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. 2005 Oct;73(10):6608-19.
doi: 10.1128/IAI.73.10.6608-6619.2005.

"V体育官网入口" Protection against hemorrhagic colitis in an animal model by oral immunization with isogeneic rabbit enteropathogenic Escherichia coli attenuated by truncating intimin

Tonia S Agin  1 , Chengru Zhu, Laura A Johnson, Timothy E Thate, Zhuolu Yang, Edgar C Boedeker
Affiliations

Protection against hemorrhagic colitis in an animal model by oral immunization with isogeneic rabbit enteropathogenic Escherichia coli attenuated by truncating intimin

Tonia S Agin et al. Infect Immun. 2005 Oct.

Abstract

Strains of Shiga toxin (Stx)-producing Escherichia coli, also called enterohemorrhagic E. coli (EHEC), are important food-borne pathogens for humans. Most EHEC strains intimately adhere to the intestinal mucosa in a characteristic attaching and effacing (A/E) pattern, which is mediated by the bacterial adhesin intimin. Subsequent release of Stx1 and/or Stx2 leads to the frequent development of hemorrhagic colitis and, less commonly, to hemolytic-uremic syndrome. The aim of the present study was to develop an attenuated A/E E. coli strain for use as a vaccine against EHEC infection encoding a truncated intimin lacking adhesive capacity, but which would still express somatic antigens, other products of the locus of enterocyte effacement pathogenicity island, and an immunogenic remnant of the intimin molecule. A single-nucleotide deletion was generated in the eae gene in the prototype rabbit A/E E. coli strain RDEC-1 (O15:H-), which resulted in truncation of intimin by 81 C-terminal residues (860 to 939 amino acids) containing a disulfide loop. Inoculation of rabbits with large doses of the truncated intimin mutant (RDEC-1Deltaeae(860-939)) was well tolerated, as observed by the absence of clinical signs of disease or evidence of intestinal A/E lesions. The efficacy of RDEC-1Deltaeae(860-939) as a vaccine was evaluated by orogastric inoculation of rabbits with RDEC-1Deltaeae(860-939) followed by challenge with the virulent strain RDEC-H19A, an Stx1-producing derivative of wild-type RDEC-1 capable of inducing hemorrhagic colitis in rabbits. Following RDEC-H19A challenge, nonimmunized control rabbits exhibited characteristic weight loss with watery to bloody diarrhea and demonstrated intimate bacterial attachment, effacement of microvilli, submucosal edema, mucosal heterophile infiltrates, and Shiga toxin-induced vascular lesions VSports手机版. In contrast, the RDEC-1Deltaeae(860-939)-immunized rabbits showed no clinical signs of disease, maintained normal weight gain, had reduced fecal shedding of challenge organisms, and showed an absence of gross or microscopic lesions in the intestinal mucosa. Serum antibodies specific to intimin were detected among rabbits immunized with RDEC-1Deltaeae(860-939), indicating that truncation of the intimin functional domain not only attenuated bacterial virulence, but also retained at least some of the immunogenicity of native intimin. Although it is not possible to gauge the exact contribution of residual intimin immunity to protection, this attenuation strategy for A/E E. coli strains shows promise for the development of effective vaccines to prevent EHEC infection in humans and animals. .

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Figures

FIG. 1.
FIG. 1.
(A) Schematic representation of RDEC-1 intimin C-terminal fragment (539 to 939 aa) showing Ig-like (Ig) and Tir-binding domains. Numbers above the diagram represent the amino acid position of intimin deduced from RDEC-1 LEE (50). The residues of the intimin Tir-binding domain (842 to 939 aa) and of the truncated intimin mutant are shown below the diagram. Asterisks indicate two conserved cysteine residues (aa 860 and 937) involved in the formation of a disulfide loop essential for intimin function. Identical amino acids in the mutant are shown by dots. (B) DNA alignment of partial sequence of the eae gene and the mutant showing a single-base deletion (arrow) which introduced a stop codon (underlined) 24 bp immediately after deletion. (C) Comparison of OMP profiles showing native intimin (denoted by a white arrowhead) produced by RDEC-1 in the left panel or truncated intimin in the right panel (arrowhead) expressed by RDEC-1Δeae860-939. Molecular mass markers are indicated in kDa.
FIG. 2.
FIG. 2.
FAS tests (corresponding actin fluorescence [A and B] and phase-contrast micrographs [C and D]) showing adherence pattern to HeLa cells of WT RDEC-1 (A and C) and RDEC-1Δeae860-939 (B and D) following 5 h of incubation.
FIG. 3.
FIG. 3.
Clinical observations in rabbits inoculated with WT RDEC-1 (open circles) or its derivative eae mutant (open triangles). Comparisons of cumulative weight change (A) and semiquantitative determination of fecal bacterial shedding of the administered strain (B) are shown. Averages were derived from six rabbits in each group. Bars designate standard errors.
FIG. 4.
FIG. 4.
Clinical observations in RDEC-1Δeae860-939-immunized (open circles) and nonimmunized (open triangles) rabbits following challenge with RDEC-H19A (day 0). Comparisons of stool consistency (A), cumulative weight change (B), and semiquantitative determination of fecal bacterial shedding of the challenge strain RDEC-H19A (C) are shown. Bars designate standard errors.
FIG. 5.
FIG. 5.
Comparison of percentages of mucosal surface (cecum) with adherent bacteria between RDEC-1Δeae860-939-immunized and nonimmunized groups following challenge with RDEC-H19A. Bars designate standard errors.
FIG. 6.
FIG. 6.
(A) Histological section from a nonimmunized rabbit challenged with RDEC-H19A showing intimate bacterial adherence and effacement of microvilli. Bacteria are adhering to the cecal enterocytes, and the mucosal surface has become irregular with a cluster of desquamating cells (white arrow). In comparison, the mucosa from an immunized and challenged animal remained normal (B). Hematoxylin and eosin staining; magnification, ×400.
FIG. 7.
FIG. 7.
Comparisons of enumeration of heterophiles per HPF (A) and scoring of edema (B) between immunized, unchallenged (day 28) animals (open columns), immunized, challenged (day 35) animals (solid columns), and nonimmunized, challenged (day 35) animals (semisolid columns). Bars designate standard errors. *, statistically significant difference versus both other groups.
FIG. 8.
FIG. 8.
Microvascular changes in mucosal, submucosal, and serosal compartments, as measured by scores for adherent heterophiles (A), endothelial denudation (B), endothelial swelling (C), and thrombus formation (D) expressed as individual parameters and as a composite vascular score (E) among immunized, unchallenged animals (open columns), immunized, challenged animals (solid columns), and nonimmunized, challenged animals (semisolid columns). Bars designate standard errors. *, statistically significant difference between the nonimmunized, challenged group and the immunized, challenged group; **, statistically significant difference versus the immunized, unchallenged group.
FIG. 9.
FIG. 9.
Rabbit serum IgG titers specific to MBP-Int280 prior to immunization (day 0), following prime (day 14) and boost (day 28) immunizations with RDEC-1Δeae860-939, and after RDEC-H19A challenge (day 35). Bars designate standard errors. *, statistically significant difference between the nonimmunized, challenged group and the immunized, challenged group.
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"VSports在线直播" References

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