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The binding of ETEC strains expressing different colonization factors to the human enterocyte-like cell line Caco-2 and to isolated human enterocytes were determined. Strains expressing CFA/I, CS2, CS4+CS6, CS5+CS6, CS7, CFA/III+CS6 and PCFO166 adhered well to both types of cells whereas bacteria producing CS1, CS6 only, or CS17 did not adhere to either Caco-2 cells or to jejunal human enterocytes, suggesting that similar binding structures are present in both cell types. However, in some instances, binding of bacteria to the two types of cells differed, e.g. bacteria expressing CS3 or PCFO9 bound well to human enterocytes but not to Caco-2 cells. Conversely, bacteria producing PCFO20 or PCFO159 only adhered to Caco-2 cells and not to jejunal enterocytes. With few exceptions, this inability to bind to human enterocytes was the same for cells from all parts of the small intestine. This study contradicts previous reports suggesting that the binding structures of Caco-2 cells are identical to those of human enterocytes.

Production of coli surface-associated antigen 1 (CS1) by Escherichia coli strain E24377 of serotype 0139.H28 was controlled by a plasmid that also encoded heat stable and heat labile enterotoxins and CS3. The presence of a regulatory sequence was detected on this plasmid by hybridization with the cfaD gene that regulates expression of colonization factor antigen I fimbriae and is at least 96% homologous with the rns sequence controlling production of CS1 or CS2 fimbriae by strains of serotype 06.H16 of appropriate biotype. A separate plasmid, pDEP20, carrying the structural genes for CS1 synthesis was identified and transformed into E. coli strain HB101 or a derivative of strain E24377 without large plasmids. Transformants carrying pDEP20 did not produce CS1 fimbrial antigen, but antigen expression was obtained when a cloned cfaD gene or a wild-type plasmid carrying the rns sequence was introduced. Transposon mutagenesis with Tn1000 identified a 3.7 kbp region of pDEP20 essential for production of CS1 fimbriae. Genes encoding production of CS1 fimbriae were cloned on a 9.9 kbp BamHI fragment and were expressed in the presence of the cfaD sequence. A strain producing both CS1 and CS2 antigens was constructed by introduction of the cloned cfaD gene into a strain of serotype 06.H16 biotype C carrying plasmid pDEP20.

A DNA sequence, homologous to the cfaD gene of CFA/I region 2, was identified on CFA/I region 1. This sequence is designated cfaD′. It differs from the cfaD gene in containing two deletions and a stop codon. The cfaD′ sequence therefore can only encode a truncated CfaD-like protein. The CfaD protein may be a DNA binding protein and functions as a positive regulator of CFA/I fimbriae expression. A regulatory function for the cfaD′ is not likely since deletion of the cfaD′ sequence does not affect production of CFA/I fimbriae in E. coli K-12 strains. That the cfaD′ sequence is present on CFA/I wild-type plasmids isolated from CFA/I strains of different serotypes, obtained at various geographical locations, suggests, however, that this DNA region is not completely without a function.

Production of the plasmid-coded fimbrial antigen CFA/I of Escherichia coli requires both CFA/I region 1 and CFA/I region 2, which are separated by about 40 kb on the wildtype plasmid. The nucleotide sequence of region 2 was determined and contains an open reading frame (cfa d), encoding a protein of 265 amino acids. The protein has no signal sequence and upon sequence analysis appeared to be a DNA-binding protein. A plasmid was constituted, with a promoterless beta-galactosidase gene preceded by the promoter of region 1. Introduction of a plasmid, carrying the cfa d gene, into a strain containing this construct enhanced expression of beta-galactosidase by at least five-fold indicating that the cfa d protein was enhancing expression from the promoter of region 1. The cfa d gene sequence differed at 28 positions from the Rns gene, which encodes a protein that is a positive regulator of the expression of CS1 or CS2 fimbriae. It was shown that the cfa d gene and the Rns gene can functionally substitute each other in regulating fimbrial synthesis.