Heterobinary adhesins based on the Escherichia coli FimH fimbrial protein

The FimH adhesin of Escherichia coli type 1 fimbriae confers the ability to bind to D-mannosides by virtue of a receptor-binding domain located in its N-terminal region. This protein was engineered into a heterobifunctional adhesin by introducing a secondary binding site in the C-terminal region. The insertion of histidine clusters into this site resulted in coordination of various metal ions by recombinant cells expressing chimeric FimH proteins. In addition, libraries consisting of random peptide sequences inserted into the FimH display system and screened by a "panning" technique were used to identify specific sequences conferring the ability to adhere to Ni2+ and Cu2+. Recombinant cells expressing heterobifunctional FimH adhesins could adhere simultaneously to both metals and saccharides. Finally, combining the metal-binding modifications with alterations in the natural receptor-binding region demonstrated the ability to independently modulate the binding of FimH to two ligands simultaneously.     

Localization of a domain in the FimH adhesin of Escherichia coli type 1 fimbriae capable of receptor recognition and use of a domain-specific antibody to confer protection against experimental urinary tract infection

The FimH subunit of type 1-fimbriated Escherichia coli has been implicated as an important determinant of bacterial adherence and colonization of the urinary tract. Here, we sought to localize the functionally important domain(s) within the FimH molecule and to determine if antibodies against this domain would block adherence of type 1-fimbriated E. coli to the bladder mucosa in situ and in vivo in an established mouse model of cystitis. We generated translational fusion proteins of disparate regions of the FimH molecule with an affinity tag MalE, and tested each of the fusion products in vitro for functional activity. The minimum region responsible for binding mouse bladder epithelial cells and a soluble mannoprotein, horseradish peroxidase, was contained within residues 1-100 of the FimH molecule. We validated and extended these findings by demonstrating that antibodies directed at the putative binding region of FimH or at synthetic peptides corresponding to epitopes within the binding domain could specifically block type 1 fimbriae-mediated bacterial adherence to bladder epithelial cells in situ and yeast cells in vitro. Next, we compared the ability of mice passively immunized intraperitoneally with antisera raised against residues 1-25 and 253-264 of FimH or 1-13 of FimA to resist bladder colonization in vivo after intravesicular challenge with type 1-fimbriated E. coli. Only the antibody directed at the putative binding region of FimH (anti- s-FimH1-25) significantly reduced E. coli bladder infections in the experimental mouse model of urinary tract infections. Similar results were obtained when the mice were actively immunized with synthetic peptides corresponding to residues 1-25 and 253-264 of FimH or 1-13 of FimA. The mechanism of protection was attributed, at least in part, to inhibition of bacterial adherence to the bladder surface by s-FimH1-25-specific antibody molecules that had filtered through the kidneys into the urine. The level of FimH antibodies entering the bladder from the circulatory system of the immunized mice was found to be markedly enhanced upon bacterial challenge. The potential broad spectrum activity of the protective FimH antibody was indicated from its serologic cross-reactivity with various urinary tract bacterial isolates bearing type 1 fimbriae. These findings could be relevant in the design of an efficacious and broadly reactive FimH vaccine against urinary tract infections.     

Oligomeric interaction of the PapB transcriptional regulator with the upstream activating region of pili adhesin gene promoters in Escherichia coli

1. Prostanoid receptor-mediated sensitization, or excitation, of sensory nerve fibres contributes to the generation of hyperalgesia. To characterize the prostanoid receptors present on sensory neurones, biochemical assays were performed on primary cultures of adult rat dorsal root ganglia (DRG) and the F-11 (embryonic rat DRG x neuroblastoma hybrid) cell line. 2. In DRG cultures, the IP receptor agonists, cicaprost and carbaprostacyclin (cPGI2) stimulated cyclic AMP accumulation. Prostaglandin E2 (PGE2) also increased cyclic AMP levels, but to a lesser extent, while carbocyclic thromboxane A2 (cTxA2), PGD2 and PGF2alpha had negligible effects. The rank order of agonist potency was cicaprost>PGE2=BMY45778=cPGI2=PGI2. In the F-11 cells, the rank order of agonist potency for the stimulation of cyclic AMP accumulation was: cicaprost>iloprost=cPGI2=PGI2=BMY45778>PGE2=cTXA2++ +. In DRG cultures, cicaprost induced significantly more accumulation of inositol phosphates than PGE2. 3. To examine the effects of prostanoids on C-fibre activity, extracellular recordings of d.c. potentials from the rat isolated vagus nerve were made with the 'grease-gap' technique. PGI2 (0.1 nM-10 microM) produced the largest depolarizations of the nerve. The rank order of agonist potency was: PGI2=cPGI2=PGE1>cTXA2>PGE2=PGD2=TXB2>PGF2alpha. 4. Prior depolarization of nerves with either forskolin (10 microM) or phorbol dibutyrate (1 microM) alone significantly reduced the response to PGI2 (10 microM), while simultaneous application of both forskolin and phorbol dibutyrate attenuated PGI2 responses almost completely. 5. Putative EP1 and/or TP receptor-selective antagonists had no effect on the responses to PGI2, cPGI2 or PGE2 in the three preparations studied. 6. Collectively, these data are consistent with a positive coupling of IP receptors to both adenylyl cyclase and phospholipase C in sensory neurones. These findings suggest that IP receptors play a major role in the sensitization of rat sensory neurones.     

