Article Figures & Data
Figures
- FIG. 1.
The C. rodentium lifA/efa1 gene. The lifA/efa1 gene from C. rodentium was identified on cosmid clone p3-B-8 by testing bacterial lysates in an inhibitory assay and cross hybridization with an internal EcoRI fragment from EPEC strain E2348/69 lifA/efa1. The D-X-D motif in C. rodentium lifA/efa1, encoding a glycosyltransferase activity and inactivating small GTP-binding proteins, is also present in large clostridial cytotoxins. The protease motif is present in avirulence proteins of plant pathogens and YopT, and it disrupts the actin cytoskeleton in host cells. The third distinct motif present in C. rodentium lifA/efa1 encodes an aminotransferase II. Areas in grey indicate domains with significant sequence similarities to known motifs present in other gram-negative bacteria. Three domains of the lifA/efa1 gene in C. rodentium were mutated, generating mutant GlM12, PrMC31, and EID3 (arrows).
- FIG. 2.
(A) Bacterial lysates from lifA/efa1 mutant strain EID3 lose IL-2 inhibitory activity. PBMC (106/ml) were preincubated for 2 h with increasing concentrations (ranging from 2.5 to 50 μg/ml) of bacterial lysates from EPEC (♦), C. rodentium wild type (▪), lifA/efa1 mutant strain EID3 (▴), and complemented mutant EI3B8 (•). Lymphocyte cultures were activated with the combination of pokeweed mitogen (10 μg/ml) and phorbol myristate acetate (10 ng/ml). After a total of 24 h of incubation, supernatants were aspirated and analyzed in duplicate by ELISA. Data from three independent experiments were pooled, and mean values with SE normalized against values for the positive control were set at 100%. Unlike lysates from generated from EPEC, C. rodentium, and EI3B8, lysates from lifA/efa1 mutant EID3 failed to inhibit expression of IL-2 protein in activated lymphocyte cultures. This difference was statistically significant (P < 0.05) at concentrations of 5 μg/ml and greater for C. rodentium EI3B8 than for lysates fromEID3. Statistical differences were determined by Student's t test and considered significant if P was ≤ 0.05. (B) Formation of attaching lesions in 3T3 cells in response to infection with EPEC, C. rodentium (CrWT), EID3, and EI3B8. 3T3 cell cultures were infected with EPEC, C. rodentium, EID3, and EI3B8 for 5 h, rinsed in PBS, fixed in paraformaldehyde, and stained for nucleic acids and filamentous actin. Images were obtained with a Zeiss Axiscope and digital imaging package. The accumulation of filamentous actin underneath the site of bacterial adherence was not affected by a mutation in lifA/efa1, as mutant and wild-type bacteria induced similar attaching lesions in 3T3 fibroblast cultures. (C) Quantification of bacterial adherence to fibroblast cultures. 3T3 cells were infected with 5 × 104 CFU of wild-type EPEC and C. rodentium (CrWT), lifA/efa1 mutant EID3, and complemented strain EI3B8. Infection was allowed to proceed for 4 h, when cell cultures were washed three times with PBS and lysed in 1% Triton X-100. Serial dilutions were plated on MacConkey agar plates, incubated overnight, and enumerated the following morning. Bacterial adherence to fibroblast cells was determined in triplicate in three independent experiments. Data are expressed as means, with error bars representing SE of the means, and were subjected to Student's t test for statistical analysis (P ≤ 0.05). Regardless of the presence or absence of an intact lifA/efa1 gene, wild-type and mutant C. rodentium adhered with equal efficiency to fibroblast cultures at 4 h of infection.
- FIG. 3.
