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S N Abraham

Publications and source records attributed to S N Abraham.

At least 55 records · Page 3Linked to original sources
PubMed1993

FimC is a periplasmic PapD-like chaperone that directs assembly of type 1 pili in bacteria.

Biogenesis of the type 1 pilus fiber in Escherichia coli requires the product of the fimC locus. We have demonstrated that FimC is a member of the periplasmic chaperone family. The deduced primary sequence of FimC shows a high degree of homology to PapD and fits well with the derived consensus sequence for periplasmic chaperones, predicting that it has an immunoglobulin-like topology. The chaperone activity of FimC was demonstrated by purifying a complex that FimC forms with the FimH adhesion. A fimC1 null allele could be complemented by the prototype member of the chaperone superfamily, PapD, resulting in the production of adhesive type 1 pili. The general mechanism of action of members of the chaperone superfamily was demonstrated by showing that the ability of PapD to assemble both P and type 1 pili was dependent on an invariant arginine residue (Arg-8), which forms part of a conserved subunit binding site in the cleft of PapD. We suggest that the conserved cleft is a subunit binding feature of all members of this protein family. These studies point out the general strategies used by Gram-negative bacteria to assemble adhesins into pilus fibers, allowing them to promote attachment to eukaryotic receptors.

Adhesins, Escherichia coli↗
PubMed1993

Neutrophil activation by nascent FimH subunits of type 1 fimbriae purified from the periplasm of Escherichia coli.

Previous studies of type 1 fimbriae of Escherichia coli have implicated FimH, a minor subunit, as the determinant of its mannose binding property. Structure-function analysis of FimH has not been possible because of the difficulty in obtaining adequate amounts of the subunit from type 1 fimbriae. We have obtained nascent FimH that has not been incorporated into the fimbrial structure from the periplasm of an E. coli strain expressing the cloned fimH gene. Nascently translocated FimH was initially degraded in the periplasm; however, when co-expressed with FimC, a putative fimbrial chaperone, the FimH molecules were stabilized and readily isolated from the periplasmic extract by fractionation on a sodium dodecyl sulfate-polyacrylamide gel followed by electroelution of the FimH band from the gel. The eluted protein was purified to homogeneity by affinity chromatography on a mannose-Sepharose column. Purified FimH displayed the same mannose-inhibitable binding to human neutrophils as type 1 fimbriated bacteria, including triggering an oxidative burst with concomitant release of reactive oxygen metabolites. In addition, inert microspheres coated with FimH, but not those coated with bovine serum albumin, were phagocytosed by neutrophils. These data provide direct evidence that FimH is the determinant on type 1 fimbriae which is responsible for mediating mannose-specific adherence and that isolated FimH is a potent activator of human neutrophils.

Adhesins, Escherichia coli↗
PubMed1992

Functional heterogeneity of type 1 fimbriae of Escherichia coli.

Escherichia coli and other members of the family Enterobacteriaceae express surface fibrillar structures, fimbriae, that promote bacterial adhesion to host receptors. Type 1 fimbriae possess a lectinlike component, FimH, that is commonly thought to cause binding to mannose-containing oligosaccharides of host receptors. Since adhesion of type 1 fimbriated organisms are inhibited by mannose, the reactions are described as mannose sensitive (MS). We have studied the adhesion of the type 1 fimbriated CSH-50 strain of E. coli (which expresses only type 1 fimbriae) to fibronectin (FN). E. coli CSH-50 does not bind detectable amounts of soluble FN but adheres well to immobilized plasma or cellular FN. This adhesion was inhibited by mannose-containing saccharides. By using purified domains of FN, it was found that E. coli CSH-50 adheres primarily to the amino-terminal and gelatin-binding domains, only one of which is glycosylated, in an MS fashion. Binding of the mannose-specific lectin concanavalin A to FN and ovalbumin was eliminated or reduced, respectively, by incubation with periodate or endoglycosidase. Adhesion of E. coli CSH-50 to ovalbumin was reduced by these treatments, but adhesion to FN was unaffected. E. coli CSH-50 also adheres to a synthetic peptide copying a portion of the amino-terminal FN domain (FNsp1) in an MS fashion. Purified CSH-50 fimbriae bound to immobilized FN and FNsp1 in an MS fashion and inhibited adhesion of intact organisms. However, fimbriae purified from HB101 (pPKL4), a recombinant strain harboring the entire type 1 fim gene locus and expressing functional type 1 fimbriae, neither bound to FN or FNsp1 nor inhibited E. coli adhesion to immobilized FN or FNsp1. These novel findings suggest that there are two forms of type 1 MS fimbriae. One form exhibits only the well-known MS lectinlike activity that requires a substratum of mannose-containing glycoproteins. The other form exhibits not only the MS lectinlike activity but also binds to nonglycosylated regions of proteins in an MS manner.

