Uropathogenic and diarrheal Escherichia coli strains expressing adhesins of the Dr family bind to decayaccelerating factor, invade epithelial cells, preferentially infect children and pregnant women, and may be associated with chronic or recurrent infections. Thus far, no fimbrial domain(s) that facilitates cell invasion has been identified. We used alanine scanning mutagenesis to replace selected amino acids in hydrophilic domain II of the structural fimbrial subunit DraE and evaluated recombinant mutant DraE for attachment, invasion, and intracellular compartmentalization. The mutation of amino acids V28, T31, G33, Q34, T36, and P40 of DraE reduced or abolished HeLa cell invasion but did not affect attachment. Electron micrographs showed a stepwise entry and fusion of vacuoles containing Escherichia coli mutants T36A and Q34A or corresponding beads with lysosomes, whereas vacuoles with wild-type Dr adhesin showed no fusion. Mutants T31A and Q34A, which were deficient in invasion, appeared to display a reduced capacity for clustering decay-accelerating factor. Our findings suggest that hydrophilic domain II may be involved in cell entry. These data are consistent with the interpretation that in HeLa cells the binding and invasion phenotypes of Dr fimbriae may be separated.

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...

Vibrio cholerae is a Gram-negative bacterium with a single polar flagellum. Motility is an important virulence factor for this non-invasive pathogen. We cloned and sequenced a locus in V. cholerae V86 (El Tor, Inaba) that contained five different structural genes of the flagellum. The cloned genes and their products were assigned names and functions based on homology with sequences of similar genes and their products from other related bacteria. All of these genes of V. cholerae V86, namely, flgI, J, M, L and flaA, were transcribed in the same direction. These genes respectively encoded the P- and L-ring proteins, the hook-associated proteins 1 and 3 and the flagellin core protein of the flagellum. Our data indicated the presence of more than one flagellar locus in V. cholerae which could provide a means of immunoavoidance during infection. When compared with homologs in other bacteria, the flagellin core protein of V. cholerae exhibited conservation in the N- and C-termini, but had diverged in the central region.

Each year cholera epidemics occur in various places around the world. Though there is no effective vaccine against cholera, people who recover from an infection usually have prolonged immunity to the disease. Sera from convalescent patients contain antibodies to a number of outer membrane proteins (OMPs) of V. cholerae. We isolated several OMPs (43, 42, 30, and 22 kDa) from V. cholerae V86 E1 Tor Inaba, sequenced their amino-termini, and generated hyperimmune sera against them in rabbits. Antisera to the 43-, 42-, and 22-kDa OMPs, but not the preimmune sera, significantly reduced V. cholerae-induced fluid secretion seen in rabbit intestinal loops challenged with the homologous strain. In addition, a combination of antisera to the different OMPs reduced the fluid secretion induced by challenge with heterologous V. cholerae Ogawa and O139 strains. These results have significance in the development of vaccines to V. cholerae, as the hyperexpression of these OMP encoding genes in vaccine strains may improve the efficacy of cholera vaccines.