Expression and detection of pap-, sfa-, and afa-encoded fimbrial adhesin systems among uropathogenic Escherichia coli

Thirty-five Escherichia coli isolates from young children and women with pyelonephritis, cystitis, or asymptomatic bacteriuria were characterized genotypically and phenotypically. The isolates were examined genotypically by using DNA probes specific for the hemolysis gene and for the pap, sfa, and afa adhesin systems. Genes for the adhesin systems were also detected by polymerase chain reaction, using multigene amplification. The isolates were serotyped, tested for hemolysin production, and classified for their adhesion specificity by hemagglutination and by binding specificity assays. Twenty-seven of the 35 isolates were pap positive. Results showed that pap-positive isolates expressing class I or class II G adhesins were more frequent in cases of pyelonephritis than in cases of cystitis and asymptomatic bacteriuria. Expression of the class III G adhesins was more frequent in isolates from cystitis and asymptomatic bacteriuria than in isolates from pyelonephritis. Multiple adhesin systems and hemolysin were more frequently found in isolates from cases of pyelonephritis than in isolates from cases of cystitis and asymptomatic bacteriuria. There was perfect correlation between the results obtained by polymerase chain reaction and and those obtained by hybridization.     

Mechanisms of gut damage by Escherichia coli

This chapter primarily concerns three main categories of diarrhoeagenic Escherichia coli, enteropathogenic (EPEC), enterohaemorrhagic (EHEC) and enteroaggregative (EAEC) E. coli. They have distinctive virulence factors and vary in the enteropathies they produce. The molecular biological approach has opened up the complex way in which they interact with the intestine. EPEC and EHEC show a subversive approach to colonization in that they adapt the host cell to their requirements in the formation of the attaching effacing lesion. EAEC appear to co-opt the host defence system to produce a biofilm-like colony and currently go unrecognized in routine laboratories.     

Transfection of Escherichia coli by Mu DNA

The total amount of noradrenaline (NA) in the male accessories increases with normal growth of the organs and also with the age of the rat. The ductus deferens of the old rat has about twice as high NA concentration as that of the young rat. Castration of the prepuberal rat or the puberal rat leads to retardation or cessation, respectively, of the increase in NA amount of the male genital tract. Only in the old rat does castration produce a definite decrease in NA amount of the tract. Castration always raises the NA concentration of the sex accessories. Testosterone treatment of the puberal rat or the old rat produces minor or no increase, respectively, in total amount of NA of the male sex accessories. Testosterone treatment of old prepuberally castrated rats produces a marked increase in NA content of the male organs. It is concluded that androgens have no substancial direct effect on the adrenergic innervation per se, but affects the transmitter levels of the male organs indirectly through changes in number, size and relative proportion of the target cells of the adrenergic nerves i.e. the smooth muscle cells.     

Reduction of aerobic acetate production by Escherichia coli

Acetate excretion by Escherichia coli during aerobic growth on glucose is a major obstacle to enhanced recombinant protein production. We report here that the fraction of carbon flux through the anaplerotic pathways is one of the factors influencing acetate excretion. Flux analysis of E. coli central metabolic pathways predicts that increasing the fraction of carbon flux through the phosphoenolpyruvate carboxylase (PPC) pathway and the glyoxylate bypass reduces acetate production. We tested this prediction by overexpressing PPC and deregulating the glyoxylate bypass by using a fadR strain. Results show that the acetate yield by the fadR strain with PPC overexpression is decreased more than fourfold compared to the control, while the biomass yield is relatively unaffected. Apparently, the fraction of carbon flux through the anaplerotic pathways is one of the factors that influence acetate excretion. These results confirm the prediction of our flux analysis and further suggest that E. coli is not fully optimized for efficient utilization of glucose.     