Bacterial Adhesion↗
PubMed1992

T-cell-independent stimulation of immunoglobulin secretion in resting human B lymphocytes by the mannose-specific adhesin of Escherichia coli type 1 fimbriae.

Purified Escherichia coli type 1 fimbriae have been shown previously to stimulate T-cell-independent proliferation of human B lymphocytes. The response is mediated by the mannose-specific, lectin-like adhesin protein FimH. Here we show that type 1 fimbriae also stimulate immunoglobulin (Ig) secretion by B cells. The response was maximal at three days of culture and consisted predominantly of the IgM isotype. It was independent of serum components, T lymphocytes, monocytes, and natural killer cells. Highly purified resting B cells were induced to proliferate and secrete Ig in response to the fimbriae. The role of FimH in the response was shown by the failure of FimH- type 1 fimbriae to stimulate and by inhibition of the response with alpha-methyl mannoside. In light of the fact that carbohydrate-binding adhesins have been found on a wide variety of microorganisms, these studies suggest the possibility that responses of other cell types to other microbial adhesins will be discovered.

Adhesins, Escherichia coli↗
PubMed1992

Glycerol-induced unraveling of the tight helical conformation of Escherichia coli type 1 fimbriae.

Glycerol was found to unravel the helical conformation of Escherichia coli type 1 fimbriae without appreciable depolymerization. The linearized fimbrial polymers have a diameter of 2 nm, react strongly with a monoclonal antibody directed at an inaccessible epitope on native fimbriae, and display greater mannose-binding activity and trypsin sensitivity than native fimbriae. Removal of glycerol by dialysis results in spontaneous reassembly of the linear polymers into structures morphologically, antigenically, and functionally indistinguishable from native fimbriae.

Escherichia coli↗
PubMed1991

Isolation and characterization of a 180-kiloDalton salivary glycoprotein which mediates the attachment of Actinomyces naeslundii to human buccal epithelial cells.

The adherence of Actinomyces naeslundii to human buccal mucosa is mediated by specific interactions between the bacterial cell surface fimbriae and complementary beta-linked galactoside receptors on the epithelial cell surface. The buccal mucosa and the bacteria that colonize its surface are constantly bathed in saliva. Several salivary components are thought to play an important role in modulating adhesive interactions between oral bacteria and the buccal epithelium. We have observed that pretreatment of isolated buccal epithelial cells (BEC) with human parotid saliva increased the attachment of three different strains of A. naeslundii. By employing affinity chromatography, ion-exchange and high-pressure liquid chromatographic techniques we have isolated a 180 kDa A. naeslundii-binding salivary glycoprotein (An-SPG). This salivary glycoprotein was capable of mediating separate but specific binding interactions with A. naeslundii and BEC. Pretreatment of BEC with increasing amounts of An-SGP resulted in a corresponding increase in the attachment of A. naeslundii. The adherence of A. naeslundii to An-SGP-coated BEC is sensitive to the same inhibitors previously shown to block adherence of A. naeslundii to uncoated BEC, namely lactose- and galactosyl-binding lectins. When a solubilized extract of freshly isolated and washed BEC was reacted on a Western blot with antibodies to An-SGP, a prominent 180 kDa immunoreactive band was detected. Furthermore, the immunoreactive component was demonstrated on the BEC surface when assayed by immunofluorescence using An-SGP-specific antibodies, suggesting that An-SGP or a protein structurally and immunologically identical to the isolated glycoprotein is present on BEC.