Weight chart for mice infected with EPEC and C. rodentium strains. Enteric infection of groups of five mice with EPEC (♦), C. rodentium (▪), EID3 (▴), and EI3B8 (•) was initiated by gavage with 3 × 108 CFU/animal in 100 μl of PBS on day 0. Animals were weighed daily until day 20 of infection, and data are expressed as means with error bars for SE. Statistical analysis was performed by Student's t test, and results were considered significant if P was ≤0.05. In comparison to mice infected with wild-type C. rodentium, EID3 did not induce signs and symptoms of systemic disease, and animals gained weight throughout the 20-day experiment. Weight gain for control mice (○) and animals infected with EID3 and EPEC was indistinguishable.
- FIG. 4.
Transmission electron microscopy of EPEC, C. rodentium wild type, EID3, and EI3B8 on day 8 of infection. Morphology of colonic tissue from infected mice at day 8 of infection was evaluated by transmission electron microscopy. Epithelial pedestal-like structures consistent with A/E lesions were observed in the apical pole of epithelial cells infected with C. rodentium wild type (CrWT), EID3, and EI3B8.
- FIG. 5.
Bacterial counts of EPEC, C. rodentium, EID3, and EI3B8 isolated from the colon of infected mice. Following inoculation with EPEC (♦), C. rodentium (▪), EID3 (▴), and EI3B8 (•) (A), wild-type C. rodentium (▪), GlM12 (▵), and PrM31 (○) (B) on day 0, animals were sacrificed on day 2, 8, 14, or 20. Fecal pellets were removed from the distal colon of infected animals, and tissue was weighed and homogenized at low speed. Serial dilutions were plated on MacConkey agar, and recovered bacteria were counted the following day. Data depict the mean CFU per gram of colon tissue of infected animals, with bars representing the SE of the mean. Nonparametric analysis of organ colonization counts was performed by the Mann-Whitney U test. (A) Wild-type C. rodentium (▪) and mutant EID3 (▴) were able to colonize the colon of infected animals with equal efficiency until day 8, after which EID3 was rapidly cleared from the colon, decreasing to levels similar to the CFU determined for EPEC (♦). (B) Mutations in the glycosyltransferase (▵) and protease motif (○) resulted in significantly reduced colonic colonization throughout the infection in comparison to wild-type C. rodentium infection (▪).
- FIG. 6.
Time course of epithelial cell regenerative and histological changes in response to infection with EPEC, C. rodentium, EID3, and EI3B8. (A) Epithelial regenerative changes for all animals per group were analyzed by two pathologists in a blinded manner on days 2, 8, 14, and 20. The analysis included control animals and mice infected with EPEC, C. rodentium (CrWT), EID3, and EI3B8. Cytological changes were graded as 1+ (no changes), 2+ (minimal, involving crypts with ≤20% increases in mucosal thickness), 3+ (mild, extending up to half crypt height with 20 to 80% increases in mucosal thickness), 4+ (moderate, extending over half of crypt height with surface maturation and with 80 to 100% increases in mucosal thickness), and 5+ (florid, involving entire crypt and surface epithelium with >100% increases in mucosal thickness). Scores determined for epithelial regenerative changes were analyzed by Student's t test (P ≤ 0.05). Interestingly, all infected animals responded with increased regenerative scores on days 2, 8, 14, and 20 of infection. A statistically significant difference inregeneration was determined on day 20 of infection in a comparison of scores obtained for EID3 and C. rodentium wild type. (B) Distal colonic sections from each animal were fixed in formalin and embedded in paraffin, and transverse 5-μm sections were stained with hematoxylin and eosin. In comparison to control mice inoculated with PBS, all groups responded with increased regenerative scores. TMCH developed only in wild-type C. rodentium (Cr WT)-infected mice by day 8 and increased in severity by day 20.
