Pet, an autotransporter enterotoxin from enteroaggregative Escherichia coli

Abstract

Enteroaggregative Escherichia coli (EAEC) is an emerging cause of diarrheal illness. Clinical data suggest that diarrhea caused by EAEC is predominantly secretory in nature, but the responsible enterotoxin has not been described. Work from our laboratories has implicated a ca. 108-kDa protein as a heat-labile enterotoxin and cytotoxin, as evidenced by rises in short-circuit current and falls in tissue resistance in rat jejunal tissue mounted in an Ussing chamber. Here we report the genetic cloning, sequencing, and characterization of this high-molecular-weight heat-labile toxin. The toxin (designated the plasmid-encoded toxin [Pet]) is encoded on the 65-MDa adherence-related plasmid of EAEC strain 042. Nucleotide sequence analysis suggests that the toxin is a member of the autotransporter class of proteins, characterized by the presence of a conserved C-terminal domain which forms a beta-barrel pore in the bacterial outer membrane and through which the mature protein is transported. The Pet toxin is highly homologous to the EspP protease of enterohemorrhagic E. coli and to EspC of enteropathogenic E. coli, an as yet cryptic protein. In addition to its potential role in EAEC infection, Pet represents the first enterotoxin within the autotransporter class of secreted proteins. We hypothesize that other closely related members of this class may also produce enterotoxic effects.

FIG. 1

Map of the cloned insert of pJPN201. The fimbrial subunit of the AAF/II antigen is encoded on the far right end of the fragment (17). At the left end of the insert is the pet gene, followed in opposite orientation by the astA gene embedded within an IS-like element and then by four other IS-homologous sequences (see text). Subclones used for sequencing and expression of pet are indicated. Restriction sites introduced by PCR are indicated by asterisks.

FIG. 2

Alignment of the predicted Pet protein with its closest homologs, EspP (accession no. X97542), EspC (U69128), and SepA (Z48219). Shaded residues represent identity. Coordinates are shown for Pet only. The asterisk at residue 53 indicates the first amino acid of the mature Pet protein. The arrowhead at residue 1018 indicates the position of cleavage of the β domain. The serine protease motif is in boldface.

FIG. 2

Alignment of the predicted Pet protein with its closest homologs, EspP (accession no. X97542), EspC (U69128), and SepA (Z48219). Shaded residues represent identity. Coordinates are shown for Pet only. The asterisk at residue 53 indicates the first amino acid of the mature Pet protein. The arrowhead at residue 1018 indicates the position of cleavage of the β domain. The serine protease motif is in boldface.

FIG. 3

(a) SDS-PAGE analysis of clone pCEFN1, encoding the complete pet gene. Lanes: A, Triton X-100-insoluble fraction of HB101(pCEFN1); B, Triton X-100-insoluble fraction of HB101 harboring the cloning vector pSPORT1; C, supernatant of HB101(pCEFN1); D, supernatant of HB101(pSPORT1). The arrow at 104 kDa represents the Pet passenger domain (the mature form of the protein); the arrow at ca. 30 kDa represents the β domain inserted into the bacterial outer membrane. (b) Western immunoblot of bacterial supernatants reacted with anti-Pet antiserum. Lanes: A, periplasmic fraction of HB101(pCEFN1); B, periplasmic fraction of HB101(pJPN205) harboring the C-terminal deletion mutant of the pet gene; C, cytoplasmic fraction of HB101(pCEFN1); D, cytoplasmic fraction of HB101(pJPN205); E, supernatant of HB101(pCEFN1); F, supernatant of HB101(pJPN205). Arrows denote expected sizes of mature Pet and the expected truncated species produced by pJPN205. Smaller species reacting with antibodies in lanes B and E most likely represent breakdown products of the mature toxin.

FIG. 4

Enterotoxic activity of the Pet protein derived from pCEFN1. Supernatants from overnight cultures were size fractionated (>50 kDa), and 5 μg of protein was added per Ussing chamber into which was mounted full-thickness rat jejunal tissue. The supernatants of HB101(pCEFN1) and HB101(pSPORT1) are illustrated in lanes C and D, respectively of Fig. 3a. Data points represent the means of at least three experiments; error bars represent standard errors of the means. The insert of pCEFN1 generates significant rises in PD and Isc compared with negative controls (P < 0.05 by Student’s t test).