Actinomyces↗
PubMed1991

Fragmentation of Escherichia coli type 1 fimbriae exposes cryptic D-mannose-binding sites.

Cells of the gram-negative bacterium Escherichia coli are able to attach to various host cells by means of a mannose-specific adhesin associated with type 1 fimbriae. Here we show that fragmentation of type 1 fimbriae by freezing and thawing results in increased mannose-binding activity as demonstrated by increased hemagglutination, increased stimulation of human lymphocyte proliferation, and increased binding of the mannose-containing enzyme horseradish peroxidase. Increased activity in all three assays was mannose sensitive and was not exhibited by FimH- mutant type 1 fimbriae lacking the adhesin. Scatchard analysis of the data from peroxidase binding assays showed that unfrozen and frozen fimbriae contain binding sites displaying two classes of affinity. Frozen and thawed fimbriae expressed an increase in the number of high-affinity binding sites. These results show that fragmentation of the fimbrial structure exposes cryptic mannose-binding activity associated with type 1 fimbriae, presumably that of internally located adhesin molecules. Our data support earlier observations that adhesin moieties of type 1 fimbriae are located both at the tips and at intervals along the length of the fimbriae. In addition, our data suggest that only the adhesin moieties that are located at the fimbrial tips are functional in binding mannose. Adhesins located along the length of the fimbriae have their mannose-binding activity buried within the fimbrial structure and hence are not functional. We propose an updated model for the structure of type 1 fimbriae that is in agreement with the above observations.

Bacterial Adhesion↗
PubMed1991

Chaperone-assisted assembly and molecular architecture of adhesive pili.

The assembly of bacterial pili as exemplified here by P and type 1 pili of E. coli is a complex process involving specific molecular interactions between structural and chaperone proteins. The assembly process occurs postsecretionally, i.e. after the subunits are translocated across the cytoplasmic membrane. In a single cell, hundreds of thousands of interactive subunits are typically surface localized and assembled into pili. Periplasmic chaperones are generally required to bind to the interactive subunits and partition them into assembly-competent complexes. The binding of the chaperone to the subunits apparently protects the interactive surfaces and prevents them from aggregating at the wrong time and place within the cell. Pili are most likely assembled into linear polymers that package into right-handed helices after their translocation through specific outer-membrane channels. Each pilus filament is a quaternary assembly of the structural subunit and several minor subunits including the adhesin moiety. Although the assembly and organization of P and type 1 pili are very similar, there are some notable differences. For example, the P pilus adhesin is located exclusively at the tips of the pilus filament and forms part of a morphologically distinct structure. In contrast, the adhesion moiety of type 1 pili is inserted into the pilus filament at intervals, but only the adhesin molecule exposed at the pilus tip is functional. The variability in isoreceptor recognition amongst P pili has been solely ascribed to structural differences in the respective adhesin molecules, whereas in type 1 pili, variability in binding specificity has been attributed to the pilus filament that influences the conformation of the adhesin moiety. Less is known about the structure or assembly of type 4 pili, which are a unique class of pili expressed by several different species of gram-negative bacteria. The phase variation of the pilC assembly gene in N. gonorrheae to the off state results in the accumulation of unassembled subunits toxic to the cells. This process exerts a strong selection pressure on the cells that triggers alterations in the pilin structural gene. Thus, antigenic variation of pili in this organism may be regulated at the level of assembly. Finally, the concept of periplasmic chaperones in postsecretional assembly is most likely a general phenomenon in the biology of gram-negative bacteria. The investigations of pilus assembly will continue to provide insight into the details of how macromolecular assembly reactions are coordinated in the bacterial cell and how the regulation of assembly genes can profoundly affect biological processes.

Adhesins, Escherichia coli↗
PubMed1990

Colonial morphology of staphylococci on Memphis agar: phase variation of slime production, resistance to beta-lactam antibiotics, and virulence.

