Molecular Pathogenesis

EspG, a Novel Type III System-Secreted Protein from Enteropathogenic Escherichia coli with Similarities to VirA of Shigella flexneri

Simon J. Elliott, Efrosinia O. Krejany, Jay L. Mellies, Roy M. Robins-Browne, Chihiro Sasakawa, James B. Kaper
DOI: 10.1128/IAI.69.6.4027-4033.2001

Article Figures & Data

Figures

  • Fig. 1.
    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.
    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.
    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.
    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.
    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 plasmidRelevant genotype or descriptionaReference or source
    Strains
     E. coli
      E2348/69E. coli O127:H7 EPEC strain E2348/6917
      E2348/69 espGE2348/69espG::pJP5603This study
      E2348/69espG(pCVD453)E2348/69espG::pJP5603(pCVD453)This study
      E2348/69 escN(pCVD453)E2348/69escN::aphA3(pCVD453)12; this study
      E2348/69 espG orf3E2348/69espG::pJP5603orf3::pJP5608This study
      85-170EHEC O157:H712
      HSNormal flora E. colistrain12
      83/39REPEC1
      SE1090REPEC 83/39espG::pJP5603This study
     S. flexneri
      YSH6000Wild-type S. flexneri27
      virA mutantS. flexneri N194527
      virA(pEspG) mutantS. flexneri N1945(pCVD453::Tp)This study
      virA(pOrf3) mutantS. flexneriN1945(pTB101:Orf3)This study
      virA(pVirA) mutantS. flexneri N1945(pKU002)27
      Inv mutantS. flexneri 2a cured of invasion plasmid22
      BL21(DE3)F−ompT hsdSB(rB− mB−) gal dcm (DE3)Novagen
    Plasmids
     pCVD4533.1-kb LEE fragment in pSPORT (Apr)19
     pEspG3.1-kb LEE fragment from pCVD453 and Tpr cassette in pBluescript SK (AprTpr)This study
     pOrf3Orf3 in pTB101 (Tpr)This study
     pQE30::His6EspGN-terminal MRGSHis6 fusion to EspG (Apr)This study
     pJP5603oriR6K mobRP4 lacZ::MCSkan (Kmr)23
     pJP5608oriR6K 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

    StrainResults for assay of:
    LAaFASbTir, EspABD secretioncHEp-2 invasion (%)dIntracellular persistence (%)e
    E2348/69+++100100
    E2348/69 espG+++8383
    E2348/69espG(pCVD453)+++ntnt
    E2348/69 espG orf3++ntf7070

    • ↵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.

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KEYWORDS

Bacterial Proteins
Escherichia coli
Escherichia coli Infections
Escherichia coli Proteins
virulence factors

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