THE STRUCTURAL GENE ENCODING HUMAN ENTEROTOXIGENIC ESCHERICHIA-COLI PCFO20 IS HOMOLOGOUS TO THAT FOR PORCINE 987P

Putative colonization factor PCFO20 was recently identified in an ente rotoxigenic Escherichia coli (ETEC) strain of serogroup O20 isolated f rom a child with diarrhea in Argentina. The gene encoding the structur al subunit of PCFO20 fimbriae, fotA, was cloned from strain ARG-2 in t he expression phage vector lambda ZAP Express. One positive clone, pGV 29, that carried a 3.3-kb fragment was identified on the basis of fimb rillin production by using a monospecific rabbit anti-PCFO20 serum, Nu cleotide sequencing of a 1.3-kb Sau3A-ClaI fragment of the subclone pG V292 containing the region coding for PCFO20 fimbrillin revealed two o pen reading frames of which one was complete. A Western blot (immunobl ot) showed that the cloned protein, FotA, migrated like the PCFO20 fim brial subunit protein did, Fimbriae were not detected on the surface o f E. coli host bacteria containing pGV292 or pGV29, suggesting that th e genes needed for assembly of PCFO20 fimbriae are lacking in both clo nes. The fotA gene encodes a 20,574-Da prefimbrillin protein which con tains a 21-amino-acid signal sequence; the mature protein has a size o f 18.1 kDa. The subunit protein FotA was found to be more homologous t o the subunit of porcine 987P than to any fimbrial subunit produced by human ETEC. Alignments of the amino acid sequences of the two protein s indicate that they are partly identical, with an overall similarity of 82%. FotA fimbrillin was shown to be transported and assembled by t he fimbria assembly machinery in porcine ETEC strain 987, PCFO20 and 9 87P may have evolved from a common ancestral gene. They are immunologi cally related but have affinity for different host cell receptors, sin ce PCFO20-producing bacteria do not bind to neonatal piglet enterocyte s.