The growth of Staphylococcus epidermidis sensu stricto and Staphylococcus saprophyticus on Memphis agar yielded up to 6 morphotypes with each strain. With S. epidermidis, one morphotype produced slime (rho) but became non-slime-producing (epsilon) at a high frequency. The slime-producing rho variants were methicillin-resistant and more virulent than methicillin-susceptible epsilon variants in an endocarditis model. With S. saprophyticus, phase variation was of higher frequency. Nitrosoguanidine mutagenesis produced a stable blue epsilon form that was more virulent than the parent in a mouse model of urinary tract infection. Mutants with the blue epsilon phenotype differed from gold epsilon parents in a variety of phenotypic properties, including increased resistance to oxacillin. These staphylococcal species have a high frequency of phase variation: Phase variants differ in antibiotic resistance and virulence, which is only partially correlated with suggested virulence factors such as slime production.

Animals↗
PubMed1989

Mitogenic stimulation of human B lymphocytes by the mannose-specific adhesin on Escherichia coli type 1 fimbriae.

Escherichia coli type 1 fimbriae contain in association with the major structural protein a lectin-like adhesin moiety that mediates attachment of E. coli to mannose-containing receptors on the surface of host cells. We have investigated the lymphocyte mitogenic activity of this mannose-specific adhesin by comparing the ability of purified wild type type 1 fimbriae containing the adhesin and mutant type 1 fimbriae lacking the adhesin to stimulate proliferation in human lymphocytes. Both fimbriae stimulated a peak of proliferation at 8 days whereas only the wild type fimbriae stimulated an additional peak of proliferation occurring at 3 days. Proliferation at 3 days but not at 8 days could be blocked by the addition of alpha-methyl-D-mannoside. Neonatal lymphocytes from umbilical cord blood responded to both wild type and mutant fimbriae in a fashion similar to adult cells. Stimulation of separated T and non-T cell populations indicated that the proliferation seen at 3 days was solely due to non-T cells whereas the 8-day response was due to T cell proliferation. The addition of gamma-irradiated T cells did not appear to enhance the 3-day response of the non-T cells. However, the 8-day response by T cells was dependent on the presence of gamma-irradiated non-T cells. In cultures of unseparated cells, wild type fimbriae stimulated more than 75% of the B cells to enter the S and G2 phase at 3 days whereas at 8 days cycling T cells were present in both wild type and mutant fimbriae-stimulated cultures. Taken together, our observations suggest that the adhesin molecule stimulates a polyclonal mitogenic response in B cells that peaks at 3 days, and other structural components of the fimbriae are responsible for evoking an 8-day (probably immune) response in T cells.

Adhesins, Escherichia coli↗
PubMed1988

Conservation of the D-mannose-adhesion protein among type 1 fimbriated members of the family Enterobacteriaceae.

A variety of genera and species of the family Enterobacteriaceae bear surface fimbriae that enable them to bind to D-mannose residues on eukaryotic cells. Until recently, it was thought that the D-mannose binding site was located in the major structural subunit (FimA), of relative molecular mass (Mr) 17,000 (17 K), of these organelles in Escherichia coli. New evidence indicates that this binding site resides instead in a minor protein Mr 28-31 K (FimH) located at the tips and at long intervals along the length of the fimbriae, and is reminiscent of the minor tip adhesion proteins of pyelonephritis-associated pili (Pap) and S fimbriae. In contrast to the antigenic heterogeneity of the major FimA subunit, the antigenic structure of FimH is conserved among different strains of E. coli. Here, we report an even broader conservation of this minor adhesion protein extending to other genera and species of type 1 fimbriated Enterobacteriaceae. Our results may have implications for the development of broadly protective vaccines against Gram-negative bacillary infections in animals and perhaps in man.

Antibodies, Monoclonal↗
PubMed1988

Isolation and characterization of a receptor for type 1 fimbriae of Escherichia coli from guinea pig erythrocytes.

