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Sample records for mannose-resistant proteus-like fimbriae

  1. Requirement of MrpH for Mannose-Resistant Proteus-Like Fimbria-Mediated Hemagglutination by Proteus mirabilis

    PubMed Central

    Li, Xin; Johnson, David E.; Mobley, Harry L. T.

    1999-01-01

    Two new genes, mrpH and mrpJ, were identified downstream of mrpG in the mrp gene cluster encoding mannose-resistant Proteus-like (MR/P) fimbriae of uropathogenic Proteus mirabilis. Since the predicted MrpH has 30% amino acid sequence identity to PapG, the Galα(1-4)Gal-binding adhesin of Escherichia coli P fimbriae, we hypothesized that mrpH encodes the functional MR/P hemagglutinin. MR/P fimbriae, expressed in E. coli DH5α, conferred on bacteria both the ability to cause mannose-resistant hemagglutination and the ability to aggregate to form pellicles on the broth surface. Both a ΔmrpH mutant expressed in E. coli DH5α and an isogenic mrpH::aphA mutant of P. mirabilis were unable to produce normal MR/P fimbriae efficiently, suggesting that MrpH was involved in fimbrial assembly. Amino acid residue substitution of the N-terminal cysteine residues (C66S and C128S) of MrpH abolished the receptor-binding activity (hemagglutinating ability) of MrpH but allowed normal fimbrial assembly, supporting the notion that MrpH was the functional MR/P hemagglutinin. Immunogold electron microscopy of P. mirabilis HI4320 revealed that MrpH was located at the tip of MR/P fimbriae, also consistent with its role in receptor binding. The isogenic mrpH::aphA mutant of HI4320 was less able to colonize the urine, bladder, and kidneys in a mouse model of ascending urinary tract infection (P < 0.01), and therefore MR/P fimbriae contribute significantly to bacterial colonization in mice. While there are similarities between P. mirabilis MR/P and E. coli P fimbriae, there are more notable differences: (i) synthesis of the MrpH adhesin is required to initiate fimbrial assembly, (ii) MR/P fimbriae confer an aggregation phenotype, (iii) site-directed mutation of specific residues can abolish receptor binding but allows fimbrial assembly, and (iv) mutation of the adhesin gene abolishes virulence in a mouse model of ascending urinary tract infection. PMID:10338487

  2. Proteus mirabilis mannose-resistant, Proteus-like fimbriae: MrpG is located at the fimbrial tip and is required for fimbrial assembly.

    PubMed Central

    Li, X; Zhao, H; Geymonat, L; Bahrani, F; Johnson, D E; Mobley, H L

    1997-01-01

    The mannose-resistant, Proteus-like (MR/P) fimbria, responsible for mannose-resistant hemagglutination, is a virulence factor for uropathogenic Proteus mirabilis. Based on known fimbrial gene organization, we postulated that MrpG, a putative minor subunit of the MR/P fimbria, functions as an adhesin responsible for hemagglutination, while MrpA serves as the major structural subunit for the filamentous structure. To test this hypothesis, an mrpG mutant was constructed by allelic-exchange mutagenesis and verified by PCR and Southern blotting. The mrpG mutant was found to be negative for hemagglutination, while wild-type strain H14320 and the complemented mutant were positive. Western blots with antiserum raised against an overexpressed MrpG'-His6 fusion protein showed that MrpG was present in the fimbrial preparations of both the wild-type strain and the complemented mutant but absent in that of the mrpG mutant. The mrpG mutant was significantly less virulent in a CBA mouse model of ascending urinary tract infection. Western blots with antiserum to whole MR/P fimbriae showed that MrpA protein was also missing from the fimbrial preparation of the mrpG mutant. Using immunogold electron microscopy, we found that the normal MR/P-fimbrial structure was absent in the mrpG mutant, suggesting that MrpG is essential for initiation of normal fimbrial formation. In the wild-type strain, MrpG protein was localized to the tips of the fimbriae or at the surface of the cell when antiserum raised against overexpressed MrpG was used. Given the tip localization, MrpG may be required for initiation of assembly of MR/P fimbriae but does not appear to be the fimbrial adhesin. PMID:9119470

  3. Proteus mirabilis fimbriae: identification, isolation, and characterization of a new ambient-temperature fimbria.

    PubMed Central

    Massad, G; Bahrani, F K; Mobley, H L

    1994-01-01

    Urinary tract infections involving Proteus mirabilis may lead to complications including bladder and kidney stones, acute pyelonephritis, and bacteremia. This bacterium produces a number of fimbriae, two of which, MR/P fimbria and P. mirabilis fimbria, have been shown to contribute to the ability of this pathogen to colonize the bladder and kidney. We have now purified and characterized a previously undescribed fimbria of P. mirabilis, named ambient-temperature fimbria (ATF). Electron microscopy of a pure preparation and immunogold labeling of cells demonstrated that ATF was fimbrial in nature. The major fimbrial subunit of ATF has an apparent molecular weight of 24,000. The N-terminal amino acid sequence, E-X-T-G-T-P-A-P-T-E-V-T-V-D-G-G-T-I-D-F, did not show significant similarity to that of any previously described fimbrial protein. ATF was expressed by all eight P. mirabilis strains examined. Culture conditions affected expression of ATF, with optimal expression observed in static broth cultures at 23 degrees C. This fimbria was not produced by cells grown at 42 degrees C or on solid medium. Expression of ATF did not correlate with mannose-resistant/Proteus-like (MR/P) or mannose-resistant/Klebsiella-like (MR/K) hemagglutination and represents a novel fimbria of P. mirabilis. Images PMID:7909538

  4. The expression of nonagglutinating fimbriae and its role in Proteus mirabilis adherence to epithelial cells.

    PubMed

    Tolson, D L; Harrison, B A; Latta, R K; Lee, K K; Altman, E

    1997-08-01

    Proteus mirabilis is a common causative agent of human urinary tract infections, especially in catheterized patients and in those patients with structural abnormalities of the urinary tract. In addition to the production of hemolysin and urease, fimbriae-mediated adherence to uroepithelial cells and kidney epithelium may be essential for virulence of P. mirabilis. A single P. mirabilis strain is capable of expressing several morphologically distinct fimbrial species, which can each be favoured by specific in vitro growth conditions. The fimbrial species reported to date include mannose-resistant/Proteus-like fimbriae, ambient temperature fimbriae, P. mirabilis fimbriae, and nonagglutinating fimbriae (NAF). Here, using intact bacteria or purified NAF as immunogens, we have generated the first reported NAF-specific monoclonal antibodies (mAbs). Bacteria expressing NAF as their only fimbrial species adhered strongly to a number of cell lines in vitro, including uroepithelial cell lines. Binding of P. mirabilis was markedly reduced following preincubation with NAF-specific mAbs and Fab fragments. The presence of NAF with highly conserved N-terminal sequences on all P. mirabilis strains so far examined, combined with the ability of both anti-NAF mAbs and purified NAF molecules to inhibit P. mirabilis adherence in vitro, suggests that NAF may contribute to the pathogenesis of P. mirabilis. PMID:9304781

  5. Proteus mirabilis fimbriae- and urease-dependent clusters assemble in an extracellular niche to initiate bladder stone formation.

    PubMed

    Schaffer, Jessica N; Norsworthy, Allison N; Sun, Tung-Tien; Pearson, Melanie M

    2016-04-19

    The catheter-associated uropathogenProteus mirabilisfrequently causes urinary stones, but little has been known about the initial stages of bladder colonization and stone formation. We found thatP. mirabilisrapidly invades the bladder urothelium, but generally fails to establish an intracellular niche. Instead, it forms extracellular clusters in the bladder lumen, which form foci of mineral deposition consistent with development of urinary stones. These clusters elicit a robust neutrophil response, and we present evidence of neutrophil extracellular trap generation during experimental urinary tract infection. We identified two virulence factors required for cluster development: urease, which is required for urolithiasis, and mannose-resistantProteus-like fimbriae. The extracellular cluster formation byP. mirabilisstands in direct contrast to uropathogenicEscherichia coli, which readily formed intracellular bacterial communities but not luminal clusters or urinary stones. We propose that extracellular clusters are a key mechanism ofP. mirabilissurvival and virulence in the bladder. PMID:27044107

  6. Interaction of Escherichia coli with Different Fimbriae and Polymorphonuclear Leukocytes

    PubMed Central

    Björkstén, Bengt; Wadström, Torkel

    1982-01-01

    The effects of Escherichia coli strains with various fimbriae on bacteria-polymorphonuclear leukocyte (PMN) interactions were studied. Strains of E. coli were cultivated at 37°C to express and at 18°C to suppress the formation of fimbriae. The presence of fimbriae was confirmed by electron microscopic studies and hemagglutination and salt aggregation tests. Fimbriated E. coli strains were more readily PMN associated than the nonfimbriated strains in the absence of opsonins, confirming the results of previous studies. However, the PMN chemiluminescence (CL) induced by the various strains in the absence of serum opsonins depended on the type of fimbriae they expressed. Strains with type 1 fimbriae expressing mannose-sensitive hemagglutination induced 5 to 15 times more CL than the same strains grown at 18°C, i.e., not expressing this type of fimbriae. For strains showing mannose-resistant hemagglutination, the differences between fimbriated and nonfimbriated variants of the same strains grown at 37 and 18°C, respectively, were less pronounced. Analysis of enterotoxigenic strains expressing colonization factor antigen I (CFA/I) fimbriae showed that these induced only 25 to 33% of the CL induced by the same E. coli strains not expressing CFA/I, whereas enterotoxigenic strains expressing CFA/II fimbriae induced 100 to 200% of the CL induced by the nonfimbriated variants. Although less CL was induced by bacteria with CFA/I fimbriae than by nonfimbriated variants, this situation was reversed when the microorganisms were opsonized. Thus, CFA/I fimbriae, while enhancing adhesion to cells, induce less activation of PMN-killing mechanisms in a serum-free environment. These findings may be relevant for the virulence in certain body sites, since CFA/I fimbriae, while facilitating adhesiveness, may protect the bacteria from PMN killing. Our findings indicate that PMN interactions with fimbriated E. coli in the host defense may be complex. Certain fimbriae may indeed be

  7. Cloning and expression of an afimbrial adhesin (AFA-I) responsible for P blood group-independent, mannose-resistant hemagglutination from a pyelonephritic Escherichia coli strain.

    PubMed Central

    Labigne-Roussel, A F; Lark, D; Schoolnik, G; Falkow, S

    1984-01-01

    The uropathogenic Escherichia coli KS52 strain expresses a mannose-resistant hemagglutinin involving an erythrocyte recognition site distinct from the alpha-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 mannose-resistant 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). Images PMID:6148308

  8. Biochemical Characterization and Agglutinating Properties of Xenorhabdus nematophilus F1 Fimbriae

    PubMed Central

    Moureaux, N.; Karjalainen, T.; Givaudan, A.; Bourlioux, P.; Boemare, N.

    1995-01-01

    Xenorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the family Steinernematidae, occur spontaneously in two phases. Only the phase I variants of Xenorhabdus nematophilus F1 expressed fimbriae when the bacteria were grown on a solid medium (nutrient agar; 24 and 48 h of growth). These appendages were purified and characterized. They were rigid, with a diameter of 6.4 (plusmn) 0.3 nm, and were composed of 16-kDa pilin subunits. The latter were synthesized and assembled during the first 24 h of growth. Phase II variants of X. nematophilus did not possess fimbriae and apparently did not synthesize pilin. Phase I variants of X. nematophilus have an agglutinating activity with sheep, rabbit, and human erythrocytes and with hemocytes of the insect Galleria mellonella. The purified fimbriae agglutinated sheep and rabbit erythrocytes. The hemagglutination by bacteria and purified fimbriae was mannose resistant and was inhibited by porcine gastric mucin and N-acetyl-lactosamine. The last sugar seems to be a specific inhibitor of hemagglutination by X. nematophilus. PMID:16535079

  9. Proteus mirabilis flagella and MR/P fimbriae: isolation, purification, N-terminal analysis, and serum antibody response following experimental urinary tract infection.

    PubMed

    Bahrani, F K; Johnson, D E; Robbins, D; Mobley, H L

    1991-10-01

    Urinary tract infection with Proteus mirabilis may lead to serious complications, including cystitis, acute pyelonephritis, fever, bacteremia, and death. In addition to the production of hemolysin and the enzyme urease, fimbriae and flagellum-mediated motility have been postulated as virulence factors for this species. We purified mannose-resistant/proteuslike (MR/P) fimbriae and flagella from strains CFT322 and HU2450, respectively. Electron microscopy revealed highly concentrated preparations of fimbriae and flagella. Fimbrial and flagellar structural subunits were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 18.5 and 41 kDa, respectively. N-terminal sequencing revealed that 10 of the first 20 amino acids of the major MR/P subunit matched the sequence of the P. mirabilis uroepithelial cell adhesin N terminus and 11 of 20 amino acids matched the predicted amino acid sequence of the Escherichia coli P fimbriae structural subunit, PapA. In addition, 90 and 80% homologies were found between the first 20 amino acids of P. mirabilis flagellin and those of Salmonella typhimurium phase-1 flagellin and the E. coli hag gene product, respectively. An enzyme-linked immunosorbent assay using purified antigens showed a strong reaction between the MR/P fimbriae or flagella and sera of CBA mice challenged transurethrally with P. mirabilis. A possible role for MR/P fimbriae in the pathogenesis of urinary tract infection is supported by (i) a strong immune response to the antigen in experimentally infected animals, (ii) amino acid sequence similarity to other enteric surface structure, and (iii) our previously reported observation that MR/P fimbriae are expressed preferentially as the sole fimbrial type in human pyelonephritis isolates. PMID:1680106

  10. Avian P1 antigens inhibit agglutination mediated by P fimbriae of uropathogenic Escherichia coli.

    PubMed Central

    Johnson, J R; Swanson, J L; Neill, M A

    1992-01-01

    Whole egg white from pigeon, dove, and cockatiel eggs, as well as the ovomucoid fraction of pigeon egg white, exhibited strong P1 antigenic activities and inhibited agglutination of human P1 erythrocytes and of digalactoside-coated latex beads by P-fimbriated Escherichia coli strains. In contrast, chicken egg white exhibited only weak P1 antigenic activity and had little impact on P-fimbrial agglutination. These preparations did not affect hemagglutination by E. coli strains expressing mannose-resistant adhesins other than P fimbriae, i.e., Dr, F1845, and S adhesins. Human anti-P1 serum diminished the P-fimbrial inhibitory activities of pigeon egg white and pigeon ovomucoid. Pigeon ovomucoid was equipotent on a molar basis with globoside, and the pigeon, dove, and cockatiel egg white preparations were equipotent with each other in P-fimbrial inhibition. Incubation of p erythrocytes in whole egg whites or in pigeon ovomucoid did not render them agglutinable by P-fimbriated bacteria, whereas incubation in globoside did. These data demonstrate that whole egg whites (and their ovomucoid fraction) from members of the families Columbidae (pigeons and doves) and Psittacidae (parrots) specifically and potently inhibit P-fimbrial agglutination, probably by providing P1 antigen as a receptor for the P-fimbrial adhesin. Avian egg white preparations may facilitate adhesin characterization of wild-type uropathogenic strains and may useful in preventing upper urinary tract infections due to P-fimbriated E. coli. PMID:1346125

  11. Helical structure of Bordetella pertussis fimbriae.

    PubMed Central

    Steven, A C; Bisher, M E; Trus, B L; Thomas, D; Zhang, J M; Cowell, J L

    1986-01-01

    The helical structures of Bordetella pertussis fimbriae of serotypes 2 and 6 were determined by optical diffraction analysis of electron micrographs of negatively stained paracrystalline bundles of purified fimbriae. The fimbrial structure is based on an axial repeat of 13 nm that contains five repeating units in two complete turns of a single-start helix. This structure was confirmed by direct measurements of mass per unit length for individual fimbriae performed by dark-field scanning transmission electron microscopy of unstained specimens. These data further established that the helically repeating unit is a monomer of fimbrial protein (Mr congruent to 22,000 for type 2 and Mr congruent to 21,500 for type 6). Radial density profiles calculated from the scanning transmission electron micrographs showed that the fimbria has peak density at its center, i.e., no axial channel, consistent with the results of conventional negative-staining electron microscopy. The radial profile gives an outermost diameter of approximately 7.5 nm, although the peripheral density is, on average, diffuse, allowing sufficient intercalation between adjacent fimbriae to give a center-to-center spacing of approximately 5.5 nm in the paracrystals. Despite serological and biochemical differences between type 2 and type 6 fimbriae, the packing arrangements of their fimbrial subunits are identical. From this observation, we infer that the respective subunits may have in common conserved regions whose packing dictates the helical geometry of the fimbria. It is plausible that a similar mechanism may underlie the phenomenon of phase variations in other systems of bacterial fimbriae. Images PMID:2875062

  12. Purification and characterization of serotype 6 fimbriae from Bordetella pertussis and comparison of their properties with serotype 2 fimbriae.

    PubMed

    Cowell, J L; Zhang, J M; Urisu, A; Suzuki, A; Steven, A C; Liu, T; Liu, T Y; Manclark, C R

    1987-04-01

    Fimbriae were removed from Bordetella pertussis (serotype 1.3.6) by mechanical shearing and purified by precipitation with ammonium sulfate, pH-dependent precipitation at pH 7.4, followed by two successive extractions of the precipitated fimbriae with 4 M urea. By electron microscopy, the precipitated fimbriae appeared as aggregated bundles of long, relatively straight filaments which were disaggregated to individual flexuous filaments at pH 10.5. These purified fimbriae were identified as serotype 6 agglutinogens, since antibody to the purified fimbriae agglutinated B. pertussis strains serotyped as 1.3.6, 1.2.3.6, or 1.2.3.4.6 but did not agglutinate strains of serotype 1.2.3.4, 1.2.3, or 1.3. In contrast, antibody to serotype 2 fimbriae only agglutinated B. pertussis strains containing serotype 2 agglutinogen. Purified type 6 and 2 fimbriae were found to be weakly cross-reactive by enzyme-linked immunosorbent assay, using polyclonal antibody to each type of fimbria. In an immunoblot assay, polyclonal antibodies to a 22,000-dalton subunit of fimbriae from B. bronchiseptica reacted strongly with the type 2 fimbrial subunit of B. pertussis, but only weakly with the type 6 subunit. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein subunit of the type 6 fimbriae migrated with a molecular weight of 21,500, whereas the type 2 fimbrial subunit had a molecular weight of 22,000. The two types of subunits had similar amino acid compositions and showed amino-terminal sequence homology in 15 of 21 amino acids. The amino-terminal amino acid sequences of the B. pertussis fimbriae were distinct from those reported for fimbriae from other gram-negative bacteria. Neither the type 6 nor the type 2 fimbriae caused hemagglutination when assayed with several types of erythrocytes. PMID:2881893

  13. Purification and characterization of serotype 6 fimbriae from Bordetella pertussis and comparison of their properties with serotype 2 fimbriae.

    PubMed Central

    Cowell, J L; Zhang, J M; Urisu, A; Suzuki, A; Steven, A C; Liu, T; Liu, T Y; Manclark, C R

    1987-01-01

    Fimbriae were removed from Bordetella pertussis (serotype 1.3.6) by mechanical shearing and purified by precipitation with ammonium sulfate, pH-dependent precipitation at pH 7.4, followed by two successive extractions of the precipitated fimbriae with 4 M urea. By electron microscopy, the precipitated fimbriae appeared as aggregated bundles of long, relatively straight filaments which were disaggregated to individual flexuous filaments at pH 10.5. These purified fimbriae were identified as serotype 6 agglutinogens, since antibody to the purified fimbriae agglutinated B. pertussis strains serotyped as 1.3.6, 1.2.3.6, or 1.2.3.4.6 but did not agglutinate strains of serotype 1.2.3.4, 1.2.3, or 1.3. In contrast, antibody to serotype 2 fimbriae only agglutinated B. pertussis strains containing serotype 2 agglutinogen. Purified type 6 and 2 fimbriae were found to be weakly cross-reactive by enzyme-linked immunosorbent assay, using polyclonal antibody to each type of fimbria. In an immunoblot assay, polyclonal antibodies to a 22,000-dalton subunit of fimbriae from B. bronchiseptica reacted strongly with the type 2 fimbrial subunit of B. pertussis, but only weakly with the type 6 subunit. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein subunit of the type 6 fimbriae migrated with a molecular weight of 21,500, whereas the type 2 fimbrial subunit had a molecular weight of 22,000. The two types of subunits had similar amino acid compositions and showed amino-terminal sequence homology in 15 of 21 amino acids. The amino-terminal amino acid sequences of the B. pertussis fimbriae were distinct from those reported for fimbriae from other gram-negative bacteria. Neither the type 6 nor the type 2 fimbriae caused hemagglutination when assayed with several types of erythrocytes. Images PMID:2881893

  14. Release and purification of fimbriae from Bordetella pertussis.

    PubMed

    Irons, L I; Ashworth, L A; Robinson, A

    1985-01-01

    A competitive ELISA has been used to monitor the release of fimbriae from 1.2.3 serotype of B. pertussis after treatment by different methods and fimbriae have been purified from homogenates or KSCN extracts by chromatography. Fimbriae purified from different serotypes have been studied by inhibition of bacterial agglutination, immunodiffusion and SDS-polyacrylamide gel electrophoresis. Fimbriae purified from a 1.2 serotype have been labelled in immunoelectron microscopy with a IgM monoclonal antibody to agglutinogen 2 and evidence is presented that fimbriae purified from a 1.3 serotype also carry the agglutinogen 3 specificity. A difference in subunit molecular weight has been found between fimbriae purified from 1.2 and 1.3 serotypes. PMID:2872101

  15. Identification of Two Laminin-Binding Fimbriae, the Type 1 Fimbria of Salmonella enterica Serovar Typhimurium and the G Fimbria of Escherichia coli, as Plasminogen Receptors

    PubMed Central

    Kukkonen, Maini; Saarela, Sirkku; Lähteenmäki, Kaarina; Hynönen, Ulla; Westerlund-Wikström, Benita; Rhen, Mikael; Korhonen, Timo K.