DNA excision repair as a component of adaptation to low doses of ionizing radiation in Escherichia coli

The effects of chronic lithium chloride infusions on consumption of, and subsequent preferences for, a novel diet were examined in rats with ablations of the area postrema (AP) and sham-lesioned control rats. Osmotic minipumps (Alza), filled with a saturated aqueous solution of LiCl (63 g/100 ml), were implanted in the peritoneal cavity of half of the lesioned rats (n = 9) and half of the control rats (n = 8). The remaining rats received empty pumps (n = 9 and n = 7 for lesioned and controls, respectively). The LiCl or sham drug phase was paired with free access to a highly palatable novel diet (AIN diet) during a 7-day conditioning period. Subsequent preferences for the novel diet relative to a familiar diet (ground Purina lab pellets) were determined using a two-food choice procedure. The only group to show a persistent and significant reduction in novel food consumption during the conditioning phase was the sham-lesioned group infused with LiCl (p < 0.01). This group also exhibited a marked aversion for the novel diet, indicative of a conditioned food aversion (CFA), during the preference tests. No significant differences in novel diet consumption or in novel diet preference were found between the two AP-lesioned groups. This study provides evidence that anorexia and CFAs to a novel diet, induced with chronic infusions of lithium, are abolished by destruction of the chemosensitive area postrema.     

Epitopes of the P-fimbrial adhesin of E. coli cause different urinary tract infections

PURPOSE: This is a study of the interaction of the tip protein of P-fimbriae E. coli, its specific urothelial adhesin, and urothelial receptors for the adhesin. This tip protein has several epitopes that adhere to different isoreceptors containing the urothelial alpha-gal-1-4 beta-gal disaccharide. Renal tubular cells of our monkey model contain the globoside isoreceptor, and thus ureteral inoculation of E. coli with the class II tip protein leads to pyelonephritis. The class III tip protein adheres to the Forssman antigen and causes cystitis in humans. MATERIALS AND METHODS: An E. coli strain, DS17 which originally caused pyelonephritis in a child, is P-fimbriated and contains a class II tip adhesin. A mutant was produced to contain a class III tip adhesin. Eight monkeys had a ureteral inoculation of E. coli DS17 and 4 monkeys with E. coli DS17-1. In addition, we studied in vitro adherence by these strains. RESULTS: We show that in vitro adherence by the tip protein of P-fimbriae to bladder cells of the monkey occurs by several mechanisms, adhering to specific receptors for the class II and III epitopes of the tip protein as well as by means of type 1 fimbriae. In addition, the PapE protein of the fibrillum of the P-fimbriae adheres to fibronectin. As always, electrostatic and hydrophobic interaction remain important contributions to adherence. E. coli DS17 caused pyelonephritis, but DS17-1 caused cystitis. Bacteriuria was prolonged by DS17 infection. CONCLUSION: The site of a urinary tract infection from P-fimbriated E. coli can be predicted by the epitope of the tip protein of P-fimbriae.     

Pilot scale exponential growth of Escherichia coli W to high cell concentration with temperature variation

An efficient method to grow Escherichia coli W to high cell concentrations on the pilot scale is described and discussed. The method involves growth linked introduction of glucose and ammonia to the culture, sparging with oxygen, and maintenance of aerobic conditions by gradually decreasing the temperature in the culture in order to keep the oxygen demand within the limits of the capacity of supply. Under these conditions the linear rate of cell mass production is actually the result of exponential growth with a gradually decreasing growth-rate constant. About 10 kg packed cells were produced in a 50 liter working-volume fermentor in one run of 13 hr. The concentration of the cells at the end of the growth was about 47 g dry cells/liter. The expenditure for nutrients was minimal and the controls were of simple automatic nature. From the determined yield constants for glucose, nitrogen, phosphorus, and oxygen it may be inferred that the cells grown by this method are similar to those grown exponentially at constant temperature.     

