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. 1999 Nov;67(11):5587-96.
doi: 10.1128/IAI.67.11.5587-5596.1999.

"V体育ios版" Characterization of pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli

I R Henderson  1 , J Czeczulin, C Eslava, F Noriega, J P Nataro
Affiliations

Characterization of pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli (V体育安卓版)

I R Henderson et al. Infect Immun. 1999 Nov.

Abstract

We have identified and characterized a secreted protein, designated Pic, which is encoded on the chromosomes of enteroaggregative Escherichia coli (EAEC) 042 and Shigella flexneri 2457T. The product of the pic gene is synthesized as a 146. 5-kDa precursor molecule which is processed at the N and C termini during secretion, allowing the release of a mature protein (109. 8 kDa) into the culture supernatant VSports手机版. The deduced amino acid sequence of Pic shows high homology to autotransporter proteins, particularly a subgroup termed the SPATEs (serine protease autotransporters of the Enterobacteriaceae). Present in all members of this subgroup is a motif similar to the active sites of certain serine proteases. Pic catalyzes gelatin degradation, which can be abolished by disruption of the predicted proteolytic active site. Functional analysis of the Pic protein implicates this factor in mucinase activity, serum resistance, and hemagglutination. Our data suggest that Pic may be a multifunctional protein involved in enteric pathogenesis. .

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Figures

FIG. 1
FIG. 1
Map of the chromosomal fragment cloned into pACYC184. At the 5′ end of the insert are the IS-homologous sequences IS911 and IS629, followed by the pic gene and subsequently by the perD IS-like element. The positions within the pic gene of the set1A and set1B genes which encode the two subunits of the ShET1 enterotoxin are also indicated.
FIG. 2
FIG. 2
SDS-PAGE analysis of Pic processing. (A) Analysis of Triton X-100-insoluble fractions from HB101(pPic1) (lane 2), HB101(pPicS258I) (lane 3), and HB101(pACYC184) (lane 4). Lane 1 contains molecular mass markers (in kilodaltons). The position of the β-domain is indicated. (B) Analysis of concentrated culture supernatants from HB101(pPic1) (lane 1), JCB517(pPic1) (lane 2), UT5600(pPic1) (lane 3), KS474(pPic1) (lane 4), and HB101(pPicS258I) (lane 5). The position of a molecular mass marker is indicated (in kilodaltons).
FIG. 3
FIG. 3
Regulation of Pic expression. An SDS-PAGE analysis of concentrated L-broth culture supernatants of E. coli 042 is shown. Culture conditions: 25 μg of EDDA ml−1 (lane 1), 0.5 mM FeSO4 (lane 2), pH 9.0 (lane 3), pH 5.0 (lane 4), no NaCl (lane 5), 0.4 M NaCl (lane 6), 24°C (lane 7), 37°C and pH 7.0 (lane 8), and 42°C (lane 9). The positions of some molecular mass markers are indicated (in kilodaltons).
FIG. 4
FIG. 4
Mucinolytic activity of the 116-kDa secreted protein. (A) Zones of mucin lysis on bovine submaxillary mucin are visible after treatment with Pic protein and staining with amido black (left). No lysis was visible in the section treated with similar preparations of the Pic protein containing a mutation in the serine protease active site (S258I) (right). (B) SDS-PAGE analysis of untreated crude mouse mucus (lane 1) and mucus treated with Pic protein (lane 2). The gel is PAS stained, and the band which is cleaved is indicated, as are the positions of some molecular mass markers. (C) Sephacryl S-400 column chromatography of untreated crude mouse mucus and mucus treated with the concentrated secreted protein. The column was calibrated with dextran blue, and the position of the excluded void volume is indicated. Fractions (0.8 ml) were collected during chromatography. PAS staining was performed on 300-μl aliquots and measured at 555 nm, and the absorbance has been graphed. OD, optical density.
FIG. 5
FIG. 5
Susceptibility of DH5α to killing by normal human serum. (A) Killing of DH5α is inhibited in the presence of Pic protein preparations but not in the presence of protein from the serine protease mutant (S258I), phenylmethylsulfonyl fluoride (PMSF), or antibodies to Pic. (B) Serum killing of DH5α occurs via the classical pathway of complement. Organisms were exposed to normal serum, antibody-depleted serum, C9-deficient serum, and serum with added EGTA and MgCl2. Data are presented as the percentage of the original inoculum remaining at each time point.
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