    1998-01-01

    Escherichia coli strains carrying recombinant plasmids encoding either the type 1 fimbria of Salmonella enterica serovar Typhimurium or the G fimbria of E. coli exhibited binding of human 125I-Glu-plasminogen and enhanced the tissue-type plasminogen activator-catalyzed conversion of plasminogen to plasmin. Purified type 1 or G fimbriae similarly bound plasminogen and enhanced its activation. The binding of plasminogen did not involve the characteristic carbohydrate-binding property of the fimbriae but was inhibited at low concentrations by the lysine analog ɛ-aminocaproic acid. Because these fimbrial types bind to laminin of basement membranes (M. Kukkonen et al., Mol. Microbiol. 7:229–237, 1993; S. Saarela et al., Infect. Immun. 64:2857–2860, 1996), the results demonstrate a structural unity in the creation and targeting of bacterium-bound proteolytic plasmin activity to basement membranes. PMID:9746604

  16. Porphyromonas gingivalis Fimbriae Bind to Cytokeratin of Epithelial Cells

    PubMed Central

    Sojar, Hakimuddin T.; Sharma, Ashu; Genco, Robert J.

    2002-01-01

    The adherence of Porphyromonas gingivalis to host cells is likely a prerequisite step in the pathogenesis of P. gingivalis-induced periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are shown to be involved in this process. Little is known regarding epithelial receptor(s) involved in binding of P. gingivalis fimbriae. Using an overlay assay with purified P. gingivalis fimbriae as a probe, two major epithelial cell proteins with masses of 50 and 40 kDa were identified by immunoblotting with fimbria-specific antibodies. Iodinated purified fimbriae also bound to the same two epithelial cell proteins. An affinity chromatography technique was utilized to isolate and purify the epithelial components to which P. gingivalis fimbriae bind. Purified fimbriae were coupled to CNBr-activated Sepharose-4B, and the solubilized epithelial cell extract proteins bound to the immobilized fimbriae were isolated from the column. A major 50-kDa component and a minor 40-kDa component were purified and could be digested with trypsin, suggesting that they were proteins. These affinity-eluted 50- and 40-kDa proteins were then subjected to amino-terminal sequencing, and no sequence could be determined, suggesting that these proteins have blocked amino-terminal residues. CNBr digestion of the 50-kDa component resulted in an internal sequence homologous to that of Keratin I molecules. Further evidence that P. gingivalis fimbriae bind to cytokeratin molecule(s) comes from studies showing that multicytokeratin rabbit polyclonal antibodies cross-react with the affinity-purified 50-kDa epithelial cell surface component. Also, binding of purified P. gingivalis fimbriae to epithelial components can be inhibited in an overlay assay by multicytokeratin rabbit polyclonal antibodies. Furthermore, we showed that biotinylated purified fimbriae bind to purified human epidermal keratin in an overlay assay. These studies suggest that the surface-accessible epithelial

  17. Simple method for purification of enterotoxigenic Escherichia coli fimbriae.

    PubMed

    Curtis, Brittany; Grassel, Christen; Laufer, Rachel S; Sears, Khandra T; Pasetti, Marcela F; Barry, Eileen M; Simon, Raphael

    2016-03-01

    Enterotoxigenic Escherichia coli (ETEC) are endemic pathogens in the developing world. They frequently cause illness in travelers, and are among the most prevalent causes of diarrheal disease in children. Pathogenic ETEC strains employ fimbriae as adhesion factors to bind the luminal surface of the intestinal epithelium and establish infection. Accordingly, there is marked interest in immunoprophylactic strategies targeting fimbriae to protect against ETEC infections. Multiple strategies have been reported for purification of ETEC fimbriae, however none is ideal. Purification has typically involved the use of highly virulent wild-type strains. We report here a simple and improved method to purify ETEC fimbriae, which was applied to obtain two different Class 5 fimbriae types of clinical relevance (CFA/I and CS4) expressed recombinantly in E. coli production strains. Following removal from cells by shearing, fimbriae proteins were purified by orthogonal purification steps employing ultracentrifugation, precipitation, and ion-exchange membrane chromatography. Purified fimbriae demonstrated the anticipated size and morphology by electron microscopy analysis, contained negligible levels of residual host cell proteins, nucleic acid, and endotoxin, and were recognized by convalescent human anti-sera. PMID:26581778

  18. Purification and characterization of fimbriae isolated from Bordetella pertussis.

    PubMed Central

    Zhang, J M; Cowell, J L; Steven, A C; Carter, P H; McGrath, P P; Manclark, C R

    1985-01-01

    Fimbriae were detached from Bordetella pertussis by mechanical shearing and purified by successive precipitations with ammonium sulfate, phosphate buffer (pH 6.0), and magnesium chloride. In each of these purification steps, the fimbriae aggregated into bundles as seen by electron microscopy. These aggregates could be disaggregated at pH 9.5. By electron microscopy, the purified fimbriae appeared as long filaments with a diameter of 5 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fimbriae showed a single protein subunit with a molecular weight of 22,000. The purified fimbriae did not have hemagglutinating activity when assayed with several types of erythrocytes, and they were antigenically, chemically, and structurally distinct from the filamentous hemagglutinin of B. pertussis. The purified fimbriae were also identified as serotype 2 agglutinogens, since antibody to the purified fimbriae agglutinated B. pertussis strains serotyped as 1.2.4, 1.2.3, or 1.2.3.6 but did not agglutinate those serotyped as 1.3.6. Images PMID:2859248

  19. Morphogenetic expression of Moraxella bovis fimbriae (pili) in Pseudomonas aeruginosa.

    PubMed Central

    Beard, M K; Mattick, J S; Moore, L J; Mott, M R; Marrs, C F; Egerton, J R

    1990-01-01

    Type 4 fimbriae (pili) are found in a wide variety of gram-negative bacteria and are composed of small structural subunits which share significant sequence homology among different species, especially at their amino-terminal ends. Previous studies demonstrating morphogenetic expression of Bacteroides nodosus fimbriae from cloned subunit genes in Pseudomonas aeruginosa suggested that there is a common mechanism for type 4 fimbriae assembly and that the structural subunits are interchangeable (J. S. Mattick et al., J. Bacteriol. 169:33-41, 1987). Here we have examined the expression of Moraxella bovis fimbrial subunits in P. aeruginosa. M. bovis subunits were assembled into extracellular fimbriae in this host, in some cases as a homopolymer but in others as a mosaic with the indigenous subunit, indicating structural equivalence. This result contrasts with other studies in which recombinant P. aeruginosa expressing different subunits produced fimbriae composed almost exclusively of one subunit or the other (T. C. Elleman and J. E. Peterson, Mol. Microbiol. 1:377-380, 1987). Both observations can be explained by reversibility of subunit-subunit interactions at the site of assembly, with the forward equilibrium favoring chain extension between compatible subunits. Images PMID:1970564

  20. Association between Bordetella pertussis agglutinogen 2 and fimbriae.

    PubMed

    Carter, E J; Preston, N W

    1984-08-01

    Fimbriae have been demonstrated on strains of Bordetella pertussis that possess agglutinogen 2 (types 1, 2, 3 and 1, 2), but not on those that lack it (types 1,3 and 1). This correlation between fimbriation and the presence of agglutinogen 2 has been found with fresh isolates from children and with laboratory strains that are virulent for mice. If fimbriae enhance the attachment of bacteria to mucosal cells, these findings offer an explanation for the predominance of serotypes 1,2,3 and 1,2 in non-vaccinated communities. The findings also suggest that agglutinogen 3 is not a fimbrial antigen, and because this is an essential component of fully effective whole-cell vaccine, a subcellular vaccine prepared from fimbriae alone may be inadequate. PMID:6146723

  1. Morphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa.

    PubMed Central

    Mattick, J S; Bills, M M; Anderson, B J; Dalrymple, B; Mott, M R; Egerton, J R

    1987-01-01

    Type 4 fimbriae are found in a range of pathogenic bacteria, including Bacteroides nodosus, Moraxella bovis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. The structural subunits of these fimbriae all contain a highly conserved hydrophobic amino-terminal sequence preceding a variable hydrophilic carboxy-terminal region. We show here that recombinant P. aeruginosa cells containing the B. nodosus fimbrial subunit gene under the control of a strong promoter (pL, from bacteriophage lambda) produced large amounts of fimbriae that were structurally and antigenically indistinguishable from those produced by B. nodosus. This was demonstrated by fimbrial isolation and purification, electrophoretic and Western transfer analyses, and immunogold labeling and electron microscopy. These results suggest that type 4 fimbriated bacteria use a common mechanism for fimbrial assembly and that the structural subunits are interchangeable, thereby providing a basis for the development of multivalent vaccines. Images PMID:2878919

  2. Agglutinogens and fimbriae of Bordetella pertussis.

    PubMed

    Ashworth, L A; Robinson, A; Funnell, S; Gorringe, A R; Irons, L I; Seabrook, R N

    1988-01-01

    Agglutinogen 2 (AGG2) of Bordetella pertussis is a fimbrial antigen and therefore a potential adhesin and acellular vaccine component. AGG2 was found to dissociate only under harsh conditions into the subunits of mol. wt. 22500 seen in SDS-PAGE. Results from studies of agglutinogen 3 (AGG3) are presented which confirm previous findings from this Laboratory that AGG3 is also a fimbrial protein but with a subunit mol. wt. of 22000. The amino acid sequence of AGG2, deduced from the nucleotide sequence of the gene encoding it, was used as a basis for synthesis of three peptides. Coupled to Keyhole Limpet Haemocyanin (KLH), the peptides were immunogenic in mice, inducing antibodies which bound well to homologous peptide in ELISA but poorly to intact fimbriae. Monoclonal and polyclonal serotype-specific antibodies failed to react significantly with the peptides or their KLH-conjugates. These results indicate that the synthetic peptides do not represent the serotype 2 epitope. Mice immunized with purified AGG2 or AGG3 were found to be protected against respiratory infection with B. pertussis. Results presented here indicate that this protection is, to a large extent, serotype-specific and that immunization of mice with AGG2 or AGG3 can lead to a change in serotype of the infecting strain. These results are analogous to findings from epidemiological studies of the protection induced in children by whole cell vaccines. They reaffirm the importance of both AGG2 and AGG3 as components of whole cell and acellular vaccines. PMID:2908520

  3. Structure and function of enterotoxigenic Escherichia coli fimbriae from differing assembly pathways.

    PubMed

    Mortezaei, Narges; Epler, Chelsea R; Shao, Paul P; Shirdel, Mariam; Singh, Bhupender; McVeigh, Annette; Uhlin, Bernt Eric; Savarino, Stephen J; Andersson, Magnus; Bullitt, Esther

    2015-01-01

    Pathogenic enterotoxigenic Escherichia coli (ETEC) are the major bacterial cause of diarrhea in young children in developing countries and in travelers, causing significant mortality in children. Adhesive fimbriae are a prime virulence factor for ETEC, initiating colonization of the small intestinal epithelium. Similar to other Gram-negative bacteria, ETEC express one or more diverse fimbriae, some assembled by the chaperone-usher pathway and others by the alternate chaperone pathway. Here, we elucidate structural and biophysical aspects and adaptations of each fimbrial type to its respective host niche. CS20 fimbriae are compared with colonization factor antigen I (CFA/I) fimbriae, which are two ETEC fimbriae assembled via different pathways, and with P-fimbriae from uropathogenic E. coli. Many fimbriae unwind from their native helical filament to an extended linear conformation under force, thereby sustaining adhesion by reducing load at the point of contact between the bacterium and the target cell. CFA/I fimbriae require the least force to unwind, followed by CS20 fimbriae and then P-fimbriae, which require the highest unwinding force. We conclude from our electron microscopy reconstructions, modeling and force spectroscopy data that the target niche plays a central role in the biophysical properties of fimbriae that are critical for bacterial pathophysiology. PMID:25355550

  4. Structure and function of Enterotoxigenic Escherichia coli fimbriae from differing assembly pathways

    PubMed Central

    Mortezaei, Narges; Epler, Chelsea R.; Shao, Paul P.; Shirdel, Mariam; Singh, Bhupender; McVeigh, Annette; Uhlin, Bernt Eric; Savarino, Stephen J.; Andersson, Magnus; Bullitt, Esther

    2014-01-01

    Pathogenic enterotoxigenic Escherichia coli (ETEC) are the major bacterial cause of diarrhea in young children in developing countries and in travelers, causing significant mortality in children. Adhesive fimbriae are a prime virulence factor for ETEC, initiating colonization of the small intestinal epithelium. Similar to other Gram-negative bacteria, ETEC express one or more diverse fimbriae, some assembled by the chaperone-usher pathway and others by the alternate chaperone pathway. Here we elucidate structural and biophysical aspects and adaptations of each fimbrial type to its respective host niche. CS20 fimbriae are compared to CFA/I fimbriae, which are two ETEC fimbriae assembled via different pathways, and to P-fimbriae from uropathogenic E. coli. Many fimbriae unwind from their native helical filament to an extended linear conformation under force, thereby sustaining adhesion by reducing load at the point of contact between the bacterium and the target cell. CFA/I fimbriae require the least force to unwind, followed by CS20 fimbriae and then P-fimbriae, which require the highest unwinding force. We conclude from our electron microscopy reconstructions, modeling, and force spectroscopy data that the target niche plays a central role in the biophysical properties of fimbriae that are critical for bacterial pathophysiology. PMID:25355550

  5. Production and characterization of recombinant pertactin, fimbriae 2 and fimbriae 3 from Bordetella pertussis

    PubMed Central

    2009-01-01

    Background Bordetella pertussis is a causative agent of pertussis or whooping cough in humans. Pertactin (Prn), fimbriae 2 (Fim2) and fimbriae 3 (Fim3) of B. pertussis are important virulence factors and immunogens which have been included in some acellular pertussis vaccines. In this present study, we cloned, expressed and purified Prn, Fim2 and Fim3, respectively. The immunogenicity and protective efficacy of the three recombinant proteins (rPrn, rFim2 and rFim3) were investigated in mouse model. Results Three recombinant proteins with amount of 12 to 25 mg/L were produced. Compared to the control mice only immunized with adjuvant, serum IgG antibody responses were significantly induced in the mice immunized with rPrn, rFim2 or rFim3 (P < 0.001 for all three proteins). Furthermore, T cell responses characteristic of increased production of IL-2 and TNF-α (only for rPrn) were elicited in the mice immunized with the three proteins (P < 0.05 for all three proteins). Immunization with rPrn, but not with rFim2 or rFim3, significantly enhanced clearance of bacteria in the lungs of mice after intranasal challenge with B. pertussis (P < 0.05). When tested in a lethal intracerebral infection model, certain protection was observed in mice immunized with rPrn. Conclusions We have developed an efficient method to produce large amounts of rPrn, rFim2, and rFim3 from B. pertussis. The three recombinant proteins induced both humoral and cellular immune responses in mice. Immunization with rPrn also conferred protection against pertussis in mouse infection models. Our results indicated that the recombinant proteins still retain their immunological properties and highlighted the potential of the recombinant proteins for the future development of the B. pertussis vaccines. PMID:20040101

  6. Fimbriae and determination of host species specificity of Bordetella bronchiseptica.

    PubMed Central

    Burns, E H; Norman, J M; Hatcher, M D; Bemis, D A

    1993-01-01

    A monoclonal antibody, designated CF8 and prepared against fimbrial protein enrichments of Bordetella bronchiseptica 110H, was determined by immunogold electron microscopy to bind to some but not all fimbrial filaments on intact bacterial cells. Comparison of the reactivity of this antibody with that of monoclonal antibody BPF2, which is specific for Bordetella pertussis serotype 2 fimbriae, indicated that CF8 recognizes an epitope similar to that recognized by BPF2. By Western blot (immunoblot), it was determined that monoclonal antibody CF8 does not react with proteins denatured by treatment with sodium dodecyl sulfate and beta-mercaptoethanol and by boiling for 5 min but that it does recognize fimbrial proteins in their native, nondenatured state. This antibody was used to compare fimbriae between strains of B. bronchiseptica isolated from different species. Strains from pigs, dogs, guinea pigs, and four other species were compared by an enzyme immunoassay. Strains isolated from pigs were found to express significantly more CF8-reactive and B. pertussis serotype 2 cross-reactive fimbriae than strains isolated from guinea pigs. Strains from dogs were more variable in reactivity than those from pigs or guinea pigs. The reactivity with antifimbrial monoclonal antibody CF8 did not correlate with enzyme electromorphotype but did correlate with the host species, suggesting a role for fimbriae in the determination of host species specificity of B. bronchiseptica. Images PMID:8102377

  7. Structure of CFA/I fimbriae from enterotoxigenic Escherichia coli

    PubMed Central

    Li, Yong-Fu; Poole, Steven; Nishio, Kazuya; Jang, Ken; Rasulova, Fatima; McVeigh, Annette; Savarino, Stephen J.; Xia, Di; Bullitt, Esther

    2009-01-01

    Adhesion pili (fimbriae) play a critical role in initiating the events that lead to intestinal colonization and diarrheal disease by enterotoxigenic Escherichia coli (ETEC), an E. coli pathotype that inflicts an enormous global disease burden. We elucidate atomic structures of an ETEC major pilin subunit, CfaB, from colonization factor antigen I (CFA/I) fimbriae. These data are used to construct models for 2 morphological forms of CFA/I fimbriae that are both observed in vivo: the helical filament into which it is typically assembled, and an extended, unwound conformation. Modeling and corroborative mutational data indicate that proline isomerization is involved in the conversion between these helical and extended forms. Our findings affirm the strong structural similarities seen between class 5 fimbriae (from bacteria primarily causing gastrointestinal disease) and class 1 pili (from bacteria that cause urinary, respiratory, and other infections) in the absence of significant primary sequence similarity. They also suggest that morphological and biochemical differences between fimbrial types, regardless of class, provide structural specialization that facilitates survival of each bacterial pathotype in its preferred host microenvironment. Last, we present structural evidence for bacterial use of antigenic variation to evade host immune responses, in that residues occupying the predicted surface-exposed face of CfaB and related class 5 pilins show much higher genetic sequence variability than the remainder of the pilin protein. PMID:19515814

  8. Partially Assembled K99 Fimbriae Are Required for Protection

    PubMed Central

    Ascón, Miguel A.; Ochoa-Repáraz, Javier; Walters, Nancy; Pascual, David W.

    2005-01-01

    Antibodies to K99 fimbriae afford protection to F5+ bovine enterotoxigenic Escherichia coli (ETEC). Previous studies show that murine dams immunized with Salmonella vaccine vectors stably expressing K99 fimbriae confer protection to ETEC-challenged neonatal pups. To begin to address adaptation of the K99 scaffold to display heterologous B- and T-cell epitopes, studies were conducted to determine how much of the assembled K99 fimbria is required to maintain protective immunity. Sequential deletions in the K99 gene clusters were made, resulting in diminished localization of the K99 fimbrial subunit in the outer membrane. As placement of the K99 fimbrial subunit became progressively contained within the vaccine vector, diminished immunoglobulin A (IgA) and IgG1 antibody titers, as well as diminished Th2-type cytokine responses, were observed in orally immunized mice. Deletion of fanGH, which greatly reduced the export of the fimbrial subunit to the outer membrane, showed only partial reduction in protective immunity. By contrast, deletion of fanDEFGH, which also reduced the export of the fimbrial subunit to the outer membrane but retained more subunit in the cytoplasm, resulted in protective immunity being dramatically reduced. Thus, these studies showed that retention of K99 fimbrial subunit as native fimbriae or with the deletion of fanGH is sufficient to confer protection. PMID:16239523

  9. Structure of CFA/I fimbriae from enterotoxigenic Escherichia coli

    SciTech Connect

    Li, Yong-Fu; Poole, Steven; Nishio, Kazuya; Jang, Ken; Rasulova, Fatima; McVeigh, Annette; Savarino, Stephen J.; Xia, Di; Bullitt, Esther

    2009-10-21

    Adhesion pili (fimbriae) play a critical role in initiating the events that lead to intestinal colonization and diarrheal disease by enterotoxigenic Escherichia coli (ETEC), an E. coli pathotype that inflicts an enormous global disease burden. We elucidate atomic structures of an ETEC major pilin subunit, CfaB, from colonization factor antigen I (CFA/I) fimbriae. These data are used to construct models for 2 morphological forms of CFA/I fimbriae that are both observed in vivo: the helical filament into which it is typically assembled, and an extended, unwound conformation. Modeling and corroborative mutational data indicate that proline isomerization is involved in the conversion between these helical and extended forms. Our findings affirm the strong structural similarities seen between class 5 fimbriae (from bacteria primarily causing gastrointestinal disease) and class 1 pili (from bacteria that cause urinary, respiratory, and other infections) in the absence of significant primary sequence similarity. They also suggest that morphological and biochemical differences between fimbrial types, regardless of class, provide structural specialization that facilitates survival of each bacterial pathotype in its preferred host microenvironment. Last, we present structural evidence for bacterial use of antigenic variation to evade host immune responses, in that residues occupying the predicted surface-exposed face of CfaB and related class 5 pilins show much higher genetic sequence variability than the remainder of the pilin protein.

