Article Figures & Data
Figures
- Fig. 1.
Alignment of EspG from EPEC O127: H7 (EPEC) with EspG from EHEC O157: H7 (EHEC), rabbit pathogen RDEC-1, REPEC strain 83/39 (REPEC), Orf3 from the EspC pathogenicity islet, and VirA fromS. flexneri. Numbers at the end of the line, amino acid numbers; period, identical amino acids; +, similar but nonidentical amino acids; blank space, nonhomologous amino acids. The DNA sequence encoding the first 22 amino acids (aa) of REPEC VirA is not known, and each of these missing amino acids is indicated with an “x.” Asterisks (within the VirA sequence), areas where VirA contains extra amino acids not found within EspG, including an insertion of 5 aa into the region corresponding to aa 210 and 211 in EspG and a 15-aa insertion between aa 320 and 321 in EspG. The predicted consensus secondary structures (struct) conserved between EspG and VirA are denoted A (α-helix), B (β sheet), and P (proline; indicating a turn). The percent identity and percent similarity (%ID and %Sim, respectively) to the sequence of EPEC EspG are listed at the end of the alignment.
- Fig. 2.
Western blot with anti-EspG antiserum on whole-cell lysates. Exposure times: lanes 1 and 2 (numbering from the left), 3 min; lanes 3 to 5, 15 s. Antiserum recognized the 44-kDa EspG protein in whole-cell lysates of wild-type EPEC E2348/69 (lanes 1 and 3), EHEC (lane 2), and EPEC E2348/69 espG(pCVD453) (lane 5) but not in that of EPEC E2348/69 espG(lane 4). Comparison of relative amounts of EspG produced by EPEC, theespG mutant, and the complemented mutant (lanes 3 to 5) demonstrates that EspG is produced in small amounts by wild-type EPEC and that the complement overproduces EspG.
- Fig. 3.
Type III secretion of EspG into supernatants and translocation into HEp-2 cells. Western blots with EspG antiserum indicate that EspG is produced by EPEC E2348/69 espG(pCVD453), EPEC E2348/69 escN (pCVD453), and in lesser amounts by wild-type EPEC E2348/69. EspG is observed in the supernatant and in the Triton X-100-soluble fraction of HEp-2 cells infected with EPEC E2348/69 espG (pCVD453) but not EPEC E2348/69escN (pCVD453), indicating that secretion and translocation are dependent on type III secretion. Intimin is observed in whole cells but not in the Triton X-100-soluble fraction, indicating that the Triton X-100-soluble fraction is not contaminated with bacteria, and so EspG present in that fraction must be due to translocation and not contamination.
- Fig. 4.
Intracellular persistence of bacteria within HeLa cells as a measure of intracellular survival and/or replication, according to the protocol of Anderson et al. (2), and expressed as percentages of that for wild-type EPEC or S. flexneri(S.flex). The plasmid-free variant of S. flexneri(S.flex-pInv) was used as a negative control. Relative scores are above the bars.
- Fig. 5.
Geometric mean of the CFU count from rectal swabs, as a measure of fecal CFU, from rabbits orally inoculated with wild-type REPEC strain 83/39 or espG mutant SE1090. Error bars, standard errors.
Tables
- Table 1.
Strains and plasmids used in this study
Strain or plasmid Relevant genotype or descriptiona Reference or source Strains E. coli E2348/69 E. coli O127:H7 EPEC strain E2348/69 17 E2348/69 espG E2348/69espG::pJP5603 This study E2348/69espG(pCVD453) E2348/69espG::pJP5603(pCVD453) This study E2348/69 escN(pCVD453) E2348/69escN::aphA3(pCVD453) 12; this study E2348/69 espG orf3 E2348/69espG::pJP5603orf3::pJP5608 This study 85-170 EHEC O157:H7 12 HS Normal flora E. colistrain 12 83/39 REPEC 1 SE1090 REPEC 83/39espG::pJP5603 This study S. flexneri YSH6000 Wild-type S. flexneri 27 virA mutant S. flexneri N1945 27 virA(pEspG) mutant S. flexneri N1945(pCVD453::Tp) This study virA(pOrf3) mutant S. flexneriN1945(pTB101:Orf3) This study virA(pVirA) mutant S. flexneri N1945(pKU002) 27 Inv mutant S. flexneri 2a cured of invasion plasmid 22 BL21(DE3) F− ompT hsdSB(rB − mB −) gal dcm (DE3) Novagen Plasmids pCVD453 3.1-kb LEE fragment in pSPORT (Apr) 19 pEspG 3.1-kb LEE fragment from pCVD453 and Tpr cassette in pBluescript SK (AprTpr) This study pOrf3 Orf3 in pTB101 (Tpr) This study pQE30::His6EspG N-terminal MRGSHis6 fusion to EspG (Apr) This study pJP5603 oriR6K mobRP4 lacZ::MCSkan (Kmr) 23 pJP5608 oriR6K mobRP4 lacZ::MCS tet(Tcr) 23 ↵a Apr, resistance to 100 μg of ampicillin/ml; Tpr, resistance to 50 μg of trimethoprim/ml; Kmr, resistance to 25 μg of kanamycin/ml; Tcr, resistance to 15 μg of tetracycline/ml.
- Table 2.
Virulence properties of wild-type and mutant EPEC strains
Strain Results for assay of: LAa FASb Tir, EspABD secretionc HEp-2 invasion (%)d Intracellular persistence (%)e E2348/69 + + + 100 100 E2348/69 espG + + + 83 83 E2348/69espG(pCVD453) + + + nt nt E2348/69 espG orf3 + + ntf 70 70 ↵a LA, localized adherence.
↵b FAS, fluorescent actin staining (an assay for attaching and effacing lesion formation).
↵c Type III secretion of EspADB and Tir.
↵d Invasion of HEp-2 cells, relative to E2348/69.
↵e Intracellular persistence within HEp-2 cells after invasion, relative to E2348/69.
↵f nt, not tested.
