Decay-accelerating factor (DAF), a complement regulatory protein, also serves as a receptor for Dr adhesinbearing Escherichia coli. The repeat three of DAF was shown to be important in Dr adhesin binding and complement regulation. However, Dr adhesins do not bind to red blood cells with the rare polymorphism of DAF, designated Dr(a ؊ ); these cells contain a point mutation (Ser165-Leu) in DAF repeat three. In addition, monoclonal antibody IH4 specific against repeat three was shown to block both Dr adhesin binding and complement regulatory functions of DAF. Therefore, to identify residues important in binding of Dr adhesin and IH4 and in regulating complement, we mutated 11 amino acids-predominantly those in close proximity to Ser165 to alanine-and expressed these mutations in Chinese hamster ovary cells. To map the mutations, we built a homology model of repeat three based on the poxvirus complement inhibitory protein, using the EXDIS, DIAMOD, and FANTOM programs. We show that perhaps Ser155, and not Ser165, is the key amino acid that interacts with the Dr adhesin and amino acids Gly159, Tyr160, and Leu162 and also aids in binding Dr adhesin. The IH4 binding epitope contains residues Phe148, Ser155, and L171. Residues Phe123 and Phe148 at the interface of repeat 2-3, and also Phe154 in the repeat three cavity, were important for complement regulation. Our results show that residues affecting the tested functions are located on the same loop (148 to 171), at the same surface of repeat three, and that the Dr adhesin-binding and complement regulatory epitopes of DAF appear to be distinct and are Ϸ20 Å apart.

We used a gentamicin protection assay to assess the ability of gestational pyelonephritis isolates of Escherichia coli to invade HeLa cells. The ability to enter HeLa cells was strongly associated with the presence of Dr operons coding for Dr adhesins. In contrast, the nonivasive isolates predominantly expressed papG, coding for P fimbriae.Pyelonephritis in pregnant patients is a serious complication that may lead to severe sequelae such as bacteremia, urosepsis, adult respiratory distress syndrome, and death (16). The course of the pregnancy may be affected, resulting in preterm labor or intrauterine growth retardation (2). Escherichia coli remains the primary cause of renal infections in pregnant patients, accounting for 65 to 80% of cases (11). E. coli strains isolated from pregnant women with pyelonephritis are genetically closely related and express gestational age-dependent profiles of virulence factors such as Dr and P fimbriae (7, 13). Interaction of recombinant E. coli bearing Dr adhesin with receptor decay-accelerating factor (DAF; CD55) on HeLa cells mediates bacterial invasion into epithelial cells (6). Moreover, Dr-positive E. coli is able to kill pregnant rats while not affecting nonpregnant animals (12). Mutation of the E. coli Dr operon results in abolishment of bacterial invasion and abrogates the development of experimental chronic interstitial nephritis in mice (5). Our general hypothesis is that an experimental lethality of Dr-positive E. coli for pregnant animals may account for their epidemiological association with pyelonephritis in pregnant patients and suggest unique gestational virulence. In this report, we evaluated the hypothesis that expression of the Dr family of adhesins in clinical gestational isolates of E. coli is associated with invasive properties. We also assessed whether other common virulence traits encountered in uropathogenic E. coli, such as P fimbriae and ␣-hemolysin, may be associated with bacterial entry into HeLa epithelial cells.In the first set of experiments, we tested the invasive properties of 73 gestational isolates of E. coli derived from pregnant patients hospitalized due to pyelonephritis at the University of Texas Medical Branch at Galveston between 1996 and 1999. Strains were selected on the basis of a positive urine culture and clinical symptoms. The standard gentamicin protection assay was performed on human cervical cell line HeLa (ATCC CCL2) as described previously to evaluate the ability of E. coli isolates to enter epithelial cells (6). The invasion rate was expressed as the percentage of the initial bacterial inoculum (1.36 ϫ 10 8 ) that was recovered after treatment of the HeLa cell monolayer with antibiotic and subsequent lysis with detergent. Isolates which yielded fewer than 0.001% survivors were characterized as noninvasive. This criterion is based on our previous studies, which revealed that a survival rate of Ͻ0.001% characterized Dr-negative mutants of clinical or laboratory recombinant strains that are not able to invade HeLa cells as furth...

E. coli target complement-protective CD55 epitopes for invasion and exploit CD55-MT complexes to escape phagolysosomal fusion, leading to a nondestructive parasitism that allows bacteria to persist intracellularly.

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