  10. Fimbriae have distinguishable roles in Proteus mirabilis biofilm formation.

    PubMed

    Scavone, Paola; Iribarnegaray, Victoria; Caetano, Ana Laura; Schlapp, Geraldine; Härtel, Steffen; Zunino, Pablo

    2016-07-01

    Proteus mirabilis is one of the most common etiological agents of complicated urinary tract infections, especially those associated with catheterization. This is related to the ability of P. mirabilis to form biofilms on different surfaces. This pathogen encodes 17 putative fimbrial operons, the highest number found in any sequenced bacterial species so far. The present study analyzed the role of four P. mirabilis fimbriae (MR/P, UCA, ATF and PMF) in biofilm formation using isogenic mutants. Experimental approaches included migration over catheter, swimming and swarming motility, the semiquantitative assay based on adhesion and crystal violet staining, and biofilm development by immunofluorescence and confocal microscopy. Different assays were performed using LB or artificial urine. Results indicated that the different fimbriae contribute to the formation of a stable and functional biofilm. Fimbriae revealed particular associated roles. First, all the mutants showed a significantly reduced ability to migrate across urinary catheter sections but neither swimming nor swarming motility were affected. However, some mutants formed smaller biofilms compared with the wild type (MRP and ATF) while others formed significantly larger biofilms (UCA and PMF) showing different bioarchitecture features. It can be concluded that P. mirabilis fimbriae have distinguishable roles in the generation of biofilms, particularly in association with catheters. PMID:27091004

  11. Isolation and characterization of fimbriae from a sparsely fimbriated strain of Porphyromonas gingivalis.

    PubMed Central

    Sojar, H T; Hamada, N; Genco, R J

    1997-01-01

    Porphyromonas gingivalis W50 (ATCC 53978) possesses the gene for fimbriae; however, the surface-expressed fimbriae are sparse and have not been previously isolated and characterized. We purified fimbriae from strain W50 to homogeneity by ammonium sulfate precipitation and reverse-phase high-performance liquid chromatography [H. T. Sojar, N. Hamada, and R. J. Genco, Protein Expr. Purif. 9(1):49-52, 1997]. Negative staining of purified fimbriae viewed by electron microscopy revealed that the fimbriae were identical in diameter to fimbriae of other P. gingivalis strains, such as 2561, but were shorter in length. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the apparent molecular weight of isolated fimbrillin from strain W50 was found to be identical to that of the fimbrillin molecule of strain 2561. Unlike 2561 fimbriae, W50 fimbriae, under reducing condition, exhibited a monomeric structure on SDS-PAGE at room temperature. However, under nonreduced conditions, even at 100 degrees C, no monomer was observed. In immunoblot analysis as well as immunogold labeling of isolated fimbriae, polyclonal antibodies against 2561 fimbriae, as well as antibodies against peptide I (V-V-M-A-N-T-G-A-M-E-V-G-K-T-L-A-E-V-K-Cys) and peptide J (A-L-T-T-E-L-T-A-E-N-Q-E-A-A-G-L-I-M-T-A-E-P-Cys), reacted. However, antifimbrial antibodies against strain 2561 reacted very weakly compared to anti-peptide I and anti-peptide J. Negative staining of whole W50 cells, as well as immunogold electron microscopy with anti-peptide I and anti-peptide J, showed fimbriae shorter in length and very few in number compared to those of strain 2561. Purified fimbriae showed no hemagglutinating activity. Amino acid composition was very similar to that of previously reported fimbriae of the 2561 strain. PMID:9172351

  12. Purification of serotype 2 fimbriae of Bordetella pertussis and their identification as a mouse protective antigen.

    PubMed

    Zhang, J M; Cowell, J L; Steven, A C; Manclark, C R

    1985-01-01

    Fimbriae were removed from Bordetella pertussis by mechanical shearing and purified by successive precipitations with ammonium sulfate, pH 6.0 phosphate buffer, and magnesium chloride. Electron microscopy showed the purified fimbriae to be long filamentous structures (5 nm in diameter) which aggregated into bundles at pH 6.0. Sodium dodecyl sulfate gel electrophoresis of the purified fimbriae gave a single protein subunit with a molecular weight of 22,000. The purified fimbriae did not have hemagglutinating activity when assayed with a variety of erythrocytes and were shown to be antigenically and structurally distinct from the filamentous hemagglutinin of B. pertussis. The purified fimbriae were identified as serotype 2 agglutinogens as antibody to the purified fimbriae agglutinated B. pertussis strains serotyped as 1.2.4, 1.2.3, or 1.2.3.6, but did not agglutinate serotypes 1.3.6. Immunization of mice with the purified fimbriae (14 micrograms) protected them from a lethal respiratory infection with strain 18323 (agglutinogen serotype 1.2.3.4.6) or with strain 432 (serotype 1.3.6). Immunization of mice with purified fimbriae containing 0.02% LPF also protected them from a lethal intracerebral infection with 18323. The ED50 was about 13 micrograms. Fimbriae containing less than 0.005% LPF did not protect mice from intracerebral challenge. PMID:2872103

  13. Purification and characterization of fimbriae from Salmonella enteritidis.

    PubMed Central

    Feutrier, J; Kay, W W; Trust, T J

    1986-01-01

    A human isolate of Salmonella enteritidis which displayed strong pellicle formation during static broth culture and mannose-sensitive hemagglutination produced fimbriae which were morphologically indistinguishable from type 1 fimbriae of members of the family Enterobacteriaceae. Fimbrin was purified to homogeneity, and the apparent molecular weight (Mr, 14,400) was markedly lower than that reported for the type 1 fimbrin of Salmonella typhimurium (Mr, 22,100). This fimbrin contained 40% hydrophobic amino acids and lacked cysteine. The sequence of the N-terminal 64 amino acids was determined, and sequence alignment revealed that although the 18 N-terminal residues of the S. enteritidis molecule shared considerable homology with Escherichia coli and S. typhimurium type 1 fimbrins, the S. enteritidis fimbrin lacked a 6- to 9-residue terminal sequence present in the other type 1 fimbrins and, after residue 18, shared little homology with the E. coli sequence. Antibodies raised to the purified S. enteritidis fimbrin bound to surface-exposed conformational epitopes on the native fimbriae and displayed pronounced serospecificity. These antibodies were used in the isolation of a nonfimbriated Tn10 insertion mutant which was unable to hemagglutinate. Images PMID:2875990

  14. Structure, Function, and Assembly of Type 1 Fimbriae

    NASA Astrophysics Data System (ADS)

    Knight, Stefan D.; Bouckaert, Julie

    Bacterial infections constitute a major global health problem, acutely accentuated by the rapid spread of antibiotic resistant bacterial strains. The widespread need for bacteria to attach - adhere - to target cells before they can initiate an infection may be used to advantage by targeting the bacterial adhesion tools such as pili and fimbriae for development of novel anti-bacterial vaccines and drugs. Type 1 fimbriae are widely expressed by Escherichia coli. and are used by uropathogenic strains to mediate attachment to specific niches in the urinary tract. These fimbriae belong to a class of fibrillar adhesion organelles assembled through the chaperone/usher pathway, one of the terminal branches of the general secretion pathway in Gram-negative bacteria. Our understanding of the assembly, structure and function of these structures has evolved significantly over the last decade. Here, we summarize current understanding of the function and biogenesis of fibrillar adhesion organelles, and provide some examples of recent progress towards interfering with bacterial adhesion as a means to prevent infection.

  15. Biomechanical and Structural Features of CS2 Fimbriae of Enterotoxigenic Escherichia coli

    PubMed Central

    Mortezaei, Narges; Singh, Bhupender; Zakrisson, Johan; Bullitt, Esther; Andersson, Magnus

    2015-01-01

    Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrhea worldwide, and infection of children in under-developed countries often leads to high mortality rates. Isolated ETEC expresses a plethora of colonization factors (fimbriae/pili), of which CFA/I and CFA/II, which are assembled via the alternate chaperone pathway (ACP), are among the most common. Fimbriae are filamentous structures whose shafts are primarily composed of helically arranged single pilin-protein subunits, with a unique biomechanical ability to unwind and rewind. A sustained ETEC infection, under adverse conditions of dynamic shear forces, is primarily attributed to this biomechanical feature of ETEC fimbriae. Recent understanding about the role of fimbriae as virulence factors points to an evolutionary adaptation of their structural and biomechanical features. In this work, we investigated the biophysical properties of CS2 fimbriae from the CFA/II group. Homology modeling of its major structural subunit, CotA, reveals structural clues related to the niche in which they are expressed. Using optical-tweezers force spectroscopy, we found that CS2 fimbriae unwind at a constant force of 10 pN and have a corner velocity (i.e., the velocity at which the force required for unwinding rises exponentially with increased speed) of 1300 nm/s. The biophysical properties of CS2 fimbriae assessed in this work classify them into a low-force unwinding group of fimbriae together with the CFA/I and CS20 fimbriae expressed by ETEC strains. The three fimbriae are expressed by ETEC, colonize in similar gut environments, and exhibit similar biophysical features, but differ in their biogenesis. Our observation suggests that the environment has a strong impact on the biophysical characteristics of fimbriae expressed by ETEC. PMID:26153701

  16. Biomechanical and structural features of CS2 fimbriae of enterotoxigenic Escherichia coli.

    PubMed

    Mortezaei, Narges; Singh, Bhupender; Zakrisson, Johan; Bullitt, Esther; Andersson, Magnus

    2015-07-01

    Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrhea worldwide, and infection of children in under-developed countries often leads to high mortality rates. Isolated ETEC expresses a plethora of colonization factors (fimbriae/pili), of which CFA/I and CFA/II, which are assembled via the alternate chaperone pathway (ACP), are among the most common. Fimbriae are filamentous structures whose shafts are primarily composed of helically arranged single pilin-protein subunits, with a unique biomechanical ability to unwind and rewind. A sustained ETEC infection, under adverse conditions of dynamic shear forces, is primarily attributed to this biomechanical feature of ETEC fimbriae. Recent understanding about the role of fimbriae as virulence factors points to an evolutionary adaptation of their structural and biomechanical features. In this work, we investigated the biophysical properties of CS2 fimbriae from the CFA/II group. Homology modeling of its major structural subunit, CotA, reveals structural clues related to the niche in which they are expressed. Using optical-tweezers force spectroscopy, we found that CS2 fimbriae unwind at a constant force of 10 pN and have a corner velocity (i.e., the velocity at which the force required for unwinding rises exponentially with increased speed) of 1300 nm/s. The biophysical properties of CS2 fimbriae assessed in this work classify them into a low-force unwinding group of fimbriae together with the CFA/I and CS20 fimbriae expressed by ETEC strains. The three fimbriae are expressed by ETEC, colonize in similar gut environments, and exhibit similar biophysical features, but differ in their biogenesis. Our observation suggests that the environment has a strong impact on the biophysical characteristics of fimbriae expressed by ETEC. PMID:26153701

  17. Comparison of type 2 and type 6 fimbriae of Bordetella pertussis by using agglutinating monoclonal antibodies.

    PubMed

    Li, Z M; Brennan, M J; David, J L; Carter, P H; Cowell, J L; Manclark, C R

    1988-12-01

    Two types of fimbriae have been identified on the pathogenic gram-negative organism Bordetella pertussis. Monoclonal antibodies to these fimbriae were produced to better understand the role of fimbriae as serotype-specific agglutinogens and to investigate the antigenic relationship between these fimbriae. Three monoclonal antibodies were identified that specifically agglutinated B. pertussis cells containing the U.S. Reference Factor 2 agglutinogen, and six monoclonal antibodies were produced that agglutinated only those strains containing the U.S. Reference Factor 6 agglutinogen. Indirect immunofluorescence studies and immunogold electron microscopy demonstrated that these monoclonal antibodies bind to an outer membrane component on serotype-specific strains of B. pertussis. All of the monoclonal antibodies reacted with native or partially assembled type-specific fimbriae but not with monomeric fimbrial subunits as indicated by Western blot (immunoblot) analysis. The fimbrial agglutinogens recognized by the monoclonal antibodies were also uniquely reactive with either U.S. Reference Factor 2 or 6 antiserum (Eldering agglutinogen 2 or 6 polyclonal antiserum) in an indirect ELISA. No cross-reactivity of the monoclonal antibodies with the unrelated fimbriae was observed in any of the comparative immunological studies. Some of the monoclonal antibodies agglutinated certain strains of B. bronchiseptica, suggesting that this closely related species can contain antigenically similar fimbriae. These monoclonal antibodies should prove useful for further structural and functional analysis of Bordetella fimbriae and for studies on the role that these antigens play in prevention of infection and disease. PMID:2903125

  18. Comparison of type 2 and type 6 fimbriae of Bordetella pertussis by using agglutinating monoclonal antibodies.

    PubMed Central

    Li, Z M; Brennan, M J; David, J L; Carter, P H; Cowell, J L; Manclark, C R

    1988-01-01

    Two types of fimbriae have been identified on the pathogenic gram-negative organism Bordetella pertussis. Monoclonal antibodies to these fimbriae were produced to better understand the role of fimbriae as serotype-specific agglutinogens and to investigate the antigenic relationship between these fimbriae. Three monoclonal antibodies were identified that specifically agglutinated B. pertussis cells containing the U.S. Reference Factor 2 agglutinogen, and six monoclonal antibodies were produced that agglutinated only those strains containing the U.S. Reference Factor 6 agglutinogen. Indirect immunofluorescence studies and immunogold electron microscopy demonstrated that these monoclonal antibodies bind to an outer membrane component on serotype-specific strains of B. pertussis. All of the monoclonal antibodies reacted with native or partially assembled type-specific fimbriae but not with monomeric fimbrial subunits as indicated by Western blot (immunoblot) analysis. The fimbrial agglutinogens recognized by the monoclonal antibodies were also uniquely reactive with either U.S. Reference Factor 2 or 6 antiserum (Eldering agglutinogen 2 or 6 polyclonal antiserum) in an indirect ELISA. No cross-reactivity of the monoclonal antibodies with the unrelated fimbriae was observed in any of the comparative immunological studies. Some of the monoclonal antibodies agglutinated certain strains of B. bronchiseptica, suggesting that this closely related species can contain antigenically similar fimbriae. These monoclonal antibodies should prove useful for further structural and functional analysis of Bordetella fimbriae and for studies on the role that these antigens play in prevention of infection and disease. Images PMID:2903125

  19. Antigenic relationship between serotype-specific agglutinogen and fimbriae of Bordetella pertussis.

    PubMed

    Ashworth, L A; Irons, L I; Dowsett, A B

    1982-09-01

    The widely held view that the filamentous hemagglutinin (FHA) of Bordetella pertussis is derived from fimbriae (pili) is not supported by studies of fimbriation and FHA content of the organism. Fimbriae do not label specifically with antibody to FHA in immuno-electron microscopy but do label with antibody to serotype-specific agglutinogen. PMID:6127315

  20. Identification, purification, and characterization of the type 4 fimbriae of Pasteurella multocida.

    PubMed Central

    Ruffolo, C G; Tennent, J M; Michalski, W P; Adler, B

    1997-01-01

    The presence of fimbriae on Pasteurella multocida has been reported, but there have been no prior studies aimed at conclusively characterizing these structures. We now report on the identification and characterization of type 4 fimbriae on serogroup A, B, and D strains of P. multocida. Under microaerophilic conditions P. multocida showed an increased expression of the fimbriae, which were observed to form bundles. Fimbriae purified by high-performance reverse-phase liquid chromatography constituted a single 18-kDa subunit, the first 21 amino acids of which shared very high similarity with the N-terminal amino acid sequence of other type 4 fimbrial subunits. Antiserum against the P. multocida 18-kDa protein immunostained the type 4 fimbrial subunit of Moraxella bovis and Dichelobacter nodosus. Based on these observations we conclude that P. multocida possesses type 4 fimbriae and have designated the P. multocida fimbrial subunit PtfA. PMID:8975936

  1. Pseudocatabolite repression of type 1 fimbriae of Escherichia coli.

    PubMed

    Eisenstein, B I; Dodd, D C

    1982-09-01

    Previous work on the control of fimbriation in bacteria has demonstrated the importance of environmental factors such as static versus shaking broth and the absence versus the presence of glucose on the degree of fimbriation. When the Pil+ K-12 strain of Escherichia coli CSH50 was grown in static broth, the bacteria grown with glucose were less fimbriate (as determined by electron microscopy) than those grown without glucose. In contrast, a derivative, the pil-lac operon fusion strain VL361, gave off similar proportions of Lac+ and Lac- colonies when grown with or without glucose. Introduction of delta cya into either CSH50 or VL361 did not affect synthesis of either fimbriae or beta-galactosidase, respectively. When total synthesis of fimbriae by strain CSH50 was assayed, using an enzyme-linked immunosorbent inhibition test, glucose-grown bacteria made less antigen when they were grown in static broth but not when they were grown in shaking broth. When results are taken together, we interpret them as showing that glucose does not suppress fimbrial synthesis by classic catabolite repression but rather merely prevents the outgrowth or fimbriate bacteria in static broth. PMID:6125501

  2. Characterization of 17 chaperone-usher fimbriae encoded by Proteus mirabilis reveals strong conservation

    PubMed Central

    Kuan, Lisa; Schaffer, Jessica N.; Zouzias, Christos D.

    2014-01-01

    Proteus mirabilis is a Gram-negative enteric bacterium that causes complicated urinary tract infections, particularly in patients with indwelling catheters. Sequencing of clinical isolate P. mirabilis HI4320 revealed the presence of 17 predicted chaperone-usher fimbrial operons. We classified these fimbriae into three groups by their genetic relationship to other chaperone-usher fimbriae. Sixteen of these fimbriae are encoded by all seven currently sequenced P. mirabilis genomes. The predicted protein sequence of the major structural subunit for 14 of these fimbriae was highly conserved (≥95 % identity), whereas three other structural subunits (Fim3A, UcaA and Fim6A) were variable. Further examination of 58 clinical isolates showed that 14 of the 17 predicted major structural subunit genes of the fimbriae were present in most strains (>85 %). Transcription of the predicted major structural subunit genes for all 17 fimbriae was measured under different culture conditions designed to mimic conditions in the urinary tract. The majority of the fimbrial genes were induced during stationary phase, static culture or colony growth when compared to exponential-phase aerated culture. Major structural subunit proteins for six of these fimbriae were detected using MS of proteins sheared from the surface of broth-cultured P. mirabilis, demonstrating that this organism may produce multiple fimbriae within a single culture. The high degree of conservation of P. mirabilis fimbriae stands in contrast to uropathogenic Escherichia coli and Salmonella enterica, which exhibit greater variability in their fimbrial repertoires. These findings suggest there may be evolutionary pressure for P. mirabilis to maintain a large fimbrial arsenal. PMID:24809384

  3. How type 1 fimbriae help Escherichia coli to evade extracellular antibiotics

    PubMed Central

    Avalos Vizcarra, Ima; Hosseini, Vahid; Kollmannsberger, Philip; Meier, Stefanie; Weber, Stefan S.; Arnoldini, Markus; Ackermann, Martin; Vogel, Viola

    2016-01-01

    To survive antibiotics, bacteria use two different strategies: counteracting antibiotic effects by expression of resistance genes or evading their effects e.g. by persisting inside host cells. Since bacterial adhesins provide access to the shielded, intracellular niche and the adhesin type 1 fimbriae increases bacterial survival chances inside macrophages, we asked if fimbriae also influenced survival by antibiotic evasion. Combined gentamicin survival assays, flow cytometry, single cell microscopy and kinetic modeling of dose response curves showed that type 1 fimbriae increased the adhesion and internalization by macrophages. This was caused by strongly decreased off-rates and affected the number of intracellular bacteria but not the macrophage viability and morphology. Fimbriae thus promote antibiotic evasion which is particularly relevant in the context of chronic infections. PMID:26728082

  4. Essential immunogens in human pertussis: the role of fimbriae.

    PubMed

    Preston, N W

    1985-01-01

    After the decline in pertussis vaccination in Britain from the mid-1970s, isolates possessing agglutinogen 2 (types 1.2.3 and 1.2) have replaced type 1.3 as the predominant serotypes. These agglutinogen-2 strains are fimbriate, and their predominance in non-vaccinated communities may result from enhanced attachment to mucosal cells. However, type 1.3 cells are not fimbriate; and, because agglutinogen 3 is essential in fully effective whole-cell vaccine, a subcellular vaccine prepared from fimbriae alone (agglutinogen 2) would probably be inadequate. The mouse can be killed with type 1 strains, devoid of agglutinogens 2 and 3, and is therefore not a suitable model for ensuring that pertussis vaccine contains these essential immunogens. PMID:2872099

  5. Proteus mirabilis ambient-temperature fimbriae: cloning and nucleotide sequence of the aft gene cluster.

    PubMed Central

    Massad, G; Fulkerson, J F; Watson, D C; Mobley, H L

    1996-01-01

    Uropathogenic Proteus mirabilis produces at least four types of fimbriae. Amino acid sequences from two peptides, derived by tryptic digestion of the structural subunit of one type of these fimbriae, the ambient-temperature fimbriae, were determined: NVVPGQPSSTQ and LIEGENQLNYNA. PCR primers, based on these sequences and that of the N terminus, were used to amplify a 359-bp fragment. A cosmid clone, isolated from a P. mirabilis genomic library by hybridization with the 359-bp PCR product, was used to determine the nucleotide sequence of the atf gene cluster. A 3,903-bp region encodes three polypeptides: AtfA, the structural subunit; AtfB, the chaperone; and AtfC, the outer membrane molecular usher. No fimbria-related genes are evident either 5' or 3' to the three contiguous genes. AtfA demonstrates significant amino acid sequence identity with type 1 major fimbrial subunits of several enteric species. The 359-bp PCR product hybridized strongly with all Proteus isolates (n = 9) and 25% of 355 Escherichia coli isolates but failed to hybridize with any of 26 isolates among nine other uropathogenic species. Ambient-temperature fimbriae of P. mirabilis may represent a novel type of fimbriae of enteric species. PMID:8926119