Tables
- TABLE 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Description Source or reference EPEC E2348/69 O126:H6 isolate from infant diarrhea 22a C. rodentium Wild type causing and TMCH and colitis ATCC pKD46 Red recombinase system 7 pKD4 PCR template for Km cassette and FRT 7 pFT-A Helper plasmid encoding for recombinase 32a p3-B-8 C. rodentium lifA/efal cosmid clone This study CrpKD46 C. rodentium wild type transformed with pKD46 This study EIB2K1 Kanamycin insertion mutation at bp position 3,496 This study EID3 Kanamycin-cured lifA/efa1 non-motif mutant This study EI3B8 EID3 complemented lifA/efa1 on plasmid p3-B-8 This study G1M9 Kanamycin insertion mutation at bp position 1,672 This study G1M12 Kanamycin-cured lifA/efa1 glycosyltransferase motif mutant This study PrMC1 Kanamycin insertion mutation at bp position 3,456 This study PrMC31 Kanamycin-cured lifA/efa1 protease motif mutant This study - TABLE 2.
PCR primer combinations for molecular methods
Primer name Sequencea Description Klapp-110 5′-ATGGATATTAATACTGAAACTGAG-3′ Gam protein Klapp-111 5′-TTATACCTCTGAATCAATATCAAC-3′ Gam protein Klapp-112 5′-ATGACACCGGACATTATCCTGCAG-3′ Exonuclease Klapp-113 5′-TCATCGCCATTGCTCCCCAAATAC-3′ Exonuclease Klapp-102 5′-ACAATAAATGGACTAGGGATAACAGGTGTACACACTGCAGAATGCCTGCTACCGGCACCAgtgtaggctggagctgcttc-3′ bp 3,496-3,555, lifA/efa1-pKD4 Klapp-103 5′-CAAATTGTCAAAACCATCAGCAGCACTGACCATCACCTGTTCTGTAATTTTTCCGTTAATcatatgaatatcctccttag-3′ bp 3,640-3,669, lifA/efa1-pKD4 Klapp-165 5′-TACTGTATTATGAAGGGAGTTACAGATATTAATGATGAGTTACGAGTAAACTATgtgtaggctggagctgcttc-3′ bp 1,297-1,350, lifA/efa1-pKD4 (glycosyltransferase motif) Klapp-167 5′-ACGTAGCTTTAAGTCTTCCAGGAAACGATTATCTCCATTTGTTTCCATCATAATcatatgaatatcctccttag-3′ bp 1,672-1,725, lifA/efa1-pKD4 (glycosyltransferase motif) Klapp-169 5′-CATGCACAGGGATGGTTCGAAGTGGCCAAAGGATATGGCAGCCAGAATATTGACgtgtaggctggagctgcttc-3′ bp 4,303-4,356, lifA/efa1-pKD4 (protease motif) Klapp-170 5′-TAATTGTATACCAGCCTCAATAAACTTCAGAGCATCCAGGGAAGAAAGGAAATCcatatgaatatcctccttag-3′ bp 4,441-4,494, lifA/efa1-pKD4 (protease motif) Klapp-187 5′-TACTGTATTATGAAGGGAGTTA-3′ bp 1,297-1,318, lifA/efa1 Klapp-188 5′-AAGCCCACGTATATTCCATCCG-3′ bp 1,938-1,959, lifA/efa1 Klapp-136 5′-AGCAGACGAGTTCAAGGGATA-3′ bp 3,363-3,383, lifA/efa1 Klapp-137 5′-CAAATTGTCAAAACCATCAGC-3′ bp 3,649-3,669, lifA/efa1 Klapp-201 5′-ATCGTTGAACACGCCAAATAT-3′ bp 4,249-4,269, lifA/efa1 Klapp-202 5′-TCTGGTCATCAGGGCATTATC-3′ bp 4,528-4,548, lifA/efa1 Klapp-256 5′-AATTCTCATGTTTGACAGCT-3′ pHC79 Klapp-257 5′-AATTCTCATGTTTGACAGCT-3′ pHC79 ↵ a Uppercase letters, nucleotides with C. rodentium lifA/efa1-specific sequences; lowercase letters, nucleotides recognizing sequences upstream and downstream of the kanamycin resistance cassette in pKD4.