The adhesion of Escherichia coli to eukaryotic cells is mediated by proteinaceous surface appendages called fimbriae and complementary receptors on host cells. Although type 1 fimbriae, which contain a D-mannose-reactive lectin, have been well studied little is known about the binding mechanism of isolated fimbriae to individual cell receptors. This report describes the isolation and purification of a guinea pig erythrocyte receptor for type 1 fimbriae. Erythrocyte membranes were dissolved in 0.5% Triton X-100 and the receptor isolated and purified by affinity chromatography using type 1 fimbriae immobilized on Sepharose. The 65-kDa receptor, which inhibits the agglutination of guinea pig erythrocytes by type 1 fimbriated E. coli, has a pI of 8.5-8.7, and binds concanavalin A and type 1 fimbriae in a dose-dependent and saturable manner. The fimbrial binding activity of the receptor was reduced when treated with sodium metaperiodate, endoglycosidase H, trypsin, and V8 protease, suggesting the isolated receptor is a glycoprotein with N-linked carbohydrate units. Isolated type 1 fimbriae inhibited the binding of fimbriated E. coli to purified receptor in a dose- and time-related fashion. The calculated binding affinity was 6 X 10(6) M-1, a value consistent with the low binding affinity expected from previous studies of the agglutination of guinea pig erythrocytes by isolated type 1 fimbriae.

Animals↗
PubMed1988

Influence of berberine sulfate on synthesis and expression of Pap fimbrial adhesin in uropathogenic Escherichia coli.

We investigated the influence of berberine sulfate, an ancient Chinese antibiotic, upon the adhesion of uropathogenic Escherichia coli to erythrocytes and epithelial cells. Although berberine sulfate in increasing concentrations had no effect on bacterial growth or on the synthesis of major outer membrane proteins of the E. coli organisms, it increasingly blocked adhesion. The decreased adhesion was accompanied by a reduction in the synthesis of fimbrial subunits and in the expression of assembled fimbriae. These results suggest that the anti-infectious activity of berberine sulfate in E. coli-induced urinary tract infections may be mediated by the selective suppression of the synthesis and assembly of fimbriae by uropathogenic organisms.

Adhesins, Escherichia coli↗
PubMed1988

Hyperadhesive mutant of type 1-fimbriated Escherichia coli associated with formation of FimH organelles (fimbriosomes).

The relationships of the genes and gene-products mediating D-mannose-specific attachment of type 1 fimbriae of Escherichia coli to eucaryotic cells were investigated by deletion mutation analysis of recombinant plasmid pSH2, which carries the genetic information for the synthesis and expression of functional type 1 fimbriae. Mutant pUT2004 was derived by a deletion remote from the structural gene encoding the 17-kilodalton (kDa) subunit protein of type 1 fimbriae. Phenotypically, the mutant demonstrated an eightfold-higher mannose-specific hemagglutination titer than the parent strain. On electron microscopy, the mutant strain expressed the same number of fimbriae as the parent strain. However, numerous 10-nm-diameter rounded structures (fimbriosomes) were observed both closely associated with fimbriae and in the culture medium. Fimbriosomes isolated from the medium agglutinated guinea pig erythrocytes in a mannose-sensitive manner. Dissociation of the fimbriosomes yielded a single 29-kDa protein, as demonstrated by sodium dodecyl sulfate gel electrophoresis. Antibodies raised against fimbriosomes reacted with a 29-kDa protein on immunoelectroblots of dissociated type 1 fimbriae and also blocked the adherence of other strains of type 1 fimbriated E. coli to eucaryotic cells. These findings suggest that the enhanced adhesive properties of the mutant pUT2004 strain are associated with overproduction of the 29-kDa FimH in the form of fimbriosomes which contain the determinant of the D-mannose-sensitive adhesion of type 1 fimbriae.

Bacterial Adhesion↗
PubMed1988

Bacterial adherence. Adhesin receptor-mediated attachment of pathogenic bacteria to mucosal surfaces.