  6. Role of Thin Fimbriae in Adherence and Growth of Acinetobacter calcoaceticus RAG-1 on Hexadecane.

    PubMed

    Rosenberg, M; Bayer, E A; Delarea, J; Rosenberg, E

    1982-10-01

    Acinetobacter calcoaceticus RAG-1, a hydrocarbon-degrading bacterium which adheres avidly to hydrocarbons and other hydrophobic surfaces, possesses numerous thin fimbriae (ca. 3.5-nm diameter) on the cell surface. MR-481, a nonadherent mutant of RAG-1 which is unable to grow on hexadecane under conditions of limited emulsification and low initial cell density, lacks these fimbriae. Prolonged incubation of MR-481 in hexadecane medium enriched for partial adherence revertants. The reappearance of thin fimbriae was observed in all such revertant strains. RAG-1 cells and partial revertant strains were agglutinated in the presence of antibody, whereas MR-481 cells were not. Another mutant, AB15, which was previously isolated on the basis of its nonagglutinability in the presence of antibody, also lacked thin fimbriae and was conditionally nonadherent. Furthermore, strain AB15 was unable to grow on hexadecane medium. Adherence of RAG-1 cells to hexadecane was considerably reduced after shearing treatment. The material removed from the cell surface by shearing of RAG-1 and the partial revertant strains yielded a single antigenic band in RAG-1 and partial revertant strains, as observed by crossed immunoelectrophoresis. This band was absent in both fimbriae-less mutants, MR-481 and AB15. The data demonstrate that the thin fimbriae of RAG-1 (i) are a major factor in adherence to polystyrene and hydrocarbon, (ii) may be crucial in enabling growth of cells on hexadecane, and (iii) constitute the major cell surface agglutinogen. PMID:16346118

  7. Role of Thin Fimbriae in Adherence and Growth of Acinetobacter calcoaceticus RAG-1 on Hexadecane

    PubMed Central

    Rosenberg, Mel; Bayer, Edward A.; Delarea, Jacob; Rosenberg, Eugene

    1982-01-01

    Acinetobacter calcoaceticus RAG-1, a hydrocarbon-degrading bacterium which adheres avidly to hydrocarbons and other hydrophobic surfaces, possesses numerous thin fimbriae (ca. 3.5-nm diameter) on the cell surface. MR-481, a nonadherent mutant of RAG-1 which is unable to grow on hexadecane under conditions of limited emulsification and low initial cell density, lacks these fimbriae. Prolonged incubation of MR-481 in hexadecane medium enriched for partial adherence revertants. The reappearance of thin fimbriae was observed in all such revertant strains. RAG-1 cells and partial revertant strains were agglutinated in the presence of antibody, whereas MR-481 cells were not. Another mutant, AB15, which was previously isolated on the basis of its nonagglutinability in the presence of antibody, also lacked thin fimbriae and was conditionally nonadherent. Furthermore, strain AB15 was unable to grow on hexadecane medium. Adherence of RAG-1 cells to hexadecane was considerably reduced after shearing treatment. The material removed from the cell surface by shearing of RAG-1 and the partial revertant strains yielded a single antigenic band in RAG-1 and partial revertant strains, as observed by crossed immunoelectrophoresis. This band was absent in both fimbriae-less mutants, MR-481 and AB15. The data demonstrate that the thin fimbriae of RAG-1 (i) are a major factor in adherence to polystyrene and hydrocarbon, (ii) may be crucial in enabling growth of cells on hexadecane, and (iii) constitute the major cell surface agglutinogen. Images PMID:16346118

  8. Construction of an MR/P fimbrial mutant of Proteus mirabilis: role in virulence in a mouse model of ascending urinary tract infection.

    PubMed Central

    Bahrani, F K; Massad, G; Lockatell, C V; Johnson, D E; Russell, R G; Warren, J W; Mobley, H L

    1994-01-01

    Proteus mirabilis, a cause of acute pyelonephritis, produces at least four types of fimbriae, including MR/P (mannose-resistant/Proteus-like) fimbriae. To investigate the contribution of MR/P fimbriae to colonization of the urinary tract, we constructed an MR/P fimbrial mutant by allelic exchange. A 4.2-kb BamHI fragment carrying the mrpA gene was subcloned into a mobilizable plasmid, pSUP202. A 1.3-kb Kanr cassette was inserted into the mrpA open reading frame, and the construct was transferred to the parent P. mirabilis strain by conjugation. Following passage on nonselective medium, 1 of 500 transconjugants screened was found to have undergone allelic exchange as demonstrated by Southern blot. Colony immunoblot, Western immunoblot, and immunogold labeling with a monoclonal antibody to MR/P fimbriae revealed that MrpA was not expressed. Complementation with cloned mrpA restored MR/P expression as shown by hemagglutination, Western blot, and immunogold electron microscopy. To assess virulence, we challenged 40 CBA mice transurethrally with 10(7) CFU of wild-type or mutant strains. After 1 week, geometric means of log10 CFU per milliliter of urine or per gram of bladder or kidney for the wild-type and mutant strains were as follows: urine, 7.79 (wild type) versus 7.02 (mutant) (P = 0.035); bladder, 6.22 versus 4.78 (P = 0.019); left kidney, 5.02 versus 3.31 (P = 0.009); and right kidney, 5.28 versus 4.46 (P = 0.039). Mice challenged with the wild-type strain showed significantly more severe renal damage than did mice challenged with the MR/P-negative mutant (P = 0.007). We conclude that MR/P fimbriae contribute significantly to colonization of the urinary tract and increase the risk of development of acute pyelonephritis. Images PMID:7913698

  9. In vivo phase variation of MR/P fimbrial gene expression in Proteus mirabilis infecting the urinary tract.

    PubMed

    Zhao, H; Li, X; Johnson, D E; Blomfield, I; Mobley, H L

    1997-03-01

    Proteus mirabilis, associated with complicated urinary tract infection, expresses mannose-resistant/Proteus-like (MR/P) fimbriae. Expression of these surface structures, which mediate haemagglutination and have a demonstrated role in virulence, undergoes phase variation. By DNA sequence analysis, a 252 bp invertible element was found in the intergenic region between mrpl, the putative site-specific recombinase gene, and mrpA, the primary structural subunit gene. The invertible segment is flanked by identical 21 bp inverted repeats and the presumptive half-sites for recombinase binding show homology to those recognized by FimB and FimE encoded by the Escherichia coli fim (Type 1 fimbriae) gene cluster. When amplified by the polymerase chain reaction (PCR) from static broth cultures expressing MR/P fimbriae, the switch region was found in both ON and OFF positions. When PCR was used to amplify agar cultures which do not express the fimbriae, the switch region was OFF only. A canonical sigma 70 promoter inside the invertible element drives the transcription of mrpA when in the ON position; in the OFF position it is directed away from mrpA but does not appear to drive expression of mrpI. The mrpI gene was able to confer inversion of the mrp switch region in trans from both ON to OFF and OFF to ON. To examine the position of the switch in vivo, urine, bladder, and kidneys from mice transurethrally infected with P. mirabilis were used to prepare template DNA for PCR amplification. In the absence of urolithiasis (urease-mediated stone formation), the switch was found 100% in the ON position, a condition never observed following in vitro culture. We conclude that MR/P phase variation is regulated at the transcriptional level by the action of MrpI on an invertible element and that there is strong selective pressure for the expression of MR/P fimbriae in vivo. PMID:9076737

  10. Role of overexpressed CFA/I fimbriae in bacterial swimming

    NASA Astrophysics Data System (ADS)

    Cao, Ling; Suo, Zhiyong; Lim, Timothy; Jun, SangMu; Deliorman, Muhammedin; Riccardi, Carol; Kellerman, Laura; Avci, Recep; Yang, Xinghong

    2012-06-01

    Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 < H683-pC < H683-pY. In the liquid culture media, H683-pC2 cells precipitated to the bottom of the tube, while those of H683-pY did not. In situ imaging revealed that H683-pC2 bacilli tended to auto-agglutinate within the semisolid agar, while H683-pY bacilli did not. When the cfaBE fimbrial fiber encoding genes were deleted from pC2, the new plasmid, pC2(-), significantly recovered bacterial swimming capability. Our study highlights the negative impact of overexpressed CFA/I fimbriae on bacterial swimming motility.

  11. Role of overexpressed CFA/I fimbriae in bacterial swimming.

    PubMed

    Cao, Ling; Suo, Zhiyong; Lim, Timothy; Jun, Sangmu; Deliorman, Muhammedin; Riccardi, Carol; Kellerman, Laura; Avci, Recep; Yang, Xinghong

    2012-06-01

    Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 < H683-pC < H683-pY. In the liquid culture media, H683-pC2 cells precipitated to the bottom of the tube, while those of H683-pY did not. In situ imaging revealed that H683-pC2 bacilli tended to auto-agglutinate within the semisolid agar, while H683-pY bacilli did not. When the cfaBE fimbrial fiber encoding genes were deleted from pC2, the new plasmid, pC2(-), significantly recovered bacterial swimming capability. Our study highlights the negative impact of overexpressed CFA/I fimbriae on bacterial swimming motility. PMID:22562964

  12. Dimethylsulfoniopropionate (DMSP) Increases Survival of Larval Sablefish, Anoplopoma fimbria.

    PubMed

    Lee, Jonathan S F; Poretsky, Rachel S; Cook, Matthew A; Reyes-Tomassini, Jose J; Berejikian, Barry A; Goetz, Frederick W

    2016-06-01

    High concentrations of dimethylsulfoniopropionate (DMSP), a chemical compound released by lysed phytoplankton, may indicate high rates of grazing by zooplankton and may thus be a foraging cue for planktivorous fishes. Previous studies have shown that some planktivorous fishes and birds aggregate or alter locomotory behavior in response to this chemical cue, which is likely adaptive because it helps them locate prey. These behavioral responses have been demonstrated in juveniles and adults, but no studies have tested for effects on larval fish. Larvae suffer from high mortality rates and are vulnerable to starvation. While larvae are generally thought to be visual predators, they actually have poor vision and cryptic prey. Thus, larval fish should benefit from a chemical cue that provides information on prey abundance. We reared larval sablefish, Anoplopoma fimbria, for one week and supplemented feedings with varying concentrations of DMSP to test the hypothesis that DMSP affects larval survival. Ecologically relevant DMSP concentrations increased larval survival by up to 70 %, which has implications for production in aquaculture and recruitment in nature. These results provide a new tool for increasing larval production in aquaculture and also suggest that larvae may use DMSP as an olfactory cue. The release of DMSP may be a previously unappreciated mechanism through which phytoplankton affect larval survival and recruitment. PMID:27306913

  13. Distinct Mutations Led to Inactivation of Type 1 Fimbriae Expression in Shigella spp.

    PubMed Central

    Bravo, Verónica; Puhar, Andrea; Sansonetti, Philippe; Parsot, Claude; Toro, Cecilia S.

    2015-01-01

    Shigella spp. are responsible for bacillary dysentery in humans. The acquisition or the modification of the virulence plasmid encoding factors promoting entry of bacteria into and dissemination within epithelial cells was a critical step in the evolution of these bacteria from their Escherichia coli ancestor(s). Incorporation of genomic islands (GI) and gene inactivation also shaped interactions between these pathogens and their human host. Sequence analysis of the GI inserted next to the leuX tRNA gene in S. boydii, S. dysenteriae, S. flexneri, S. sonnei and enteroinvasive E. coli (EIEC) suggests that this region initially carried the fec, yjhATS and fim gene clusters. The fim cluster encoding type I fimbriae is systematically inactivated in both reference strains and clinical isolates and distinct mutations are responsible for this inactivation in at least three phylogenetic groups. To investigate consequences of the presence of fimbriae on the outcome of the interaction of Shigella with host cells, we used a S. flexneri strain harboring a plasmid encoding the E. coli fim operon. Production of fimbriae by this recombinant strain increased the ability of bacteria to adhere to and enter into epithelial cells and had no effect on their ability to disseminate from cell to cell. The observations that production of type I fimbriae increases invasion of epithelial cells and that independent mutations abolish fimbriae production in Shigella suggest that these mutations correspond to pathoadaptive events. PMID:25811616

  14. Effective assembly of fimbriae in Escherichia coli depends on the translocation assembly module nanomachine.

    PubMed

    Stubenrauch, Christopher; Belousoff, Matthew J; Hay, Iain D; Shen, Hsin-Hui; Lillington, James; Tuck, Kellie L; Peters, Kate M; Phan, Minh-Duy; Lo, Alvin W; Schembri, Mark A; Strugnell, Richard A; Waksman, Gabriel; Lithgow, Trevor

    2016-01-01

    Outer membrane proteins are essential for Gram-negative bacteria to rapidly adapt to changes in their environment. Intricate remodelling of the outer membrane proteome is critical for bacterial pathogens to survive environmental changes, such as entry into host tissues(1-3). Fimbriae (also known as pili) are appendages that extend up to 2 μm beyond the cell surface to function in adhesion for bacterial pathogens, and are critical for virulence. The best-studied examples of fimbriae are the type 1 and P fimbriae of uropathogenic Escherichia coli, the major causative agent of urinary tract infections in humans. Fimbriae share a common mode of biogenesis, orchestrated by a molecular assembly platform called 'the usher' located in the outer membrane. Although the mechanism of pilus biogenesis is well characterized, how the usher itself is assembled at the outer membrane is unclear. Here, we report that a rapid response in usher assembly is crucially dependent on the translocation assembly module. We assayed the assembly reaction for a range of ushers and provide mechanistic insight into the β-barrel assembly pathway that enables the rapid deployment of bacterial fimbriae. PMID:27572967

  15. Distinct mutations led to inactivation of type 1 fimbriae expression in Shigella spp.

    PubMed

    Bravo, Verónica; Puhar, Andrea; Sansonetti, Philippe; Parsot, Claude; Toro, Cecilia S

    2015-01-01

    Shigella spp. are responsible for bacillary dysentery in humans. The acquisition or the modification of the virulence plasmid encoding factors promoting entry of bacteria into and dissemination within epithelial cells was a critical step in the evolution of these bacteria from their Escherichia coli ancestor(s). Incorporation of genomic islands (GI) and gene inactivation also shaped interactions between these pathogens and their human host. Sequence analysis of the GI inserted next to the leuX tRNA gene in S. boydii, S. dysenteriae, S. flexneri, S. sonnei and enteroinvasive E. coli (EIEC) suggests that this region initially carried the fec, yjhATS and fim gene clusters. The fim cluster encoding type I fimbriae is systematically inactivated in both reference strains and clinical isolates and distinct mutations are responsible for this inactivation in at least three phylogenetic groups. To investigate consequences of the presence of fimbriae on the outcome of the interaction of Shigella with host cells, we used a S. flexneri strain harboring a plasmid encoding the E. coli fim operon. Production of fimbriae by this recombinant strain increased the ability of bacteria to adhere to and enter into epithelial cells and had no effect on their ability to disseminate from cell to cell. The observations that production of type I fimbriae increases invasion of epithelial cells and that independent mutations abolish fimbriae production in Shigella suggest that these mutations correspond to pathoadaptive events. PMID:25811616

  16. P-fimbriae in the presence of anti-PapA antibodies: new insight of antibodies action against pathogens

    NASA Astrophysics Data System (ADS)

    Mortezaei, Narges; Singh, Bhupender; Bullitt, Esther; Uhlin, Bernt Eric; Andersson, Magnus

    2013-12-01

    Uropathogenic strains of Escherichia coli establish urinary tract infections by attaching to host epithelial cells using adhesive organelles called fimbriae. Fimbriae are helix-like structures with a remarkable adaptability, offering safeguarding for bacteria exposed to changing fluid forces in the urinary tract. We challenged this property of P-fimbriae by cross-linking their subunits with shaft-specific antibodies and measuring the corresponding force response at a single organelle level. Our data show compromised extension and rewinding of P-fimbriae in the presence of antibodies and reduced fimbrial elasticity, which are important properties of fimbriae contributing to the ability of bacteria to cause urinary tract infections. The reduced elasticity found by cross-linking fimbrial subunits could thus be another assignment for antibodies; in addition to marking bacteria as foreign, antibodies physically compromise fimbrial function. We suggest that our assay and results will be a starting point for further investigations aimed at inhibiting sustained bacterial adhesion by antibodies.

  17. Functional differences of Porphyromonas gingivalis Fimbriae in determining periodontal disease pathogenesis: a literature review

    PubMed Central

    Contreras, Adolfo

    2013-01-01

    Porphyromonas gingivalis is implicated in chronic and aggressive periodontitis. This bacterium has numerous virulence factors and one is the Fimbriae, which is quite important for bacterial colonization. Fimbriae are appendices that anchor to the bacterial wall and are comprised of the protein FimBriline encoded by the FimA gene. Thus far, six genotypes have been identified, FimA I to V and Ib. Genotypes II and IV are associated with periodontal disease, while genotype I is related to gingival health. Genotype identification of P. gingivalis FimA in periodontitis would be important to confirm the pathogenic genotypes and to establish risk at population level. This review is about the P. gingivalis FimA genotype prevalence worldwide. A systematic search using Pubmed, Hinary, and Science Direct within the following descriptors: Porphyromonas gingivalis, bacterial adhesion, periodontitis, Fimbriae, FimA, genotipification was performed to April 2011. PMID:24892323

  18. Immuno-electronmicroscopy of fimbriae-like structures on Bordetella pertussis serotype 1.3.

    PubMed

    Fredriksen, J H; Namork, E; Frøholm, L O

    1988-04-01

    Fimbriae-like filaments were demonstrated on the surface of Bordetella pertussis, serotype 1.3, by negative staining and electronmicroscopy. Immunoelectronmicroscopy with a monoclonal antibody specific for strains possessing agglutinogen 3, and colloidal gold, gave strong labelling of these structures. However, incubation with adsorbed polyclonal anti-agglutinogen 3 serum gave only weak labelling of the distal parts of the filaments and of the bacterial surface. The different binding patterns of the two antisera suggested that the epitopes involved were dissimilar. Thus, agglutinogen 3, as defined by conventional adsorbed sera, appeared to be associated with the fimbriae-like structures but was not necessarily identical to the fimbrial subunit protein. The monoclonal antibody, however was more likely directed against the subunits of the fimbriae-like structures on serotype 1.3 bacteria. PMID:2895814

  19. Streptococcus salivarius Fimbriae Are Composed of a Glycoprotein Containing a Repeated Motif Assembled into a Filamentous Nondissociable Structure

    PubMed Central

    Lévesque, Céline; Vadeboncoeur, Christian; Chandad, Fatiha; Frenette, Michel

    2001-01-01

    Streptococcus salivarius, a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of S. salivarius fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of S. salivarius ATCC 25975) with glass beads. Preliminary experiments showed that S. salivarius fimbriae did not dissociate when they were incubated at 100°C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 × 106 to 40 × 106 Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 μm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that S. salivarius fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/φ, where X represents a modified amino acid residue and φ represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species, Streptococcus mitis and Streptococcus constellatus. PMID:11292790

  20. Role of the Amino-Terminal Region of Porphyromonas gingivalis Fimbriae in Adherence to Epithelial Cells

    PubMed Central

    Sojar, Hakimuddin T.; Han, Yiping; Hamada, Nobushiro; Sharma, Ashu; Genco, Robert J.