Proteins induced in Escherichia coli by benzoic acid

Proteins induced by benzoic acid in Escherichia coli were observed on two-dimensional electrophoretic gels (2-D gels). Cultures were grown in glucose-rich medium in the presence or absence of 20 mM benzoate at an external pH of 6.5, where the pH gradient (deltapH) is large and benzoate accumulates, and at an external pH of 8.0, where deltapH is inverted and little benzoate is taken up. Radiolabeled proteins were separated on 2-D gels and were identified on the basis of the index of VanBogelen and Neidhardt. In the absence of benzoic acid, little difference was seen between pH 6.5 and pH 8.0; this confirms that the mechanisms of protein homeostasis in this range are constitutive, including the transition between positive and inverted deltapH. Addition of benzoate at pH 6.5 increased the expression of 33 proteins. Twelve of the benzoate-induced proteins were induced at pH 8.0 as well, and nine of these matched proteins induced by the uncoupler dinitrophenol. Eighteen proteins were induced by benzoate only at pH 6.5, not at pH 8.0, and were not induced by dinitrophenol. One may be the iron and pH regulator Fur, which regulates acid tolerance in Salmonella spp. The other 13 proteins had not been identified previously. The proteins induced by benzoate only at a low pH may reflect responses to internal acidification or to accumulation of benzoate.     

Two-dimensional gel electrophoresis of Escherichia coli homogenates: the Escherichia coli SWISS-2DPAGE database

We report a patient, MT, who presented a specific, though not isolated, deficit in written calculation. Despite a preserved knowledge of simple arithmetic - single-digit addition and subtraction - he failed systematically in multi-digit subtraction. The nature of errors was consistent across problems and reflected the application of a disturbed underlying algorithm. Moreover, the pattern of error observed mimies a very common finding in developmental studies on arithmetical procedure acquisition (Fuson, 1990, 1992, Young and O'Shea, 1981; VanLehn, 1986, 1990). The data suggest that, within calculation skills, syntax may exist as a system of stable, but inappropriate, rules which are independent of any underlying conceptual knowledge.     

Inhibition of Escherichia coli RecA coprotease activities by DinI

In Escherichia coli, the SOS response is induced upon DNA damage and results in the enhanced expression of a set of genes involved in DNA repair and other functions. The initial step, self-cleavage of the LexA repressor, is promoted by the RecA protein which is activated upon binding to single-stranded DNA. In this work, induction of the SOS response by the addition of mitomycin C was found to be prevented by overexpression of the dinI gene. dinI is an SOS gene which maps at 24.6 min of the E.coli chromosome and encodes a small protein of 81 amino acids. Immunoblotting analysis with anti-LexA antibodies revealed that LexA did not undergo cleavage in dinI-overexpressed cells after UV irradiation. In addition, the RecA-dependent conversion of UmuD to UmuD' (the active form for mutagenesis) was also inhibited in dinI-overexpressed cells. Conversely, a dinI-deficient mutant showed a slightly faster and more extensive processing of UmuD and hence higher mutability than the wild-type. Finally, we demonstrated, by using an in vitro reaction with purified proteins, that DinI directly inhibits the ability of RecA to mediate self-cleavage of UmuD.     

Transformation of Escherichia coli with foreign DNA by electroporation

A new alkaloid, named desmodimine, C12H15NO4, gum, and a new natural product, named desmodilactone, C8H13NO3, mp 84-85 degrees C have been isolated from the aerial parts of Desmodium styracifolium (Osbeck) Merr. belonging Leguminosae. On the basis of spectral analysis their structures were deduced as formula I and II. In addition, lupenone (III), lupeol (IV), tritriacontane (V), stearic acid (VI), eicosanoic acid eicosyl ester (VIII) and beta-sitosterol (VII) were isolated for the first time from this plant.     

Modulation of P-fimbriation by ciprofloxacin in uropathogenic Escherichia coli

BACKGROUND: The aim of this study was to determine the effect of ciprofloxacin at subinhibitory concentrations on the expression of P fimbriae of uropathogenic Escherichia coli. Thirty-nine strains of Escherichia coli isolated from out patients with urinary tract infection were studied. Thirty-nine of these strains had been previously characterized as P-fimbriated and the remaining non fimbriated strain was used as a negative control. METHODS: Fimbriation was quantitatively studied by electron microscope observation of the strains before and after treatment. To determine possible qualitative variations in the fimbrial proteins and in the external membrane (OMPs), extraction and electrophoretic separation was performed in polyacrylamide gels. RESULTS: No qualitative differences were observed in the OMPs profile and fimbrial proteins induced by ciprofloxacin in any of the strains studied. However, electron microscopic observation generally showed a decrease in the percentage fimbriated bacterial cells by the antimicrobial effect. CONCLUSIONS: The mechanism of action of ciprofloxacin at subinhibitory doses may correspond to a process of fimbrial protein synthesis inhibition secondary to the initiation of general repair mechanism of the cell exposed to the antimicrobial and not to a process of specific mutations which qualitatively affect fimbrial protein composition.