Pathogenic bacteria adhere to and colonize mucosal surfaces of the susceptible host in a highly selective manner. After the organisms penetrate the nonspecific mechanical and cleansing forces, ligands (or adhesins) on the surface of the bacteria interact in a lock-and-key fashion with complementary receptors on mucosal surfaces of the host. The adhesins are usually composed of proteins in the form of fimbriae or fibrillae and the receptors of glycolipids or glycoproteins. At the epithelial cell surfaces, two classic examples of bacterial adherence are the lipoteichoic acid-mediated attachments of group A streptococcal and the type 1 fimbriae-mediated attachment of Escherichia coli. In group A streptococci, the adhesin, lipoteichoic acid (LTA), is anchored to a protein(s) on the surface of the bacterial cells and interacts through its lipid moiety with fibronectin molecules deposited on and bound to the epithelial cells. In type 1 fimbriated E. coli, a minor 29-kDa protein located at the tip of the fimbriae interacts with D-mannose residues of glycoprotein receptors on host cells. Similar adhesin-receptor interactions have now been described for a number of pathogenic microbial agents, and undoubtedly play a central role in the early steps of the infectious process.

Bacterial Adhesion↗
PubMed1987

Use of monoclonal antibodies to probe subunit- and polymer-specific epitopes of 987P fimbriae of Escherichia coli.

The relationship between the structure and biological function of 987P fimbriae of a strain of enterotoxigenic Escherichia coli (O9:K103:H-) from piglets was investigated. A set of four monoclonal antibodies was prepared from the spleen cells of mice immunized with isolated 987P fimbriae. Antibodies E11, D5, and C3, but not G10, reacted in enzyme-linked immunosorbent assays with 987P fimbriae-bearing E. coli. Electron microscopy showed that E11 and D5 reacted in a discrete periodic pattern forming a spiral motif along the length of the fimbriae. The results of enzyme-linked immunosorbent assays were in agreement with these results; antibodies E11 and D5 reacted at a high dilution (1:12,000) with native fimbriae on the surface of E. coli, whereas antibody C3 reacted at an intermediate dilution (1:3,000) and G10 failed to react at all (less than 1:250). In contrast, C3 and G10 reacted at a dilution of 1:3,276,000 with the fimbrial subunits derived by treating the isolated fimbriae with 6 M guanidine hydrochloride, whereas E11 and D5 reacted with the subunits at much lower dilutions of 1:800 and 1:6,400, respectively. Moreover, fimbriae reassembled from the subunits regained reactivity with antibodies D5 and E11, indicating that these antibodies are directed against quaternary conformational epitopes. Only the three antibodies (D5, E11, and C3) that recognized epitopes accessible on intact fimbriae were able to efficiently block the adhesion of 987P fimbriated E. coli to piglet enterocytes. These results indicate that certain epitopes of 987P fimbriae are dependent on quaternary structural conformation, whereas others are present on monomeric subunits; some of the latter appear to remain accessible on fully assembled fimbriae.

Animals↗
PubMed1987

Identification of two ancillary subunits of Escherichia coli type 1 fimbriae by using antibodies against synthetic oligopeptides of fim gene products.

We have chemically synthesized oligopeptides corresponding to the NH2-terminal stretch of two gene products, designated FimG and FimH, of the fim gene cluster of Escherichia coli. These synthetic peptides, designated S-T1FimG(1-16) and S-T1FimH(1-25)C, evoked antibodies in rabbits that reacted with 14- and 29-kilodalton subunits, respectively, of dissociated fimbriae encoded by the recombinant plasmid pSH2 carrying the genetic information for the synthesis and expression of functional type 1 fimbriae. Neither of these fimbrial proteins was detected in dissociated fimbrial preparations from nonadhesive E. coli cells carrying the mutant plasmid pUT2002, containing a restriction site-specific deletion of fimG and fimH. Anti-S-T1FimH(1-25)C inhibited the adherence of type 1 fimbriated E. coli to epithelial cells. Immunoelectron microscopy revealed that anti-S-T1FimH(1-25)C, but not anti-S-T1FimG(1-16), bound to intact type 1 fimbriae of E. coli at the fimbrial tips and at long intervals along the fimbrial filaments. Anti-S-T1FimG(1-16) appeared to be directed at epitopes not accessible on the intact fimbriae and consequently failed to bind to intact fimbriae or to block fimbrial attachment. Our results suggest that the fimG and fimH gene products are components of type 1 fimbriae and that FimH may be the tip adhesin mediating the binding of type 1 fimbriated E. coli to D-mannose residues on mucosal surfaces.

Antibodies, Bacterial↗