    1999-01-01

    Porphyromonas gingivalis fimbriae elicit many responses in eukaryotic cells, including mitogenicity, cytokine production, epithelial cell invasion, and cellular immune response. Specific domains of the major fimbrial protein (FimA) have been shown to be important in triggering some of these functions. The goal of the present study was to identify the domain(s) of P. gingivalis FimA responsible for specific interaction with human mucosal epithelial cells. Fimbriated P. gingivalis strains have been shown to bind to buccal epithelial cells, whereas nonfimbriated strains bind at low levels or not at all. This and other studies provide evidence that FimA mediates the adherence of P. gingivalis to oral epithelial cells. To determine the specific region(s) of P. gingivalis FimA involved in epithelial cell binding, specific antipeptide antibodies were used to inhibit the binding of iodinated purified fimbriae as well as the binding of P. gingivalis cells to epithelial cells. Antibodies directed against peptides 49 to 68 (VVMANTAGAMELVGKTLAEVK) and 69 to 90 (ALTTELTAENQEAAGLIMTAEP) were found to highly inhibit both the binding of fimbriae and the binding of P. gingivalis cells to epithelial cells. The antibody against FimA peptides 69 to 90 also reacted with P. gingivalis fimbriae in immunogold labeling and immunoblot analysis, thereby indicating that this peptide domain is exposed on the surface of fimbriae. Our results suggest that the amino-terminal domain corresponding to amino acid residues 49 to 90 of the fimbrillin protein is a major epithelial cell binding domain of P. gingivalis fimbriae. PMID:10531284

  1. Oral Escherichia coli colonization factor antigen I fimbriae ameliorate arthritis via IL-35, not IL-27.

    PubMed

    Kochetkova, Irina; Thornburg, Theresa; Callis, Gayle; Holderness, Kathryn; Maddaloni, Massimo; Pascual, David W

    2014-01-15

    A Salmonella therapeutic expressing enterotoxigenic Escherichia coli colonization factor Ag I (CFA/I) fimbriae protects against collagen-induced arthritis (CIA) by eliciting two regulatory T cell (Treg) subsets: TGF-β-producing Foxp3(-)CD39(+)CD4(+) T cells and IL-10-producing Foxp3(+)CD39(+)CD4(+) T cells. However, it is unclear whether CFA/I fimbriae alone are protective and whether other regulatory cytokines are involved, especially in the context for the EBI3-sharing cytokines, Treg-derived IL-35 and APC-derived IL-27, both capable of suppressing Th17 cells and regulating autoimmune diseases. Subsequent evaluation revealed that a single oral dose of purified, soluble CFA/I fimbriae protected against CIA as effectively as did Salmonella-CFA/I and found that Foxp3(+)CD39(+)CD4(+) T cells were the source of secreted IL-35, whereas IL-27 production by CD11c(+) cells was inhibited. Inquiring into their relevance, CFA/I fimbriae-treated IL-27R-deficient (WSX-1(-/-)) mice were equally protected against CIA as were wild-type mice, suggesting a limited role for IL-27. In contrast, CFA/I fimbriae-mediated protection was abated in EBI3(-/-) mice, accompanied by the loss of TGF-β- and IL-10-producing Tregs. Adoptive transfer of C57BL/6 CD39(+)CD4(+) T cells to EBI3(-/-) mice with concurrent CFA/I plus IL-35 treatment effectively stimulated Tregs suppressing proinflammatory collagen II-specific Th cells. In contrast, recipients cotransferred with C57BL/6 and EBI3(-/-) CD39(+)CD4(+) T cells and treated with CFA/I plus IL-35 were not protected, implicating the importance of endogenous IL-35 for conferring CFA/I-mediated protection. Thus, CFA/I fimbriae stimulate IL-35 required for the coinduction of TGF-β and IL-10. PMID:24337375

  2. Proteus mirabilis MR/P fimbrial operon: genetic organization, nucleotide sequence, and conditions for expression.

    PubMed Central

    Bahrani, F K; Mobley, H L

    1994-01-01

    Proteus mirabilis, an agent of urinary tract infection, expresses at least four fimbrial types. Among these are the MR/P (mannose-resistant/Proteus-like) fimbriae. MrpA, the structural subunit, is optimally expressed at 37 degrees C in Luria broth cultured statically for 48 h by each of seven strains examined. Genes encoding this fimbria were isolated, and the complete nucleotide sequence was determined. The mrp gene cluster encoded by 7,293 bp predicts eight polypeptides: MrpI (22,133 Da), MrpA (17,909 Da), MrpB (19,632 Da), MrpC (96,823 Da), MrpD (27,886 Da), MrpE (19,470 Da), MrpF (17,363 Da), and MrpG (13,169 Da). mrpI is upstream of the gene encoding the major structural subunit gene mrpA and is transcribed in the direction opposite to that of the rest of the operon. All predicted polypeptides share > or = 25% amino acid identity with at least one other enteric fimbrial gene product encoded by the pap, fim, smf, fan, or mrk gene clusters. Images PMID:7910820

  3. Development of an intranasal vaccine to prevent urinary tract infection by Proteus mirabilis.

    PubMed

    Li, Xin; Lockatell, C Virginia; Johnson, David E; Lane, M Chelsea; Warren, John W; Mobley, Harry L T

    2004-01-01

    Proteus mirabilis commonly infects the complicated urinary tract and is associated with urolithiasis. Stone formation is caused by bacterial urease, which hydrolyzes urea to ammonia, causing local pH to rise, and leads to the subsequent precipitation of magnesium ammonium phosphate (struvite) and calcium phosphate (apatite) crystals. To prevent these infections, we vaccinated CBA mice with formalin-killed bacteria or purified mannose-resistant, Proteus-like (MR/P) fimbriae, a surface antigen expressed by P. mirabilis during experimental urinary tract infection, via four routes of immunization: subcutaneous, intranasal, transurethral, and oral. We assessed the efficacy of vaccination using the CBA mouse model of ascending urinary tract infection. Subcutaneous or intranasal immunization with formalin-killed bacteria and intranasal or transurethral immunization with purified MR/P fimbriae significantly protected CBA mice from ascending urinary tract infection by P. mirabilis (P < 0.05). To investigate the potential of MrpH, the MR/P fimbrial tip adhesin, as a vaccine, the mature MrpH peptide (residues 23 to 275, excluding the signal peptide), and the N-terminal receptor-binding domain of MrpH (residues 23 to 157) were overexpressed as C-terminal fusions to maltose-binding protein (MBP) and purified on amylose resins. Intranasal immunization of CBA mice with MBP-MrpH (residues 23 to 157) conferred effective protection against urinary tract infection by P. mirabilis (P < 0.002). PMID:14688082

  4. Active sites of salivary proline-rich protein for binding to Porphyromonas gingivalis fimbriae.

    PubMed Central

    Kataoka, K; Amano, A; Kuboniwa, M; Horie, H; Nagata, H; Shizukuishi, S

    1997-01-01

    Porphyromonas gingivalis fimbriae specifically bind salivary acidic proline-rich protein 1 (PRP1) through protein-protein interactions. The binding domains of fimbrillin (a subunit of fimbriae) for PRP1 were analyzed previously (A. Amano, A. Sharma, J.-Y. Lee, H. T. Sojar, P. A. Raj, and R. J. Genco, Infect. Immun. 64:1631-1637, 1996). In this study, we investigated the sites of binding of the PRP1 molecules to the fimbriae. PRP1 (amino acid residues 1 to 150) was proteolysed to three fragments (residues 1 to 74 [fragment 1-74], 75 to 129, and 130 to 150). 125I-labeled fimbriae clearly bound fragments 75-129 and 130-150, immobilized on a polyvinylidene difluoride membrane; both fragments also inhibited whole-cell binding to PRP1-coated hydroxyapatite (HAP) beads by 50 and 83%, respectively. However, the N-terminal fragment failed to show any effect. Analogous peptides corresponding to residues 75 to 89, 90 to 106, 107 to 120, 121 to 129, and 130 to 150 of PRP1 were synthesized. The fimbriae significantly bound peptide 130-150, immobilized on 96-well plates, and the peptide also inhibited binding of 125I-labeled fimbriae to PRP1-coated HAP beads by almost 100%. Peptides 75-89, 90-106, and 121-129, immobilized on plates, showed considerable ability to bind fimbriae. For further analysis of active sites in residues 130 to 150, synthetic peptides corresponding to residues 130 to 137, 138 to 145, and 146 to 150 were prepared. Peptide 138-145 (GRPQGPPQ) inhibited fimbrial binding to PRP1-coated HAP beads by 97%. This amino acid sequence was shared in the alignment of residues 75 to 89, 90 to 106, and 107 to 120. Six synthetic peptides were prepared by serial deletions of individual residues from the N and C termini of peptide GRPQGPPQ. Peptide PQGPPQ was as inhibitory as peptide GRPQGPPQ. Further deletions of the dipeptide Pro-Gln from the N and C termini of peptide PQGPPQ resulted in significant loss of the inhibitory effect. These results strongly suggest that PQGPPQ

  5. Fimbriae and lipopolysaccharides are necessary for co-aggregation between Lactobacilli and Escherichia coli.

    PubMed

    Mizuno, Kouhei; Furukawa, Soichi; Usui, Yumi; Ishiba, Madoka; Ogihara, Hirokazu; Morinaga, Yasushi

    2014-01-01

    Cells of Lactobacilli co-aggregated with Escherichia coli K-12 cells to form co-aggregates under mixed-culture conditions at 37 °C for 24 h. Co-aggregation was inhibited by sodium dodecyl sulfate but not by protease. E. coli deletion mutants of fimbriae formation and lipopolysaccharide (LPS) formation did not co-aggregate with Lactobacilli. These results showed that fimbriae and LPS are necessary for co-aggregation between Lactobacilli and E. coli. PMID:25209514

  6. Identification of Bacterial Factors Involved in Type 1 Fimbria Expression using an Escherichia coli K12 Proteome Chip*

    PubMed Central

    Chen, Yi-Wen; Teng, Ching-Hao; Ho, Yu-Hsuan; Jessica Ho, Tien Yu; Huang, Wen-Chun; Hashimoto, Masayuki; Chiang, I-Yuan; Chen, Chien-Sheng

    2014-01-01

    Type 1 fimbriae are filamentous structures on Escherichia coli. These structures are important adherence factors. Because binding to the host cells is the first step of infection, type 1 fimbria is an important virulence factor of pathogenic E. coli. Expression of type 1 fimbria is regulated by a phase variation in which each individual bacterium can alternate between fimbriated (phase-ON) and nonfimbriated (phase-OFF) states. The phase variation is regulated by the flipping of the 314-bp fimS fragment, which contains the promoter driving the expression of the genes required for the synthesis of type 1 fimbria. Thus, the bacterial proteins able to interact with fimS are likely to be involved in regulating the expression of type 1 fimbria. To identify novel type 1 fimbria-regulating factors, we used an E. coli K12 proteome chip to screen for the bacterial factors able to interact with a 602-bp DNA fragment containing fimS and its adjacent regions. The Spr protein was identified by the proteome chip-based screening and further confirmed to be able to interact with fimS by electrophoretic mobility shift assay. Deletion of spr in the neonatal meningitis E. coli strain RS218 significantly increased the ratio of the bacterial colonies that contained the type 1 fimbria phase-ON cells on agar plates. In addition, Spr interfered with the interactions of fimS with the site-specific recombinases, FimB and FimE, which are responsible for mediating the flipping of fimS. These results suggest that Spr is involved in the regulation of type 1 fimbria expression through direct interaction with the invertible element fimS. These findings facilitate our understanding of the regulation of type 1 fimbria. PMID:24692643

  7. A role for fimbriae in Porphyromonas gingivalis invasion of oral epithelial cells.

    PubMed Central

    Njoroge, T; Genco, R J; Sojar, H T; Hamada, N; Genco, C A

    1997-01-01

    Isogenic mutants of Porphyromonas gingivalis which differ in the expression of fimbriae were used to examine the contribution of fimbriae in invasion of a human oral epithelial cell line (KB). At a multiplicity of infection of 100, the wild-type P. gingivalis strains 33277, 381, and A7436 exhibited adherence efficiencies of 5.5, 0.11, and 5.0%, respectively, and invasion efficiencies of 0.15, 0.03, and 0.10%, respectively. However, adherence to and invasion of KB cells was not detected with the P. gingivalis fimA mutants, DPG3 and MPG1. Adherence of P. gingivalis wild-type strains to KB cells was completely inhibited by the addition of hyperimmune sera raised to the major fimbriae. Examination by electron microscopy of invasion of epithelial cells by the P. gingivalis wild-type strain 381 revealed microvillus-like extensions around adherent bacteria; this was not observed with P. gingivalis fim mutants. Taken together, these results indicate that the P. gingivalis major fimbriae are required for adherence to and invasion of oral epithelial cells. PMID:9125593

  8. Transcriptional profiling of human smooth muscle cells infected with gingipain and fimbriae mutants of Porphyromonas gingivalis.

    PubMed

    Zhang, Boxi; Sirsjö, Allan; Khalaf, Hazem; Bengtsson, Torbjörn

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) is considered to be involved in the development of atherosclerosis. However, the role of different virulence factors produced by P. gingivalis in this process is still uncertain. The aim of this study was to investigate the transcriptional profiling of human aortic smooth muscle cells (AoSMCs) infected with wild type, gingipain mutants or fimbriae mutants of P. gingivalis. AoSMCs were exposed to wild type (W50 and 381), gingipain mutants (E8 and K1A), or fimbriae mutants (DPG-3 and KRX-178) of P. gingivalis. We observed that wild type P. gingivalis changes the expression of a considerable larger number of genes in AoSMCs compare to gingipain and fimbriae mutants, respectively. The results from pathway analysis revealed that the common differentially expressed genes for AoSMCs infected by 3 different wild type P. gingivalis strains were enriched in pathways of cancer, cytokine-cytokine receptor interaction, regulation of the actin cytoskeleton, focal adhesion, and MAPK signaling pathway. Disease ontology analysis showed that various strains of P. gingivalis were associated with different disease profilings. Our results suggest that gingipains and fimbriae, especially arginine-specific gingipain, produced by P. gingivalis play important roles in the association between periodontitis and other inflammatory diseases, including atherosclerosis. PMID:26907358

  9. Antibody-mediated disruption of the mechanics of CS20 fimbriae of enterotoxigenic Escherichia coli

    PubMed Central

    Singh, Bhupender; Mortezaei, Narges; Uhlin, Bernt Eric; Savarino, Stephen J.; Bullitt, Esther; Andersson, Magnus

    2015-01-01

    Preventive vaccines against enterotoxigenic Escherichia coli (ETEC) are being developed, many of which target common fimbrial colonization factors as the major constituent, based on empirical evidence that these function as protective antigens. Particularly, passive oral administration of ETEC anti-fimbrial antibodies prevent ETEC diarrhea. Little is, however, known regarding the specific mechanisms by which intestinal antibodies against ETEC fimbriae function to prevent disease. Using coli surface antigen 20 (CS20) fimbriae as a model ETEC colonization factor, we show using force spectroscopy that anti-fimbrial antibodies diminish fimbrial elasticity by inhibiting their natural capacity to unwind and rewind. In the presence of anti-CS20 antibodies the force required to unwind a single fimbria was increased several-fold and the extension length was shortened several-fold. Similar measurements in the presence of anti-CS20 Fab fragments did not show any effect, indicating that bivalent antibody binding is required to reduce fimbrial elasticity. Based on these findings, we propose a model for an in-vivo mechanism whereby antibody-mediated disruption of the biomechanical properties of CS20 fimbriae impedes sustained adhesion of ETEC to the intestinal mucosal surface. Further elucidation of the role played by intestinal antibodies in mechanical disruption of fimbrial function may provide insights relevant to ETEC vaccine development. PMID:26411657

  10. Receptor for the F4 fimbriae of enterotoxigenic Escherichia coli (ETEC).

    PubMed

    Xia, Pengpeng; Zou, Yajie; Wang, Yiting; Song, Yujie; Liu, Wei; Francis, David H; Zhu, Guoqiang

    2015-06-01

    Infection with F4(+) enterotoxigenic Escherichia coli (ETEC) responsible for diarrhea in neonatal and post-weaned piglets leads to great economic losses in the swine industry. These pathogenic bacteria express either of three fimbrial variants F4ab, F4ac, and F4ad, which have long been known for their importance in host infection and initiating protective immune responses. The initial step in infection for the bacterium is to adhere to host enterocytes through fimbriae-mediated recognition of receptors on the host cell surface. A number of receptors for ETEC F4 have now been described and characterized, but their functions are still poorly understood. The current review summarizes the latest research addressing the characteristics of F4 fimbriae receptors and the interactions of F4 fimbriae and their receptors on host cells. These include observations that as follows: (1) FaeG mediates the binding activities of F4 and is an essential component of the F4 fimbriae, (2) the F4 fimbrial receptor gene is located in a region of chromosome 13, (3) the biochemical properties of F4 fimbrial receptors that form the binding site of the bacterium are now recognized, and (4) specific receptors confer susceptibility/resistance to ETEC F4 infection in pigs. Characterizing the host-pathogen interaction will be crucial to understand the pathogenicity of the bacteria, provide insights into receptor activation of the innate immune system, and develop therapeutic strategies to prevent this illness. PMID:25967654

  11. Genetic variability between complete mitochondrion genomes of the sablefish, Anoplopoma fimbria (Pallas, 1814).

    PubMed

    Galván-Tirado, Carolina; Del Río-Portilla, Miguel Angel; Delgado-Vega, Rigoberto; García-De León, Francisco J

    2016-07-01

    The complete mitochondrial genome of the sablefish, Anoplopoma fimbria (Genbank accession KP777542) is 16,507 bp in size and contains the typical 37 genes (13 protein-coding, 2 ribosomal RNA, and 22 transfer RNA) found in teleosts mitogenomes. The genome varies in 118 positions with respect to another mitogenome sablefish specimen. PMID:26065847

  12. Fimbria-mediated adherence of Candida albicans to glycosphingolipid receptors on human buccal epithelial cells.

    PubMed Central

    Yu, L; Lee, K K; Sheth, H B; Lane-Bell, P; Srivastava, G; Hindsgaul, O; Paranchych, W; Hodges, R S; Irvin, R T

    1994-01-01

    Candida albicans is an opportunist fungal pathogen that has the ability to adhere to host cell surface receptors via a number of adhesins. Yu et al. (L. Yu, K. K. Lee, K. Ens, P. C. Doig, M. R. Carpenter, W. Staddon, R. S. Hodges, W. Paranchych, and R. T. Irvin, Infect. Immun. 62:2834-2842, 1994) described the purification and initial characterization of a fimbrial adhesin from C. albicans. In this paper, we show that C. albicans fimbriae also bind to asialo-GM1 [gangliotetraosylceramide: beta Gal(1-3)beta GalNAc(1-4) beta Gal(1-4)beta Glc(1-1)Cer] immobilized on microtiter plates in a saturable and concentration-dependent manner. C. albicans fimbrial binding to exfoliated human buccal epithelial cells (BECs) was inhibited by asialo-GM1 in in vitro binding assays. The fimbriae interact with the glycosphingolipid receptors via the carbohydrate portion of the receptors, since fimbriae were observed to bind to synthetic beta GalNAc(1-4)beta Gal-protein conjugates and the disaccharide was able to inhibit binding of fimbriae to BECs in in vitro binding assays. We conclude from these results that the C. albicans yeast form expresses a fimbrial adhesin that binds to glycosphingolipids displayed on the surface of human BECs. Images PMID:8005674

  13. Antibody-mediated disruption of the mechanics of CS20 fimbriae of enterotoxigenic Escherichia coli.

    PubMed

    Singh, Bhupender; Mortezaei, Narges; Uhlin, Bernt Eric; Savarino, Stephen J; Bullitt, Esther; Andersson, Magnus

    2015-01-01

    Preventive vaccines against enterotoxigenic Escherichia coli (ETEC) are being developed, many of which target common fimbrial colonization factors as the major constituent, based on empirical evidence that these function as protective antigens. Particularly, passive oral administration of ETEC anti-fimbrial antibodies prevent ETEC diarrhea. Little is, however, known regarding the specific mechanisms by which intestinal antibodies against ETEC fimbriae function to prevent disease. Using coli surface antigen 20 (CS20) fimbriae as a model ETEC colonization factor, we show using force spectroscopy that anti-fimbrial antibodies diminish fimbrial elasticity by inhibiting their natural capacity to unwind and rewind. In the presence of anti-CS20 antibodies the force required to unwind a single fimbria was increased several-fold and the extension length was shortened several-fold. Similar measurements in the presence of anti-CS20 Fab fragments did not show any effect, indicating that bivalent antibody binding is required to reduce fimbrial elasticity. Based on these findings, we propose a model for an in-vivo mechanism whereby antibody-mediated disruption of the biomechanical properties of CS20 fimbriae impedes sustained adhesion of ETEC to the intestinal mucosal surface. Further elucidation of the role played by intestinal antibodies in mechanical disruption of fimbrial function may provide insights relevant to ETEC vaccine development. PMID:26411657

  14. Transcriptional profiling of human smooth muscle cells infected with gingipain and fimbriae mutants of Porphyromonas gingivalis

    PubMed Central

    Zhang, Boxi; Sirsjö, Allan; Khalaf, Hazem; Bengtsson, Torbjörn

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) is considered to be involved in the development of atherosclerosis. However, the role of different virulence factors produced by P. gingivalis in this process is still uncertain. The aim of this study was to investigate the transcriptional profiling of human aortic smooth muscle cells (AoSMCs) infected with wild type, gingipain mutants or fimbriae mutants of P. gingivalis. AoSMCs were exposed to wild type (W50 and 381), gingipain mutants (E8 and K1A), or fimbriae mutants (DPG-3 and KRX-178) of P. gingivalis. We observed that wild type P. gingivalis changes the expression of a considerable larger number of genes in AoSMCs compare to gingipain and fimbriae mutants, respectively. The results from pathway analysis revealed that the common differentially expressed genes for AoSMCs infected by 3 different wild type P. gingivalis strains were enriched in pathways of cancer, cytokine-cytokine receptor interaction, regulation of the actin cytoskeleton, focal adhesion, and MAPK signaling pathway. Disease ontology analysis showed that various strains of P. gingivalis were associated with different disease profilings. Our results suggest that gingipains and fimbriae, especially arginine-specific gingipain, produced by P. gingivalis play important roles in the association between periodontitis and other inflammatory diseases, including atherosclerosis. PMID:26907358

  15. Identification and characterization of S fimbria-binding sialoglycoproteins on brain microvascular endothelial cells.

