Identification of a third metalloprotease toxin gene in extraintestinal isolates of Bacteroides fragilis

Abstract

To further understand the epidemiology of enterotoxigenic Bacteroides fragilis (ETBF), 89 extraintestinal B. fragilis strains from Seoul, Korea, were examined for secretion of B. fragilis toxin (BFT) by the HT29/C1 biologic assay and for the B. fragilis toxin gene (bft) by colony blot hybridization and PCR. Complete agreement between the three techniques was found. Overall, 34 B. fragilis strains (38%) were identified as ETBF. Eleven of the 34 ETBF strains (32%) expressed a new isoform of BFT (Korea-BFT). This new isoform is more related to BFT-2 than to BFT-1. Like BFT-1 and BFT-2, Korea-BFT cleaves E-cadherin, the zonula adherens protein.

FIG. 1

Representative results of PCR with primers 1 and 2 (sequences in text) for detection of ETBF from extraintestinal samples. All B. fragilis strains tested in this gel were identified as ETBF by cell culture assay and colony blot hybridization. Lanes: 1, DNA molecular weight marker (1-kb DNA ladder [Gibco BRL]); 2, ETBF 86-5443-2-2 (bft-2 positive control); 3, B. fragilis 419 (blood isolate); 4, B. fragilis 536 (pus isolate); 5, B. fragilis 723 (pleural fluid isolate); 6, B. fragilis 713 (right foot isolate); 7, B. fragilis 529 (blood isolate); 8, B. fragilis 586 (blood isolate); 9, B. fragilis 502 (blood isolate); 10, ETBF VPI 13784 (bft-1 positive control); 11, NTBF 077225-2 (negative control). The arrows show the predicted 1.2-kb product and the nonspecific 0.6-kb product found in strains 419 and 586.

FIG. 2

Alignment of BFT-1, Korea-BFT (BFT-K), and BFT-2 sequences from ETBF VPI 13784, 419, and 86-5443-2, respectively. The signal peptide sequences are underlined, the arrowhead shows the start of the mature protein, residues forming the zinc-binding signature motif are in boldface type and marked with asterisks, and the carboxy-terminal 20 residues which are predicted to form an amphipathic region in BFT-2 are enclosed in a box. Modeling of this region in Korea-BFT did not yield an amphipathic domain.

FIG. 3

Agarose gel electrophoresis of fragments produced by Sau3AI digestion of the bft gene amplified by PCR with primers 1 and 5 (sequences in text). Lanes: 1, DNA molecular weight marker (φX174 RF DNA/HaeIII fragments [Gibco BRL]); 2, bft-1 from ETBF VPI 13784; 3, bft-2 from ETBF 86-5443-2-2; 4, Korea-bft from ETBF 419.

FIG. 4

(A) Electrophoretic mobility of purified BFT-1 from ETBF VPI 13784 (lane 1) and purified Korea-BFT from ETBF 419 (lane 2) on SDS-PAGE, analyzed by a Western blot probed with polyclonal anti-BFT-2 serum. (B) Cleavage of E-cadherin by BFT. HT29/C1 cells were treated with BFT (100 ng/ml) for 3 h, lysed in 1× SDS gel loading buffer, and examined by Western blotting with E2 antibodies against the cytoplasmic domain of E-cadherin. Lane 1, untreated HT29/C1 cells; lane 2, HT29/C1 cells treated with BFT-2 purified from ETBF 86-5443-2; lane 3, HT29/C1 cells treated with Korea-BFT purified from ETBF 419. Loss of staining intensity of intact E-cadherin (120 kDa) is observed in HT29/C1 cells treated with BFT-2 and Korea-BFT.