days 1, 3, 5 and 8. Results are presented as medians.

Figure 2

Histopathology in E. coli O157:H7-infected mice. Pathological changes were found in untreated E. coli O157:H7-infected mice. (A) Intestinal inflammatory infiltrates (white arrow) and mucus-depleted goblet cells (white arrowhead) in a mouse sacrificed on day 10 after inoculation in the infected and untreated group (corresponding to pathological score 2 for inflammatory infiltrates and pathological score 3 for mucus-depleted goblet cells). (B) Renal tubular desquamation (black arrow) in a mouse sacrificed on day 10 after inoculation in the infected and untreated group (corresponding to pathological score 3). (C) Normal intestinal histology in infected and IgG treated mouse on day 3 sacrificed on day 14 (corresponding to pathological score 0). (D) Normal renal histology in infected and IgG treated mouse on day 3 and 6 sacrificed on day 14 (corresponding to pathological score 0). (E) Normal intestinal histology in a control mouse sacrificed on day 14 (corresponding to pathological score 0). (F) Normal renal histology in a control mouse sacrificed on day 14 (corresponding to pathological score 0). Scale bar 100 μm.

Figure 3

Blood urea nitrogen levels in E. coli O157:H7-infected mice. Blood urea nitrogen measured in plasma from E. coli O157:H7 (EHEC) infected and uninfected mice. Two mice in the infected and IgG-treated day 3 and 6 group developed symptoms and these are the two in this group with high BUN values. Data presented as median and individual values representing individual mice. *P < 0.05.

Figure 4

IgG binding to E. coli O157:H7. The binding of IgG to E. coli O157:H7 culture filtrate was analyzed by ELISA. (A) Mouse IgG exhibited dose-dependent binding to E. coli O157:H7 culture filtrate. (B) Human IgG showed dose-dependent binding to E. coli O157:H7 culture filtrate. The bar represents the median. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Mouse and human IgG bind specific E. coli O157:H7 proteins. E. coli O157:H7 proteins in culture filtrate were blotted onto membranes for detection of IgG binding. (A) E. coli O157:H7 proteins incubated with mouse IgG and human IgG. The blot detected with mouse IgG shows six bands and the blot detected with human IgG shows seven bands (arrows). Both lanes were run on the same gel. (B) E. coli O157:H7 proteins detected with mouse Fab, Fc and whole IgG showing that the binding to E. coli O157:H7 proteins is mediated via the Fc fragment. All lanes were run on the same gel and visualized by immunoblotting. (C) Silver stained gel of eluted proteins corresponding to the seven E. coli O157:H7 proteins detected with mouse and human IgG in panel (A). (D) Immunoblot of eluted proteins [according to panel (C)] detected with mouse IgG. (E) Immunoblot of eluted proteins [according to panel (C)] detected with human IgG. A faint band in lane 3 was not seen in (D). MW, molecular weight.

Figure 6

Mouse IgG and human IgG bind to EspP. Purified EspP loaded on to a membrane was analyzed for IgG binding. (A) Mouse whole IgG and the Fc fragment, but not the Fab fragment, bound to EspP. All lanes were run on the same gel. (B) Human whole IgG and the Fc fragment, but not the Fab fragment, bound to EspP. All lanes were run on the same gel. (C) Detection of EspP in culture filtrate from wild-type E. coli O157:H7 but not in the ΔespP strain run alongside purified EspP bound to mouse IgG. Full-length EspP detected at 104 kDa with mouse or human IgG, marked with a black arrowhead. Bands detected at different molecular weights than the full-length EspP marked with an open arrowhead representing proteins other than EspP that bind IgG.

Figure 7

Mouse and human IgG inhibit EspP-mediated pepsin cleavage. Pepsin incubated alone or with wild-type or ΔespP E. coli O157:H7 culture filtrates was loaded onto a membrane for detection of pepsin cleavage. Supernatant pre-incubated with mouse IgG (A) or human IgG (B) partially inhibited EspP activity. Full-length pepsin was detected at approximately 40kDa, marked with a black arrowhead, and the cleavage product was detected at approximately 20kDa marked with an open arrowhead. WT, culture filtrate from the wild-type E. coli O157:H7 strain; ΔespP, culture filtrate from the EspP mutant; IgG, immunoglobulin G; PMSF, phenylmethylsulfonyl fluoride.