    PubMed Central

    Prasadarao, N V; Wass, C A; Kim, K S

    1997-01-01

    We have previously shown that S-fimbriated Escherichia coli binds brain microvascular endothelial cells (BMEC) via a lectin-like activity of SfaS adhesin specific for NeuAc alpha2,3-galactose; however, BMEC molecules bearing these epitopes have not been identified. In the present study, we showed that the expression of S fimbriae conferred a three-fold increase in adhesion of E. coli to cow, human, and rat BMEC but did not enhance E. coli adhesion to systemic vascular endothelial cells such as human umbilical vein endothelial cells and human aortic arterial endothelial cells. Two BMEC-binding molecules for S fimbriae were identified as 65 (major)- and 130 (minor)-kDa sialoglycoproteins by S fimbria immunoblotting and were purified from bovine BMEC by wheat germ agglutinin and Maackia amurensis lectin (specific to NeuAc alpha2,3-galactose) affinity chromatography. The 65-kDa BMEC glycoprotein showed effective inhibition of S fimbria-mediated binding of E. coli to BMEC. Polyclonal antibodies raised against the mixture of 65- and 130-kDa proteins reacted to 65-kDa protein present only on BMEC, not on systemic vascular endothelial cells. Immunoprecipitation of biotinylated BMEC membrane proteins and immunocytochemistry studies of BMEC with anti-S fimbria-binding protein antibodies revealed that the 65-kDa protein is a surface protein. The N-terminal amino acid sequence of 65- and 130-kDa proteins showed no significant sequence homology with any other known proteins. These findings suggest that 65- and 130-kDa proteins represent novel sialoglycoproteins involved in the binding of S-fimbriated E. coli to BMEC. PMID:9199459

  16. DNA-based diagnostic tests for Salmonella species targeting agfA, the structural gene for thin, aggregative fimbriae.

    PubMed Central

    Doran, J L; Collinson, S K; Burian, J; Sarlós, G; Todd, E C; Munro, C K; Kay, C M; Banser, P A; Peterkin, P I; Kay, W W

    1993-01-01

    Salmonella enteritidis 27655-3b and a few diarrheagenic Escherichia coli strains produce morphologically and antigenically related, thin, aggregative fimbriae, collectively named GVVPQ fimbriae (S. K. Collinson, L. Emödy, T. J. Trust, and W. W. Kay, J. Bacteriol. 174:4490-4495, 1992). To determine whether GVVPQ fimbriae are common to Salmonella spp. and other enteropathogenic members of the family Enterobacteriaceae, 113 isolates were phenotypically screened for Congo red binding and aggregative colony morphology. Presumptive positive and representative negative strains were examined by Western blotting (immunoblotting) by using antiserum to SEF 17, the native GVVPQ fimbria of S. enteritidis. Only four S. enteritidis strains and six E. coli isolates possessed substantial amounts of GVVPQ fimbriae after 24 h of incubation on T medium. Following 5 days of incubation, 56 of 93 Salmonella isolates (60%) and 1 of 7 additional E. coli clinical isolates possessed detectable levels of GVVPQ fimbriae. Since variable expression of GVVPQ fimbriae was observed among Salmonella isolates and some E. coli strains produced scant amounts, as revealed by immunoelectron microscopy, the ability to produce these fimbriae was evaluated by genotypic screening. The structural gene for the SEF 17 fimbrin, agfA, was amplified by the polymerase chain reaction, cloned, and sequenced to provide a characterized DNA probe. An agfA DNA fragment hybridized strongly to 603 of 604 (99.8%) Salmonella isolates but very weakly to 31 of 266 other members of the family Enterobacteriaceae including 26 of 137 E. coli strains, 3 of 14 Citrobacter spp., and single isolates of Shigella sonnei and Enterobacter cloacae. The agfA DNA probe proved to be a valuable diagnostic tool for Salmonella isolates arrayed on hydrophobic grid membrane filters. Unique agfA sequences were targeted in the development of a polymerase chain reaction assay specific for Salmonella spp. Images PMID:8104955

  17. Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2

    PubMed Central

    Hasegawa, Yoshiaki; Iwami, Jun; Sato, Keiko; Park, Yoonsuk; Nishikawa, Kiyoshi; Atsumi, Tatsuo; Moriguchi, Keiichi; Murakami, Yukitaka; Lamont, Richard J.; Nakamura, Hiroshi; Ohno, Norikazu; Yoshimura, Fuminobu

    2009-01-01

    Porphyromonas gingivalis, a causative agent of periodontitis, has at least two types of thin, single-stranded fimbriae, termed FimA and Mfa1 (according to the names of major subunits), which can be discriminated by filament length and by the size of their major fimbrilin subunits. FimA fimbriae are long filaments that are easily detached from cells, whereas Mfa1 fimbriae are short filaments that are tightly bound to cells. However, a P. gingivalis ATCC 33277-derived mutant deficient in mfa2, a gene downstream of mfa1, produced long filaments (10 times longer than those of the parent), easily detached from the cell surface, similar to FimA fimbriae. Longer Mfa1 fimbriae contributed to stronger autoaggregation of bacterial cells. Complementation of the mutant with the wild-type mfa2 allele in trans restored the parental phenotype. Mfa2 is present in the outer membrane of P. gingivalis, but does not co-purify with the Mfa1 fimbriae. However, co-immunoprecipitation demonstrated that Mfa2 and Mfa1 are associated with each other in whole P. gingivalis cells. Furthermore, immunogold microscopy, including double labelling, confirmed that Mfa2 was located on the cell surface and likely associated with Mfa1 fimbriae. Mfa2 may therefore play a role as an anchor for the Mfa1 fimbriae and also as a regulator of Mfa1 filament length. Two additional downstream genes (pgn0289 and pgn0290) are co-transcribed with mfa1 (pgn0287) and mfa2 (pgn0288), and proteins derived from pgn0289, pgn0290 and pgn0291 appear to be accessory fimbrial components. PMID:19589838

  18. Targeting aminopeptidase N, a newly identified receptor for F4ac fimbriae, enhances the intestinal mucosal immune response.

    PubMed

    Melkebeek, V; Rasschaert, K; Bellot, P; Tilleman, K; Favoreel, H; Deforce, D; De Geest, B G; Goddeeris, B M; Cox, E

    2012-11-01

    Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrhea in human and animal. In piglets, ETEC having F4 fimbriae (F4(+) ETEC) induce severe diarrhea, dependent on the presence of receptors for F4 (F4R). In this study, porcine aminopeptidase N (pAPN) was identified as an F4R by comparative proteomic analysis of brush border proteins of F4R(+) and F4R(-) pigs and by adherence/internalization experiments on pAPN-transfected cells. Binding of F4 fimbriae to pAPN depended on sialic acid containing carbohydrate moieties, and resulted in clathrin-mediated endocytosis of the fimbriae. Endocytosis via pAPN was not restricted to F4 fimbriae, but was also observed for anti-pAPN antibodies. Both F4 fimbriae- and pAPN-specific antibodies were taken up in vivo by porcine enterocytes and induced subsequently a rapid immunoglobulin A and G response. In conclusion, we identified pAPN as an endocytotic receptor for F4 fimbriae and highlight the opportunity to target vaccine antigens to this epithelial receptor. PMID:22669578

  19. Platelet-Derived Growth Factor in the Ovarian Follicle Attracts the Stromal Cells of the Fallopian Tube Fimbriae

    PubMed Central

    Chen, Chiu-Hua; Hsu, Che-Fang; Huang, Rui-Len; Ding, Dah-Ching; Chu, Tang-Yuan

    2016-01-01

    During human ovulation, the fallopian tube fimbriae must move to the ovulation site to catch the oocyte. As the tissue-of-origin of the majority of ovarian high-grade serous carcinoma (HGSC), the fallopian tube fimbriae carrying a precursor cancer lesion may also approach the ovulatory site for metastasis. We hypothesize that platelet-derived growth factor (PDGF) in mature follicle fluid (FF) attracts the migration of PDGFR-expressing fimbriae toward the ovulating follicle. We observed that more PDGFR-β was expressed in the distal part than in the proximal parts of the fallopian tube, particularly in stromal cells in the lamina propria. The stromal cells, but not the epithelial cells, from normal fimbriae and fallopian tube HGSC were highly chemotactic to mature FF. The chemotactic activities were positively correlated with PDGF-BB and estradiol levels in FF and were abolished by a blocking antibody of PDGFR-β and by tyrosine kinase inhibitor imatinib. When PDGF-BB/AB was depleted from the FF, more than 80% of chemotaxis activities were diminished. This study suggests an ovarian follicle-directed and PDGF-dependent attraction of fallopian tube fimbriae before ovulation. The same mechanism may also be crucial for the ovarian homing of HGSC, which largely originates in the fimbriae. PMID:27379403

  20. S fimbriae of uropathogenic Escherichia coli bind to primary human renal proximal tubular epithelial cells but do not induce expression of intercellular adhesion molecule 1.

    PubMed Central

    Kreft, B; Placzek, M; Doehn, C; Hacker, J; Schmidt, G; Wasenauer, G; Daha, M R; van der Woude, F J; Sack, K

    1995-01-01

    We have recently reported an increase of expression of the intercellular adhesion molecule 1 by renal carcinoma cells in response to S fimbriae of Escherichia coli. Now we demonstrate that E. coli expressing S and P fimbriae strongly binds to human proximal tubular epithelial cells. However, in primary and simian virus 40-transfected renal tubular epithelial cells S fimbriae do not enhance the expression of intercellular adhesion molecule 1. PMID:7622256

  1. Twitching motility and possession of polar fimbriae in spreading Streptococcus sanguis isolates from the human throat.

    PubMed

    Henriksen, S D; Henrichsen, J

    1975-04-01

    A collection of 19 strains of alpha haemolytic streptococci, isolated from throat swabs and characterized by production of spreading zones around colonies on blood agar, was found to constitute a very homogeneous group with morphological, physiological and biochemical characters corresponding to those of streptococci of ser-group H, or Streptococcus sanguis, and they all appeared to possess the group H antigen. They all had a common agglutinogen and, in addition, heterogeneous agglutinogens. The spreading growth, which appears to be a common property of S. sanguis, was due to twitching motility, and the spreading cultures possessed polar fimbriae. tneither twitching motility nor the possession of polar fimbriae have been observed in gram-positive bacteria before. PMID:1171576

  2. Streptococcus salivarius strains carry either fibrils or fimbriae on the cell surface.

    PubMed Central

    Handley, P S; Carter, P L; Fielding, J

    1984-01-01

    Strains of Streptococcus salivarius were screened by negative staining for the presence of surface structures. Two structural subgroups were found, carrying either fibrils or fimbriae, projecting from the cell surface. Eight strains carried a very dense peritrichous array of fibrils of two distinct lengths. Long fibrils had an average length of 175 nm, and short fibrils had an average length of 95 nm. Two strains carried only long fibrils, one strain carried only short fibrils, and another strain carried a lateral tuft of very prominent fibrils of two lengths, with a fibrillar fuzz covering the remainder of the cell surface. In all the strains in which they were present, the long fibrils were unaffected by protease or trypsin treatment. In contrast, the short fibrils were completely digested by protease and partially removed by trypsin. Neither long nor short fibrils were affected structurally by mild pepsin digestion or by lipase. The Lancefield extraction procedure removed both long and short fibrils. These twelve fibrillar strains were therefore divisible into four structural subgroups. Extracts of all the fibrillar strains reacted with group K antiserum. The second main structural subgroup consisted of nine strains of S. salivarius, all of which carried morphologically identical, flexible fimbriae arranged peritrichously over the cell surface. The fimbriae were structurally distinct from fibrils and measured 0.5 to 1.0 micron long and 3 to 4 nm wide, with an irregular outline and no obvious substructure. There was no obvious reduction in the number of fimbriae after protease or trypsin treatment. Extracts of the fimbriated strains did not react with the group K antiserum. The two serological and structural subgroups could also be distinguished by colony morphology. Images PMID:6197404

  3. Promoting crystallisation of the Salmonella enteritidis fimbriae 14 pilin SefD using deuterium oxide.

    PubMed

    Liu, Bing; Garnett, James A; Lee, Wei-chao; Lin, Jing; Salgado, Paula; Taylor, Jonathan; Xu, Yingqi; Lambert, Sebastian; Cota, Ernesto; Matthews, Steve

    2012-05-01

    The use of heavy water (D(2)O) as a solvent is commonplace in many spectroscopic techniques for the study of biological macromolecules. A significant deuterium isotope effect exists where hydrogen-bonding is important, such as in protein stability, dynamics and assembly. Here we illustrate the use of D(2)O in additive screening for the production of reproducible diffraction-quality crystals for the Salmonella enteritidis fimbriae 14 (SEF14) putative tip adhesin, SefD. PMID:22497887

  4. Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae.

    PubMed

    Maeda, Kazuhiko; Nagata, Hideki; Nonaka, Aya; Kataoka, Kosuke; Tanaka, Muneo; Shizukuishi, Satoshi

    2004-11-01

    Interaction of Porphyromonas gingivalis with plaque-forming bacteria is necessary for its colonization in periodontal pockets. Participation of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P. gingivalis fimbriae in this interaction has been reported. In this investigation, the contribution of various oral streptococcal GAPDHs to interaction with P. gingivalis fimbriae was examined. Streptococcal cell surface GAPDH activity was measured by incubation of a constant number of streptococci with glyceraldehyde-3-phosphate and analysis for the conversion of NAD+ to NADH based on the absorbance at 340 nm. Coaggregation activity was measured by a turbidimetric assay. Cell surface GAPDH activity was correlated with coaggregation activity (r = 0.854, P < 0.01) with Spearman's rank correlation coefficient. S. oralis ATCC 9811 and ATCC 10557, Streptococcus gordonii G9B, Streptococcus sanguinis ATCC 10556, and Streptococcus parasanguinis ATCC 15909 exhibited high cell surface GAPDH activity and coaggregation activity; consequently, their cell surface GAPDHs were extracted with mutanolysin and purified on a Cibacron Blue Sepharose column. Subsequently, their DNA sequences were elucidated. Purified GAPDHs bound P. gingivalis recombinant fimbrillin by Western blot assay, furthermore, their DNA sequences displayed a high degree of homology with one another. Moreover, S. oralis recombinant GAPDH inhibited coaggregation between P. gingivalis and the aforementioned five streptococcal strains in a dose-dependent manner. These results suggest that GAPDHs of various plaque-forming streptococci may be involved in their attachment to P. gingivalis fimbriae and that they may contribute to P. gingivalis colonization. PMID:15488735

  5. Altered fimbria-fornix white matter integrity in anorexia nervosa predicts harm avoidance

    PubMed Central

    Kazlouski, Demitry; Rollin, Michael D.H.; Tregellas, Jason; Shott, Megan E.; Jappe, Leah M.; Hagman, Jennifer O.; Pryor, Tamara; Yang, Tony T.; Frank, Guido K.W.

    2011-01-01

    The eating disorder anorexia nervosa (AN) is associated with high anxiety. The brain mechanisms that drive those behaviors are unknown. In this study we wanted to test whether brain WM integrity is altered in AN, and related to heightened anxiety. Sixteen adult women with AN (mean age 24±7 years) and 17 healthy control women (CW, mean age 25±4 years) underwent diffusion tensor imaging (DTI) of the brain. The DTI brain images were used to calculate the fractional anisotropy (FA) of WM tracts, which is a measure for WM integrity. AN individuals compared to CW showed clusters of significantly reduced FA (p<0.05, corrected) in the bilateral fimbria-fornix, fronto-occipital fasciculus, as well as posterior cingulum WM. In the AN group, Harm Avoidance was predicted by left (F=5.8, Beta=−0.54, p<0.03) and right (F=6.0, Beta=−0.55, p<0.03) fimbria-fornix FA. Those findings were not due to WM volume deficits in AN. This study indicates that WM integrity is abnormal in AN in limbic and association pathways, which could contribute to disturbed feeding, emotion processing and body perception in AN. The prediction of Harm Avoidance in AN by fimbria-fornix WM integrity suggests that this pathway may be mechanistically involved in high anxiety in AN. PMID:21498054

  6. F4+ ETEC infection and oral immunization with F4 fimbriae elicits an IL-17-dominated immune response.

    PubMed

    Luo, Yu; Van Nguyen, Ut; de la Fe Rodriguez, Pedro Y; Devriendt, Bert; Cox, Eric

    2015-01-01

    Enterotoxigenic Escherichia coli (ETEC) are an important cause of post-weaning diarrhea (PWD) in piglets. Porcine-specific ETEC strains possess different fimbrial subtypes of which F4 fimbriae are the most frequently associated with ETEC-induced diarrhea in piglets. These F4 fimbriae are potent oral immunogens that induce protective F4-specific IgA antibody secreting cells at intestinal tissues. Recently, T-helper 17 (Th17) cells have been implicated in the protection of the host against extracellular pathogens. However, it remains unknown if Th17 effector responses are needed to clear ETEC infections. In the present study, we aimed to elucidate if ETEC elicits a Th17 response in piglets and if F4 fimbriae trigger a similar response. F4(+) ETEC infection upregulated IL-17A, IL-17F, IL-21 and IL-23p19, but not IL-12 and IFN-γ mRNA expression in the systemic and mucosal immune system. Similarly, oral immunization with F4 fimbriae triggered a Th17 signature evidenced by an upregulated mRNA expression of IL-17F, RORγt, IL-23p19 and IL-21 in the peripheral blood mononuclear cells (PBMCs). Intriguingly, IL-17A mRNA levels were unaltered. To further evaluate this difference between systemic and mucosal immune responses, we assayed the cytokine mRNA profile of F4 fimbriae stimulated PBMCs. F4 fimbriae induced IL-17A, IL-17F, IL-22 and IL-23p19, but downregulated IL-17B mRNA expression. Altogether, these data indicate a Th17 dominated response upon oral immunization with F4 fimbriae and F4(+) ETEC infection. Our work also highlights that IL-17B and IL-17F participate in the immune response to protect the host against F4(+) ETEC infection and could aid in the design of future ETEC vaccines. PMID:26490738

  7. Identification and Regulation of a Novel Citrobacter rodentium Gut Colonization Fimbria (Gcf)

    PubMed Central

    Caballero-Flores, Gustavo G.; Croxen, Matthew A.; Martínez-Santos, Verónica I.; Finlay, B. Brett

    2015-01-01

    ABSTRACT The Gram-negative enteric bacterium Citrobacter rodentium is a natural mouse pathogen that has been extensively used as a surrogate model for studying the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli. All three pathogens produce similar attaching and effacing (A/E) lesions in the intestinal epithelium. During infection, these bacteria employ surface structures called fimbriae to adhere and colonize the host intestinal epithelium. For C. rodentium, the roles of only a small number of its genome-carried fimbrial operons have been evaluated. Here, we report the identification of a novel C. rodentium colonization factor, called gut colonization fimbria (Gcf), which is encoded by a chaperone-usher fimbrial operon. A gcfA mutant shows a severe colonization defect within the first 10 days of infection. The gcf promoter is not active in C. rodentium under several in vitro growth conditions; however, it is readily expressed in a C. rodentium Δhns1 mutant lacking the closest ortholog of the Escherichia coli histone-like nucleoid structuring protein (H-NS) but not in mutants with deletion of the other four genes encoding H-NS homologs. H-NS binds to the regulatory region of gcf, further supporting its direct role as a repressor of the gcf promoter that starts transcription 158 bp upstream of the start codon of its first open reading frame. The gcf operon possesses interesting novel traits that open future opportunities to expand our knowledge of the structure, regulation, and function during infection of these important bacterial structures. IMPORTANCE Fimbriae are surface bacterial structures implicated in a variety of biological processes. Some have been shown to play a critical role during host colonization and thus in disease. Pathogenic bacteria possess the genetic information for an assortment of fimbriae, but their function and regulation and the interplay between them have not been studied in detail. This work provides new insights

  8. The Shaft of the Type 1 Fimbriae Regulates an External Force to Match the FimH Catch Bond

    PubMed Central

    Zakrisson, Johan; Wiklund, Krister; Axner, Ove; Andersson, Magnus

    2013-01-01

    Type 1 fimbriae mediate adhesion of uropathogenic Escherichia coli to host cells. It has been hypothesized that due to their ability to uncoil under exposure to force, fimbriae can reduce fluid shear stress on the adhesin-receptor interaction by which the bacterium adheres to the surface. In this work, we develop a model that describes how the force on the adhesin-receptor interaction of a type 1 fimbria varies as a bacterium is affected by a time-dependent fluid flow mimicking in vivo conditions. The model combines in vivo hydrodynamic conditions with previously assessed biomechanical properties of the fimbriae. Numerical methods are used to solve for the motion and adhesion force under the presence of time-dependent fluid profiles. It is found that a bacterium tethered with a type 1 pilus will experience significantly reduced shear stress for moderate to high flow velocities and that the maximum stress the adhesin will experience is limited to ∼120 pN, which is sufficient to activate the conformational change of the FimH adhesin into its stronger state but also lower than the force required for breaking it under rapid loading. Our model thus supports the assumption that the type 1 fimbria shaft and the FimH adhesin-receptor interaction are optimized to each other, and that they give piliated bacteria significant advantages in rapidly changing fluidic environments. PMID:23708354

  9. P-fimbriae in the presence of anti-PapA antibodies: new insight of antibodies action against pathogens

    PubMed Central

    Mortezaei, Narges; Singh, Bhupender; Bullitt, Esther; Uhlin, Bernt Eric; Andersson, Magnus

    2013-01-01

    Uropathogenic strains of Escherichia coli establish urinary tract infections by attaching to host epithelial cells using adhesive organelles called fimbriae. Fimbriae are helix-like structures with a remarkable adaptability, offering safeguarding for bacteria exposed to changing fluid forces in the urinary tract. We challenged this property of P-fimbriae by cross-linking their subunits with shaft-specific antibodies and measuring the corresponding force response at a single organelle level. Our data show compromised extension and rewinding of P-fimbriae in the presence of antibodies and reduced fimbrial elasticity, which are important properties of fimbriae contributing to the ability of bacteria to cause urinary tract infections. The reduced elasticity found by cross-linking fimbrial subunits could thus be another assignment for antibodies; in addition to marking bacteria as foreign, antibodies physically compromise fimbrial function. We suggest that our assay and results will be a starting point for further investigations aimed at inhibiting sustained bacterial adhesion by antibodies. PMID:24292100

  10. pap-2-encoded fimbriae adhere to the P blood group-related glycosphingolipid stage-specific embryonic antigen 4 in the human kidney.

