The uropathogenic Escherichia coli KS52 strain expresses a mannose-resistant hemagglutinin involving an erythrocyte recognition site distinct from the at-digalactoside glycosphingolipid receptor identified for the uropathogenic E. coli strains specifying a P adhesin. The KS52 strain showed three major properties. (i) It agglutinated human erythrocytes of all tested blood groups. (ii) Hemagglutinin activity was found both in the supernatant fluid L-broth cultures and in cells grown on L-agar plates. (iii) No fimbriae in organisms grown on L-agar plates were detected by electron microscopy. Whole-cell DNA from the KS52 strain was size fractionated and cloned into the pHC79 cosmid vector. Three recombinant cosmids expressing a mannoseresistant hemagglutination (MRHA) phenotype were characterized and used to subclone the smallest DNA fragment able to confer the same MRHA properties as the parent strain. A 6.7-kilobase chromosomal DNA fragment cloned in pBR322 (pIL14) was shown to be necessary for host-cell MRHA expression and uroepithelial cell adherence. The insert encoded the production of a 16,000-dalton hemagglutinin. This polypeptide could be detected in culture supernatant fluids, in E. coli minicells harboring the pIL14 plasmid, and, by immunoblotting, in the KS52 strain and E. coli whole cells harboring the pIL14 plasmid. No homology was detected by Southern hybridization between the cloned insert and the DNA of the operon responsible for MRHA in the P-specifying, fimbriate strains (pap operon).

To determine whether uropathogenic strains of Escherichia coli exhibit a distinctive constellation of phenotypes, we examined 44 urinary isolates from women with radiologically normal urinary tracts and pyelonephritis, cystitis, or asymptomatic bacteriuria and 73 fecal isolates from healthy control subjects. The strains were characterized by their O serogroup, by their binding specificity (as determined by adhesins), and by their production of hemolysin and colicin V. In addition, the strains were assessed for homologous gene sequences by means of DNA-hybridization probes prepared from cistrons that encode hemolysin and the Gal-Gal binding adhesin--two determinants of virulence, which cause tissue injury and promote bacterial colonization of uroepithelia, respectively. In contrast to most isolates from normal feces and from the urine of patients with asymptomatic bacteriuria, pyelonephritis strains belong to a small number of O serogroups; all express the Gal--Gal binding adhesin and 75 per cent are hemolytic. A gene probe for the Gal--Gal binding adhesin, derived from the chromosome of one strain from a patient with pyelonephritis, hybridized with the DNA of all other pyelonephritis strains. The probe for the hemolysin gene hybridized with DNA from all other hemolytic strains. These data indicate that most cases of pyelonephritis are due to a small number of pathogenic clones that express critical determinants of virulence, and that the nucleotide sequences for hemolysin and the Gal--Gal binding adhesin in heterologous strains share homology. We are tempted to speculate that the gene products of these shared regions of the genome might form the basis for a vaccine against pyelonephritis.

A chromosomal DNA fragment which mediates Pap (pili associated with pyelonephritis) pili formation, mannose‐resistant hemagglutination (MRHA) and binding to uroepithelial cells has been isolated from the uropathogenic Escherichia coli clinical isolate J96, and genetically studied. Analysis of polypeptides expressed by the Pap DNA led to detection of a number of polypeptides ranging in mol. wt. from 13 000 to 81 000 daltons. The gene order and transcriptional orientation for four of the corresponding cistrons was: 13 000 (papB) 19 500 (papA, structural gene for the Pap pilus subunit), 81 000 (papC) and 28 500 (papD). Analyses of a lacZ‐ papA gene fusion located a promoter upstream from papA within the cloned DNA. Transposon Tn5 insertions in any of these four cistrons decreased or eliminated Pap pili formation. A number of transposon Tn5 mutations were identified in a region distal to papD that expressed normal levels of the papA protein on the cell surface in the form of recognizable pili structures but did not agglutinate human erythrocytes or adhere to uroepithelial cells. This region expressed polypeptides of 15 000, 24 000, 26 000 and 35 000 daltons. This finding shows that Pap pili formation and binding properties can be genetically dissociated.

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The uropathogenic strain Escherichia coli J96 mediates mannose-resistant hemagglutination owing to production of a digalactoside-binding adhesin. A cosmid clone from this strain has been isolated that, when harbored in E. coli K-12, expressed Pap pili and this adhesin (R. Hull et al., Infect. Immun. 33:933-938, 1981). By transposon mutagenesis and by the construction of a number of hybrid plasmid derivatives, we have demonstrated that about 8.5 kilobases of DNA is required to generate a mannose-resistant hemagglutination-positive phenotype in E. coli K-12 strain P678-54. The structural gene for the Pap pili monomer, papA, has been identified and mapped close to the promotor-proximal end of the Pap operon. Although strain P678-54 that harbored a Tn5 insertion within papA showed a mannose-resistant hemagglutination-positive phenotype, it was negative in a competitive enzyme-linked immunosorbent assay with anti-Pap pilus serum. This could mean that a Pap adhesin is encoded by a region on the Pap operon that is distinct from papA.

The papA gene of the uropathogenic strain Escherichia coli J96, coding for the Pap pili subunit, was subjected to DNA sequencing, and found to code for an 185-amino acid-long polypeptide with a 22-amino acid-long signal peptide. Here we present the primary sequence, the hydrophilicity profile, and the predicted polypeptide secondary structure of the Pap pili subunit.

The genes encoding alpha-hemolysin and mannose-resistant hemagglutination were shown to be closely linked in cloned DNA from two Escherichia coli urinary tract isolates of serotypes 04 (J96) and 06 (C1212). DNA hybridization experiments demonstrated that the hly and mrh gene clusters of other E. coli 06 serotypes were also linked. Colony hybridizations showed that most normal fecal E. coli do not contain hly and mrh DNA but much of the intervening DNA between these two gene clusters is common among all E. coli. We have further demonstrated that there is a small (about 1 kilobase) region of homology located on both sides of the hly sequence and present elsewhere in the C1212 strain. We suggest that linkage of hly and mrh occurred through a transposition event, and we discuss the potential significance of this linkage in the acquisition of virulence determinants by these bacteria.

Chromosomal genes encoding the MS and Gal-Gal binding properties have been cloned into separate recombinants and their respective pili characterized. Hapten inhibition of hemagglutination with synthetic carbohydrate receptor analogues and carbohydrate-adsorbed latex agglutination studies indicate that Gal-Gal and MS pili collectively exhibit the binding properties of the parent strain. MS pili migrated in SDS-PAGE with an Mr of 19 kdaltons and 17 kdaltons; the Mr of Gal-Gal pili was 17.5 kdaltons. The pili are chemically similar by amino acid composition and when the N-terminal cysteines are aligned, 8 of the 13 residues between positions 9 and 22 are homologous. Further, carboxy-terminal sequence homology was inferred from the carboxypeptidase digestion of a MS pili and the sequence of a carboxy-terminal tryptic peptide from Gal-Gal pili.