Article Figures & Data
Figures
- 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.
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.
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.
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.
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.
(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.
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.
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.
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.
Tables
- TABLE 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Relevant feature(s) Source or reference Strains DH5α Laboratory E. coli strain RDEC-1 O15:H−, Nalr 7 RDEC-H19A RDEC-1 transduced with phage H19A, Nalr Tetr 46 SM10 SM10 λpir, recipient for suicide vector pCVD442 13 TSA01 SM10 containing pM381 This study RDEC-1Δeae860-939 RDEC-1Δeae860-939, Nalr This study Plasmids pALT417-3 pALTER-1::RDEC-1eae, Ampr p368 Derived from pALT417-3 with 1-bp deletion in eae, Ampr This study pCVD442 Suicide vector, Ampr 13 pM381 pCVD442::Δeae, Kanr Ampr This study