    PubMed Central

    Karr, J F; Nowicki, B J; Truong, L D; Hull, R A; Moulds, J J; Hull, S I

    1990-01-01

    A subtype of P fimbriae, encoded by the pap-2 gene cluster, has been analyzed for agglutination of erythrocytes and for binding to cryostat sections of the human kidney. We have demonstrated that pap-2-encoded fimbriae are capable of binding to erythrocytes from some animal species and to human erythrocytes which express globoside and the LKE (stage-specific embryonic antigen 4 [SSEA-4]) antigen. The pap-2 fimbriae bind to Bowman's capsule in the human kidney. Monoclonal antibodies directed against glycosphingolipids were used for the detection of specific P blood group-related antigens in the human kidney and on erythrocytes. Preincubation of kidney sections with monoclonal antibody MC813-70, which binds to the SSEA-4 antigen, inhibited adherence of purified pap-2-encoded fimbriae to Bowman's capsule. We suggest that one receptor for pap-2-encoded fimbriae is the antigen known as LKE (Luke) on human erythrocytes or SSEA-4 in the tissues. Images PMID:1979319

  11. Evolutionary and Functional Relationships of Colonization Factor Antigen I and Other Class 5 Adhesive Fimbriae of Enterotoxigenic Escherichia coli

    PubMed Central

    Anantha, Ravi P.; McVeigh, Annette L.; Lee, Lanfong H.; Agnew, Mary K.; Cassels, Frederick J.; Scott, Daniel A.; Whittam, Thomas S.; Savarino, Stephen J.

    2004-01-01

    Colonization factor antigen I (CFA/I) is the archetype of eight genetically related fimbriae of enterotoxigenic Escherichia coli (ETEC) designated class 5 fimbriae. Assembled by the alternate chaperone pathway, these organelles comprise a rigid stalk of polymerized major subunits and an apparently tip-localized minor adhesive subunit. We examined the evolutionary relationships of class 5-specific structural proteins and correlated these with functional properties. We sequenced the gene clusters encoding coli surface antigen 4 (CS4), CS14, CS17, CS19, and putative colonization factor antigen O71 (PCFO71) and analyzed the deduced proteins and the published homologs of CFA/I, CS1, and CS2. Multiple alignment and phylogenetic analysis of the proteins encoded by each operon define three subclasses, 5a (CFA/I, CS4, and CS14), 5b (CS1, CS17, CS19, and PCFO71), and 5c (CS2). These share distant evolutionary relatedness to fimbrial systems of three other genera. Subclass divisions generally correlate with distinguishing in vitro adherence phenotypes of strains bearing the ETEC fimbriae. Phylogenetic comparisons of the individual structural proteins demonstrated greater intrasubclass conservation among the minor subunits than the major subunits. To correlate this with functional attributes, we made antibodies against CFA/I and CS17 whole fimbriae and maltose-binding protein fusions with the amino-terminal half of the corresponding minor subunits. Anti-minor subunit Fab preparations showed hemagglutination inhibition (HAI) of ETEC expressing homologous and intrasubclass heterologous colonization factors while anti-fimbrial Fab fractions showed HAI activity limited to colonization factor-homologous ETEC. These results were corroborated with similar results from the Caco-2 cell adherence assay. Our findings suggest that the minor subunits of class 5 fimbriae may be superior to whole fimbriae in inducing antiadhesive immunity. PMID:15557644

  12. Regulation and binding properties of S fimbriae cloned from E. coli strains causing urinary tract infection and meningitis.

    PubMed

    Morschhäuser, J; Vetter, V; Korhonen, T; Uhlin, B E; Hacker, J

    1993-04-01

    S fimbriae are able to recognize receptor molecules containing sialic acid and are produced by pathogenic E. coli strains causing urinary tract infection and menigitis. In order to characterize the corresponding genetic determinant, termed S fimbrial adhesin (sfa) gene cluster, we have cloned the S-specific genes from a urinary pathogen and from a meningitis isolate. Nine genes are involved in the production of S fimbriae, two of these, sfaB and sfaC code for regulatory proteins being necessary for the expression of S fimbriae. Two promoters, PB and PC, are located in front of these genes. Transcription of the sfa determinant is influenced by activation of the promoters via SfaB and SfaC, the action of the H-NS protein and an RNaseE-specific mRNA processing. In addition, a third promoter, PA, located in front of the major subunit gene sfaA, can be activated under special circumstances. Four genes of the sfa determinant code for the subunit-specific proteins, SfaA (16 kda), SfaG (17 kda), SfaS (14 kda) and SfaH (29 kda). It was demonstrated that the protein SfaA is the major subunit protein while SfaS is identical to the sialic-acid-specific adhesin of S fimbriae. The introduction of specific mutations into sfaS revealed that a region of six amino acids of the adhesin which includes two lysine and one arginine residues is involved in the receptor specific interaction of S fimbriae. Additionally, it has been shown that SfaS is necessary for the induction of fimbriation while SfaH plays a role in the stringency of binding of S fimbriae to erythrocytes. PMID:8102267

  13. Transcriptome of Proteus mirabilis in the murine urinary tract: virulence and nitrogen assimilation gene expression.

    PubMed

    Pearson, Melanie M; Yep, Alejandra; Smith, Sara N; Mobley, Harry L T

    2011-07-01

    The enteric bacterium Proteus mirabilis is a common cause of complicated urinary tract infections. In this study, microarrays were used to analyze P. mirabilis gene expression in vivo from experimentally infected mice. Urine was collected at 1, 3, and 7 days postinfection, and RNA was isolated from bacteria in the urine for transcriptional analysis. Across nine microarrays, 471 genes were upregulated and 82 were downregulated in vivo compared to in vitro broth culture. Genes upregulated in vivo encoded mannose-resistant Proteus-like (MR/P) fimbriae, urease, iron uptake systems, amino acid and peptide transporters, pyruvate metabolism enzymes, and a portion of the tricarboxylic acid (TCA) cycle enzymes. Flagella were downregulated. Ammonia assimilation gene glnA (glutamine synthetase) was repressed in vivo, while gdhA (glutamate dehydrogenase) was upregulated in vivo. Contrary to our expectations, ammonia availability due to urease activity in P. mirabilis did not drive this gene expression. A gdhA mutant was growth deficient in minimal medium with citrate as the sole carbon source, and loss of gdhA resulted in a significant fitness defect in the mouse model of urinary tract infection. Unlike Escherichia coli, which represses gdhA and upregulates glnA in vivo and cannot utilize citrate, the data suggest that P. mirabilis uses glutamate dehydrogenase to monitor carbon-nitrogen balance, and this ability contributes to the pathogenic potential of P. mirabilis in the urinary tract. PMID:21505083

  14. Effects of guanidinium hydrochloride on the structure and immunological properties of Bordetella pertussis fimbriae.

    PubMed Central

    Pearce, A M; Irons, L I; Robinson, A; Seabrook, R N

    1992-01-01

    Denaturation of Bordetella pertussis fimbrial preparations by guanidinium hydrochloride (GdnHCl) has been characterized using static light scattering, c.d., fluorescence and antibody recognition. The susceptibility of Fim2 + 3 (a mixed preparation of two fimbrial types) to GdnHCl was found to be highly dependent on pH; as the pH was increased from pH 7.2 to 10.5, the concentration of GdnHCl required to induce 50% denaturation was decreased. At pH 10.5, Fim2 + 3 was denatured by GdnHCl in a three-step pathway comprising: (1) formation of a pre-denaturational intermediate at less than 1.0 M-GdnHCl; (2) dissociation of the fimbrial polymer into subunits between 2 M- and 3.2 M-GdnHCl; and (3) subunit unfolding between 2.8 M- and 3.6 M-GdnHCl. A similar pathway was also found for the denaturation of the individual fimbrial types, Fim2 and Fim3, except that unfolding of either subunit commenced at a lower GdnHCl concentration (2.2 M) than that found for the mixture of fimbriae, Fim2 + 3. The second step in the denaturation pathway, dissociation into subunits, was partially reversible, but the renaturation and reassociation of fully unfolded subunits to form fimbriae-like structures was not achieved. These findings demonstrate that the GdnHCl denaturation of complex polymeric proteins is unlikely to follow a reversible two-state denaturation pathway, and support the involvement of a chaperone-like protein in the folding and assembly of the fimbriae in vivo. Measurement of the ability of anti-fimbrial monoclonal antibodies to recognize intermediates in the denaturation pathway enabled the identification of two types of epitope which were dependent on different aspects of fimbrial tertiary/quaternary structure. PMID:1375451

  15. sRNA-Mediated Regulation of P-Fimbriae Phase Variation in Uropathogenic Escherichia coli.

    PubMed

    Khandige, Surabhi; Kronborg, Tina; Uhlin, Bernt Eric; Møller-Jensen, Jakob

    2015-08-01

    Uropathogenic Escherichia coli (UPEC) are capable of occupying physiologically distinct intracellular and extracellular niches within the urinary tract. This feat requires the timely regulation of gene expression and small RNAs (sRNAs) are known to mediate such rapid adjustments in response to changing environmental cues. This study aimed to uncover sRNA-mediated gene regulation in the UPEC strain UTI89, during infection of bladder epithelial cells. Hfq is an RNA chaperone known to facilitate and stabilize sRNA and target mRNA interactions with bacterial cells. The co-immunoprecipitation and high throughput RNA sequencing of Hfq bound sRNAs performed in this study, revealed distinct sRNA profiles in UPEC in the extracellular and intracellular environments. Our findings emphasize the importance of studying regulatory sRNAs in a biologically relevant niche. This strategy also led to the discovery of a novel virulence-associated trans-acting sRNA-PapR. Deletion of papR was found to enhance adhesion of UTI89 to both bladder and kidney cell lines in a manner independent of type-1 fimbriae. We demonstrate PapR mediated posttranscriptional repression of the P-fimbriae phase regulator gene papI and postulate a role for such regulation in fimbrial cross-talk at the population level in UPEC. Our results further implicate the Leucine responsive protein (LRP) as a transcriptional activator regulating PapR expression. Our study reports, for the first time, a role for sRNAs in regulation of P-fimbriae phase variation and emphasizes the importance of studying pathogenesis-specific sRNAs within a relevant biological niche. PMID:26291711

  16. Identification of Type 3 Fimbriae in Uropathogenic Escherichia coli Reveals a Role in Biofilm Formation▿

    PubMed Central

    Ong, Cheryl-Lynn Y.; Ulett, Glen C.; Mabbett, Amanda N.; Beatson, Scott A.; Webb, Richard I.; Monaghan, Wayne; Nimmo, Graeme R.; Looke, David F.; McEwan, Alastair G.; Schembri, Mark A.

    2008-01-01

    Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States. Uropathogenic Escherichia coli (UPEC), the most common cause of CAUTI, can form biofilms on indwelling catheters. Here, we identify and characterize novel factors that affect biofilm formation by UPEC strains that cause CAUTI. Sixty-five CAUTI UPEC isolates were characterized for phenotypic markers of urovirulence, including agglutination and biofilm formation. One isolate, E. coli MS2027, was uniquely proficient at biofilm growth despite the absence of adhesins known to promote this phenotype. Mini-Tn5 mutagenesis of E. coli MS2027 identified several mutants with altered biofilm growth. Mutants containing insertions in genes involved in O antigen synthesis (rmlC and manB) and capsule synthesis (kpsM) possessed enhanced biofilm phenotypes. Three independent mutants deficient in biofilm growth contained an insertion in a gene locus homologous to the type 3 chaperone-usher class fimbrial genes of Klebsiella pneumoniae. These type 3 fimbrial genes (mrkABCDF), which were located on a conjugative plasmid, were cloned from E. coli MS2027 and could complement the biofilm-deficient transconjugants when reintroduced on a plasmid. Primers targeting the mrkB chaperone-encoding gene revealed its presence in CAUTI strains of Citrobacter koseri, Citrobacter freundii, Klebsiella pneumoniae, and Klebsiella oxytoca. All of these mrkB-positive strains caused type 3 fimbria-specific agglutination of tannic acid-treated red blood cells. This is the first description of type 3 fimbriae in E. coli, C. koseri, and C. freundii. Our data suggest that type 3 fimbriae may contribute to biofilm formation by different gram-negative nosocomial pathogens. PMID:18055599

  17. Radial maze performance in three strains of mice - Role of the fimbria/fornix

    NASA Technical Reports Server (NTRS)

    Reinstein, D. K.; Deboissiere, T.; Robinson, N.; Wurtman, R. J.

    1983-01-01

    Three strains of mice were tested on an 8-arm radial maze, an index of hippocampus-dependent spatial memory. Levels of performance differed betweens strains with C57Br/cdj greater than Balb/cj greater than C57B1/6j. Lesions of the fimbria/fornix disrupted performance in the C57Br and Balb strains: the C57Bl mice never performed better than chance before or after surgery. Choline acetyltransferase activity in hippocampus was not correlated with radial maze performance. These findings suggest a possible genetic contribution towards radial maze behavior.

  18. Variation in the structural subunit and basal protein antigens of Bacteroides nodosus fimbriae.

    PubMed Central

    Anderson, B J; Kristo, C L; Egerton, J R; Mattick, J S

    1986-01-01

    The fimbriae of Bacteroides nodosus play a major role in protective immunity against ovine footrot and are an important determinant in the serological classification system that divides field isolates into at least eight serogroups and 16 serotypes. Purified fimbriae contain two polypeptide antigens, the structural subunit of the fimbrial strand (molecular weight about 17,000) and a basal protein (molecular weight about 80,000), both of which exhibit structural variation. Fimbriae were prepared from all prototype strains, as well as from a number of other isolates representative of each of the B. nodosus serotypes, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Substantial variation was observed in the electrophoretic mobility of the fimbrial subunits from the prototypes of each of the eight serogroups. With the exception of serogroup H, which is an unusual case, the apparent molecular weights of the fimbrial subunits ranged from about 16,500 in serogroup D to 19,000 in serogroup F (serotype 1); in serogroup A, B, C and E, the apparent molecular weights were clustered in the range of 17,000 to 17,500, whereas serogroup G was about 18,500. Serogroup H fimbriae appeared to consist of two smaller polypeptides, which in the prototype (H1) had apparent molecular weights of about 6,000 and 10,000 and which seem to have arisen as a consequence of an internal proteolytic nick in the original subunit. Electrophoretic variation in the fimbrial subunit was also observed between different serotypes, although with the exceptions of serogroups F and H, this was not as pronounced as between the serogroups. Examination of a number of isolates classified within the same serotypes showed that some variation, although minor, also occurred at this level. The basal antigen exhibited significant variation at all levels of the serotypic hierarchy in a manner apparently unrelated to the classification system. Among the range of isolates examined, the apparent

  19. Age-dependent competition of porcine enterotoxigenic E. coli (ETEC) with different fimbria genes - short communication.

    PubMed

    Hur, Jin; Lee, Kyeong Min; Lee, John Hwa

    2011-12-01

    To investigate the association of pathogenic Escherichia coli fimbrial adhesins with the development of diarrhoea in piglets of different age groups and to test their relative competitiveness, piglets were orally inoculated with a mixture of E. coli strains harbouring F4, F5, F6, F18 and F41 fimbrial genes. A total of 537 E. coli strains with haemolytic activity were isolated from 36 diarrhoeic piglets. The F4 fimbrial gene was observed in 98.5%, 97.6% and 80.6% strains carrying fimbrial genes isolated from diarrhoeic piglets that were infected at 1, 3 and 5 weeks of age, respectively. These data demonstrate that F4 fimbriae are highly associated with diarrhoea in piglets of all age groups. Interestingly, the F18 fimbrial gene was observed in 2.4% and 25.4% strains carrying fimbrial genes isolated from the 3- and 5-week-old groups, respectively, which confirms that F18 fimbriae are associated with diarrhoea in piglets from late stages of suckling to post-weaning, and are more related to diarrhoea in weaned than in unweaned piglets. PMID:22079701

  20. Regulation and production of Tcf, a cable-like fimbriae from Salmonella enterica serovar Typhi.

    PubMed

    Leclerc, Jean-Mathieu; Quevillon, Eve-Lyne; Houde, Yoan; Paranjape, Kiran; Dozois, Charles M; Daigle, France

    2016-05-01

    tcf (Typhi colonization factor) is one of the 12 putative chaperone/usher fimbrial clusters present in the Salmonella enterica serovar Typhi genome. We investigated the production, expression and regulation of tcf as well as its role during interaction with human cells. The tcf gene cluster was cloned and induced in Escherichia coli and S. Typhi, and the production of intertwined fibres similar to the Cbl (cable) pili of Burkholderia cepacia was observed on the bacterial surface by electron microscopy. In S. Typhi, tcf was expressed more after growth in M63 minimal medium than in standard Luria-Bertani medium. Analysis of the promoter region identified putative binding sites for the global regulators RcsB, ArgR and Fur. The expression of tcf was measured in isogenic strains lacking these global regulators. Under the conditions tested, the results showed that tcf expression was higher in the fur mutant and was regulated by iron concentration. Fur may regulate these fimbriae indirectly via the small RNAs RyhB1 and RyhB2. An isogenic mutant harbouring a deletion of the tcf cluster did not demonstrate any defect in adhesion or invasion of human epithelial cells, or in phagocytosis or survival in macrophages, when compared to the WT serovar Typhi strain. However, the tcf cluster contributed to adherence to human epithelial cells when introduced into E. coli. Thus, tcf genes encode functional fimbriae that can act as an adhesin and may contribute to colonization during typhoid fever. PMID:26944792

  1. Inversion-independent phase variation of type 1 fimbriae in Escherichia coli.

    PubMed Central

    McClain, M S; Blomfield, I C; Eberhardt, K J; Eisenstein, B I

    1993-01-01

    The roles of fimB and fimE in the phase-variable expression of type 1 fimbriae in Escherichia coli were examined. A method was developed to study the effects of fimB and fimE on both recombination of the fim invertible element and fimbrial expression. The method used an allelic exchange procedure consisting of two steps. The first step, construction of intermediate strains, deleted fimB and fimE. This step locked the invertible element in either the on or the off orientation. The second step of the exchange procedure introduced either wild-type or mutant alleles of fimB and/or fimE into the chromosome of the intermediate strains. Analysis of the resulting strains supported the current, plasmid-based model of recombination. Unexpectedly, strains in which the invertible element was locked in the on orientation (either by mutation of both fimB and fimE or, in a control strain, by mutation of the left inverted repeat sequence of the invertible element) continued to exhibit phase-variable expression of type 1 fimbriae. A strain in which fimA was transcribed from the tac promoter continued to exhibit phase-variable fimbrial expression, suggesting that inversion-independent phase variation cannot be explained by variable transcription initiation of fimA. Images PMID:8101185

  2. Crystallization of the FaeE chaperone of Escherichia coli F4 fimbriae

    SciTech Connect

    Van Molle, Inge Buts, Lieven; Coppens, Fanny; Qiang, Liu; Wyns, Lode; Loris, Remy; Bouckaert, Julie; De Greve, Henri

    2005-04-01

    The periplasmic chaperone FaeE of E. coli F4 fimbriae crystallizes in three crystal forms. F4 (formerly K88) fimbriae from enterotoxigenic Escherichia coli are assembled via the FaeE/FaeD chaperone/usher pathway. The chaperone FaeE crystallizes in three crystal forms, all belonging to space group C2. Crystals of form 1 diffract to 2.3 Å and have unit-cell parameters a = 195.7, b = 78.5, c = 184.6 Å, β = 102.2°. X-ray data for crystal form 2 were collected to 2.7 Å using an SeMet variant of FaeE. The crystals have unit-cell parameters a = 136.4, b = 75.7, c = 69.4 Å, β = 92.8°. Crystals of form 3 were formed in a solution containing the FaeE–FaeG complex and diffract to 2.8 Å. Unit-cell parameters are a = 109.7, b = 78.6, c = 87.8 Å, β = 96.4°.

  3. Curli fimbriae are conditionally required in Escherichia coli O157:H7 for initial attachment and biofilm formation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several species of enteric pathogens produce curli fimbriae, which may affect their interaction with surfaces and other microbes in nonhost environments. Here we used two E. coli O157:H7 outbreak strains with distinct genotypes to understand the role of curli in surface attachment and biofilm format...

  4. Structural and functional insight into the carbohydrate receptor binding of F4 fimbriae-producing enterotoxigenic Escherichia coli.

    PubMed

    Moonens, Kristof; Van den Broeck, Imke; De Kerpel, Maia; Deboeck, Francine; Raymaekers, Hanne; Remaut, Han; De Greve, Henri

    2015-03-27

    Enterotoxigenic Escherichia coli (ETEC) strains are important causes of intestinal disease in humans and lead to severe production losses in animal farming. A range of fimbrial adhesins in ETEC strains determines host and tissue tropism. ETEC strains expressing F4 fimbriae are associated with neonatal and post-weaning diarrhea in piglets. Three naturally occurring variants of F4 fimbriae (F4ab, F4ac, and F4ad) exist that differ in the primary sequence of their major adhesive subunit FaeG, and each features a related yet distinct receptor binding profile. Here the x-ray structure of FaeGad bound to lactose provides the first structural insight into the receptor specificity and mode of binding by the poly-adhesive F4 fimbriae. A small D'-D″-α1-α2 subdomain grafted on the immunoglobulin-like core of FaeG hosts the carbohydrate binding site. Two short amino acid stretches Phe(150)-Glu(152) and Val(166)-Glu(170) of FaeGad bind the terminal galactose in the lactosyl unit and provide affinity and specificity to the interaction. A hemagglutination-based assay with E. coli expressing mutant F4ad fimbriae confirmed the elucidated co-complex structure. Interestingly, the crucial D'-α1 loop that borders the FaeGad binding site adopts a different conformation in the two other FaeG variants and hints at a heterogeneous binding pocket among the FaeG serotypes. PMID:25631050

  5. Production of cellulose and curli fimbriae by members of the family Enterobacteriaceae isolated from the human gastrointestinal tract.

    PubMed

    Zogaj, Xhavit; Bokranz, Werner; Nimtz, Manfred; Römling, Ute

    2003-07-01

    Citrobacter spp., Enterobacter spp., and Klebsiella spp. isolated from the human gut were investigated for the biosynthesis of cellulose and curli fimbriae (csg). While Citrobacter spp. produced curli fimbriae and cellulose and Enterobacter spp. produced cellulose with various temperature-regulatory programs, Klebsiella spp. did not show pronounced expression of those extracellular matrix components. Investigation of multicellular behavior in two Citrobacter species and Enterobacter sakazakii showed an extracellular matrix, cell clumping, pellicle formation, and biofilm formation associated with the expression of cellulose and curli fimbriae. In those three strains, the csgD-csgBA region and the cellulose synthase gene bcsA were conserved. PCR screening for the presence of csgD, csgA and bcsA revealed that besides Klebsiella pneumoniae and Klebsiella oxytoca, all species investigated harbored the genetic information for expression of curli fimbriae and cellulose. Since Citrobacter spp., Enterobacter spp., and Klebsiella spp. are frequently found to cause biofilm-related infections such as catheter-associated urinary tract infections, the human gut could serve as a reservoir for dissemination of biofilm-forming isolates. PMID:12819107

  6. Production of Cellulose and Curli Fimbriae by Members of the Family Enterobacteriaceae Isolated from the Human Gastrointestinal Tract

    PubMed Central

    Zogaj, Xhavit; Bokranz, Werner; Nimtz, Manfred; Römling, Ute

    2003-01-01

    Citrobacter spp., Enterobacter spp., and Klebsiella spp. isolated from the human gut were investigated for the biosynthesis of cellulose and curli fimbriae (csg). While Citrobacter spp. produced curli fimbriae and cellulose and Enterobacter spp. produced cellulose with various temperature-regulatory programs, Klebsiella spp. did not show pronounced expression of those extracellular matrix components. Investigation of multicellular behavior in two Citrobacter species and Enterobacter sakazakii showed an extracellular matrix, cell clumping, pellicle formation, and biofilm formation associated with the expression of cellulose and curli fimbriae. In those three strains, the csgD-csgBA region and the cellulose synthase gene bcsA were conserved. PCR screening for the presence of csgD, csgA and bcsA revealed that besides Klebsiella pneumoniae and Klebsiella oxytoca, all species investigated harbored the genetic information for expression of curli fimbriae and cellulose. Since Citrobacter spp., Enterobacter spp., and Klebsiella spp. are frequently found to cause biofilm-related infections such as catheter-associated urinary tract infections, the human gut could serve as a reservoir for dissemination of biofilm-forming isolates. PMID:12819107

  7. FIMBRIA-FORNIX TRANSECTIONS DISRUPT THE ONTOGENY OF DELAYED ALTERNATION BUT NOT POSITION DISCRIMINATION IN THE RAT

    EPA Science Inventory

    In Experiment 1, Long-Evans rat pups received fimbria-fornix transections or sham surgery on postnatal day 10 (PND10) and were then trained on PND23 to perform either a discrete-trials delayed alternation or a simple position discrimination (position habit) in a T-maze. at pups i...

  8. Downregulation of the DNA-Binding Activity of Nuclear Factor-κB p65 Subunit in Porphyromonas gingivalis Fimbria-Induced Tolerance

    PubMed Central

    Hajishengallis, George; Genco, Robert J.

    2004-01-01

    Porphyromonas gingivalis fimbriae induce high levels of nuclear factor-κB (NF-κB)-dependent cytokine release upon primary but not secondary stimulation of monocytic cells (FimA tolerance). In this study, fimbriae induced Toll-like receptor-mediated activation of both p50 and p65 subunits of NF-κB upon primary cellular activation. However, activation of the transactivating p65 subunit (but not of the transcriptionally inactive p50 subunit) was significantly inhibited in fimbria-restimulated cells. Moreover, expression of a NF-κB-dependent reporter gene was inhibited upon secondary stimulation with fimbriae. NF-κB p65 downregulation may thus contribute to induction of FimA tolerance. PMID:14742573

  9. Involvement of polyphosphate kinase in virulence and stress tolerance of uropathogenic Proteus mirabilis.

    PubMed

    Peng, Liang; Jiang, Qiao; Pan, Jia-Yun; Deng, Cong; Yu, Jing-Yi; Wu, Xiao-Man; Huang, Sheng-He; Deng, Xiao-Yan

    2016-04-01

    Proteus mirabilis (P. mirabilis), a gram-negative enteric bacterium, frequently causes urinary tract infections. Many virulence factors of uropathogenic P. mirabilis have been identified, including urease, flagella, hemolysin and fimbriae. However, the functions of polyphosphate kinase (PPK), which are related to the pathogenicity of many bacteria, remain entirely unknown in P. mirabilis. In this study, a ppk gene encoding the PPK insertional mutant in P. mirabilis strain HI4320 was constructed, and its biological functions were examined. The results of survival studies demonstrated that the ppk mutant was deficient in resistance to oxidative, hyperosmotic and heat stress. The swarming and biofilm formation abilities of P. mirabilis were also attenuated after the ppk interruption. In vitro and in vivo experiments suggested that ppk was required for P. mirabilis to invade the bladder. The negative phenotypes of the ppk mutant could be restored by ppk gene complementation. Furthermore, two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry were used to analyze the proteomes of the wild-type strain and the ppk mutant. Compared with the wild-type strain, seven proteins including TonB-dependent receptor, universal stress protein G, major mannose-resistant/Proteus-like fimbrial protein (MR/P fimbriae), heat shock protein, flagellar capping protein, putative membrane protein and multidrug efflux protein were down-regulated, and four proteins including exported peptidase, repressor protein for FtsI, FKBP-type peptidyl-prolyl cis-trans isomerase and phosphotransferase were up-regulated in the ppk mutant. As a whole, these results indicate that PPK is an important regulator and plays a crucial role in stress tolerance and virulence in uropathogenic P. mirabilis. PMID:26233310

  10. [Fimbriae of animal-originated enterotoxigenic Escherichia coli--a review].

    PubMed

    Zhou, Hong; Zhu, Jun; Zhu, Guoqiang

    2012-06-01

    Animal-originated enterotoxigenic Escherichia coli (ETEC) are major pathogens resulting in newborn and young animal diarrhea. Adhesins and enterotoxins, both are essential for the pathogenicity of ETEC, are two major virulent factors of ETEC. Adhesion of animal-originated ETEC fimbrial adhesins (mainly including K88, K99, 987P, F18, F17 and F41) to intestinal epithelial cells is the initial and most important step involved in the ETEC infection. From the 1960s, studies on ETEC fimbrial genes, structure, biosynthesis, regulation of expression, interaction between fimbriae and host receptors have helped to better understand the biology and role of these organelles in pathogenesis. These studies also provide insight into new diagnostic tools and development of vaccines and inhibitors of ETEC colonization. PMID:22934347

  11. Detection of antibodies against fimbria type 3 (Fim3) is useful diagnostic assay for pertussis.

    PubMed

    Oguchi, Kaoru; Miyata, Akiko; Kazuyama, Yukimasa; Noda, Atsuya; Suzuki, Eri; Watanabe, Mineo; Nakayama, Tetsuo

    2015-09-01

    Isolation of Bordetella pertussis and detection of the pertussis genome are not always successful because of low bacterial loads in adult patients with pertussis. Antibodies against pertussis toxin (PT) are measured but have low sensitivity in vaccinated subjects. There is no reliable diagnostic method at present. In this study, a fluorescent-EIA against several pertussis antigens and genome detection were investigated to establish clinical laboratory diagnostic methods for pertussis. The study was conducted in an outpatient clinic between September 2007 and 2013. Subjects consisted of 209 patients including adults suspected of pertussis and 35 staff members of the clinic. Loop-mediated isothermal amplification (LAMP) was performed to detect the pertussis genome in 5' UTR of the pertussis toxin (PT) gene. The catalytic region of the adenylate cyclase toxin (catACT), C-terminal of filamentous hemagglutinin (cFHA), and type 3 fimbria (Fim3) were selected, which are not pertussis vaccine component. Conventional PT and FHA antibodies were examined together with type 2 fimbria (Fim2) antibodies, and these are vaccine antigens. Pertussis DNA was detected in 23 (11%) out of 209. Detection sensitivity was high in young infants. Antibodies against Fim3 showed a higher positive rate in all age groups. Staff members at the pediatric outpatient clinic showed serological booster responses in Fim2 and Fim3 antibodies more sensitively than those in PT antibodies during outbreaks. LAMP was useful for detecting the pertussis genome in young infants, whereas a serological assay for fluorescent-EIA against Fim2 and Fim3 was preferable for adolescents and adults. PMID:26134278

  12. F9 Fimbriae of Uropathogenic Escherichia coli Are Expressed at Low Temperature and Recognise Galβ1-3GlcNAc-Containing Glycans

    PubMed Central

    Wurpel, Daniël J.; Totsika, Makrina; Allsopp, Luke P.; Hartley-Tassell, Lauren E.; Day, Christopher J.; Peters, Kate M.; Sarkar, Sohinee; Ulett, Glen C.; Yang, Ji; Tiralongo, Joe; Strugnell, Richard A.; Jennings, Michael P.; Schembri, Mark A.

    2014-01-01

    Uropathogenic Escherichia coli (UPEC) is the leading causative agent of urinary tract infections (UTI) in the developed world. Among the major virulence factors of UPEC, surface expressed adhesins mediate attachment and tissue tropism. UPEC strains typically possess a range of adhesins, with type 1 fimbriae and P fimbriae of the chaperone-usher class the best characterised. We previously identified and characterised F9 as a new chaperone-usher fimbrial type that mediates biofilm formation. However, the regulation and specific role of F9 fimbriae remained to be determined in the context of wild-type clinical UPEC strains. In this study we have assessed the distribution and genetic context of the f9 operon among diverse E. coli lineages and pathotypes and demonstrated that f9 genes are significantly more conserved in a UPEC strain collection in comparison to the well-defined E. coli reference (ECOR) collection. In the prototypic UPEC strain CFT073, the global regulator protein H-NS was identified as a transcriptional repressor of f9 gene expression at 37°C through its ability to bind directly to the f9 promoter region. F9 fimbriae expression was demonstrated at 20°C, representing the first evidence of functional F9 fimbriae expression by wild-type E. coli. Finally, glycan array analysis demonstrated that F9 fimbriae recognise and bind to terminal Galβ1-3GlcNAc structures. PMID:24671091

  13. Volunteer challenge with enterotoxigenic Escherichia coli that express intestinal colonization factor fimbriae CS17 and CS19.

    PubMed

    McKenzie, Robin; Porter, Chad K; Cantrell, Joyce A; Denearing, Barbara; O'Dowd, Aisling; Grahek, Shannon L; Sincock, Stephanie A; Woods, Colleen; Sebeny, Peter; Sack, David A; Tribble, David R; Bourgeois, A Louis; Savarino, Stephen J

    2011-07-01

    Human challenges with enterotoxigenic Escherichia coli (ETEC) have broadened our understanding of this important enteropathogen. We report findings from the first challenge studies using ETEC-expressing colonization factor fimbria CS17 and CS19. LSN03-016011/A (LT, CS17) elicited a dose-dependent effect, with the upper dose (6 × 10(9) organisms) causing diarrhea in 88% of recipients. WS0115A (LTSTp, CS19) also showed a dose response, with a 44% diarrhea rate at 9 × 10(9) organisms. Both strains elicited homologous antifimbrial and anti-LT antibody seroconversion. These studies establish the relative pathogenicity of ETEC expressing newer class 5 fimbriae and suggest suitability of the LT|CS17-ETEC challenge model for interventional trials. PMID:21628659

  14. Volunteer Challenge With Enterotoxigenic Escherichia coli That Express Intestinal Colonization Factor Fimbriae CS17 and CS19

    PubMed Central

    McKenzie, Robin; Porter, Chad K.; Cantrell, Joyce A.; DeNearing, Barbara; O’Dowd, Aisling; Grahek, Shannon L.; Sincock, Stephanie A.; Woods, Colleen; Sebeny, Peter; Sack, David A.; Tribble, David R.; Bourgeois, A. Louis

    2011-01-01

    Human challenges with enterotoxigenic Escherichia coli (ETEC) have broadened our understanding of this important enteropathogen. We report findings from the first challenge studies using ETEC-expressing colonization factor fimbria CS17 and CS19. LSN03-016011/A (LT, CS17) elicited a dose-dependent effect, with the upper dose (6 × 109 organisms) causing diarrhea in 88% of recipients. WS0115A (LTSTp, CS19) also showed a dose response, with a 44% diarrhea rate at 9 × 109 organisms. Both strains elicited homologous antifimbrial and anti-LT antibody seroconversion. These studies establish the relative pathogenicity of ETEC expressing newer class 5 fimbriae and suggest suitability of the LT|CS17-ETEC challenge model for interventional trials. PMID:21628659

  15. Role of Fimbriae, Flagella and Cellulose on the Attachment of Salmonella Typhimurium ATCC 14028 to Plant Cell Wall Models

    PubMed Central

    Tan, Michelle S. F.; White, Aaron P.; Rahman, Sadequr

    2016-01-01

    Cases of foodborne disease caused by Salmonella are frequently associated with the consumption of minimally processed produce. Bacterial cell surface components are known to be important for the attachment of bacterial pathogens to fresh produce. The role of these extracellular structures in Salmonella attachment to plant cell walls has not been investigated in detail. We investigated the role of flagella, fimbriae and cellulose on the attachment of Salmonella Typhimurium ATCC 14028 and a range of isogenic deletion mutants (ΔfliC fljB, ΔbcsA, ΔcsgA, ΔcsgA bcsA and ΔcsgD) to bacterial cellulose (BC)-based plant cell wall models [BC-Pectin (BCP), BC-Xyloglucan (BCX) and BC-Pectin-Xyloglucan (BCPX)] after growth at different temperatures (28°C and 37°C). We found that all three cell surface components were produced at 28°C but only the flagella was produced at 37°C. Flagella appeared to be most important for attachment (reduction of up to 1.5 log CFU/cm2) although both cellulose and fimbriae also aided in attachment. The csgD deletion mutant, which lacks both cellulose and fimbriae, showed significantly higher attachment as compared to wild type cells at 37°C. This may be due to the increased expression of flagella-related genes which are also indirectly regulated by the csgD gene. Our study suggests that bacterial attachment to plant cell walls is a complex process involving many factors. Although flagella, cellulose and fimbriae all aid in attachment, these structures are not the only mechanism as no strain was completely defective in its attachment. PMID:27355584

  16. Role of Fimbriae, Flagella and Cellulose on the Attachment of Salmonella Typhimurium ATCC 14028 to Plant Cell Wall Models.

    PubMed

    Tan, Michelle S F; White, Aaron P; Rahman, Sadequr; Dykes, Gary A

    2016-01-01

    Cases of foodborne disease caused by Salmonella are frequently associated with the consumption of minimally processed produce. Bacterial cell surface components are known to be important for the attachment of bacterial pathogens to fresh produce. The role of these extracellular structures in Salmonella attachment to plant cell walls has not been investigated in detail. We investigated the role of flagella, fimbriae and cellulose on the attachment of Salmonella Typhimurium ATCC 14028 and a range of isogenic deletion mutants (ΔfliC fljB, ΔbcsA, ΔcsgA, ΔcsgA bcsA and ΔcsgD) to bacterial cellulose (BC)-based plant cell wall models [BC-Pectin (BCP), BC-Xyloglucan (BCX) and BC-Pectin-Xyloglucan (BCPX)] after growth at different temperatures (28°C and 37°C). We found that all three cell surface components were produced at 28°C but only the flagella was produced at 37°C. Flagella appeared to be most important for attachment (reduction of up to 1.5 log CFU/cm2) although both cellulose and fimbriae also aided in attachment. The csgD deletion mutant, which lacks both cellulose and fimbriae, showed significantly higher attachment as compared to wild type cells at 37°C. This may be due to the increased expression of flagella-related genes which are also indirectly regulated by the csgD gene. Our study suggests that bacterial attachment to plant cell walls is a complex process involving many factors. Although flagella, cellulose and fimbriae all aid in attachment, these structures are not the only mechanism as no strain was completely defective in its attachment. PMID:27355584

  17. Structural and Functional Insight into the Carbohydrate Receptor Binding of F4 Fimbriae-producing Enterotoxigenic Escherichia coli *

    PubMed Central

    Moonens, Kristof; Van den Broeck, Imke; De Kerpel, Maia; Deboeck, Francine; Raymaekers, Hanne; Remaut, Han; De Greve, Henri

    2015-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are important causes of intestinal disease in humans and lead to severe production losses in animal farming. A range of fimbrial adhesins in ETEC strains determines host and tissue tropism. ETEC strains expressing F4 fimbriae are associated with neonatal and post-weaning diarrhea in piglets. Three naturally occurring variants of F4 fimbriae (F4ab, F4ac, and F4ad) exist that differ in the primary sequence of their major adhesive subunit FaeG, and each features a related yet distinct receptor binding profile. Here the x-ray structure of FaeGad bound to lactose provides the first structural insight into the receptor specificity and mode of binding by the poly-adhesive F4 fimbriae. A small D′-D″-α1-α2 subdomain grafted on the immunoglobulin-like core of FaeG hosts the carbohydrate binding site. Two short amino acid stretches Phe150–Glu152 and Val166–Glu170 of FaeGad bind the terminal galactose in the lactosyl unit and provide affinity and specificity to the interaction. A hemagglutination-based assay with E. coli expressing mutant F4ad fimbriae confirmed the elucidated co-complex structure. Interestingly, the crucial D′-α1 loop that borders the FaeGad binding site adopts a different conformation in the two other FaeG variants and hints at a heterogeneous binding pocket among the FaeG serotypes. PMID:25631050

  18. Regulation of type 1 fimbriae synthesis and biofilm formation by the transcriptional regulator LrhA of Escherichia coli.

    PubMed

    Blumer, Caroline; Kleefeld, Alexandra; Lehnen, Daniela; Heintz, Margit; Dobrindt, Ulrich; Nagy, Gábor; Michaelis, Kai; Emödy, Levente; Polen, Tino; Rachel, Reinhard; Wendisch, Volker F; Unden, Gottfried

    2005-10-01

    Type 1 fimbriae of Escherichia coli facilitate attachment to the host mucosa and promote biofilm formation on abiotic surfaces. The transcriptional regulator LrhA, which is known as a repressor of flagellar, motility and chemotaxis genes, regulates biofilm formation and expression of type 1 fimbriae. Whole-genome expression profiling revealed that inactivation of lrhA results in an increased expression of structural components of type 1 fimbriae. In vitro, LrhA bound to the promoter regions of the two fim recombinases (FimB and FimE) that catalyse the inversion of the fimA promoter, and to the invertible element itself. Translational lacZ fusions with these genes and quantification of fimE transcript levels by real-time PCR showed that LrhA influences type 1 fimbrial phase variation, primarily via activation of FimE, which is required for the ON-to-OFF transition of the fim switch. Enhanced type 1 fimbrial expression as a result of lrhA disruption was confirmed by mannose-sensitive agglutination of yeast cells. Biofilm formation was stimulated by lrhA inactivation and completely suppressed upon LrhA overproduction. The effects of LrhA on biofilm formation were exerted via the changed levels of surface molecules, most probably both flagella and type 1 fimbriae. Together, the data show a role for LrhA as a repressor of type 1 fimbrial expression, and thus as a regulator of the initial stages of biofilm development and, presumably, bacterial adherence to epithelial host cells also. PMID:16207912

  19. Inhibition of S-fimbria-mediated adhesion to human ileostomy glycoproteins by a protein isolated from bovine colostrum.

    PubMed Central

    Ouwehand, A C; Conway, P L; Salminen, S J

    1995-01-01

    The aim of this study was to isolate and purify the component in bovine colostrum which is responsible for the inhibition of S-fimbria-mediated adhesion of Escherichia coli. Whey from defatted colostrum was fractionated by ultrafiltration, and the < 100K, < 30K, and < 10K fractions and the colostral whey were tested for inhibition of in vitro adhesion of radiolabelled S-fimbria-bearing E. coli to human ileostomy glycoproteins, which provide a model for human intestinal mucus. The inhibiting compound was purified from a dialyzed < 30K fraction with an anion exchange column which was eluted with a NaCl gradient (0 to 1.0 M). The compound was found to be a heat-resistant but pepsin-sensitive protein with an Mr of approximately 18,000 and an isoelectric point of approximately 5.75. The protein appears to block receptor sites for S-fimbriae on ileostomy glycoproteins, with steric hindrance being the most likely mechanism. Analysis of the amino acid sequence of the amino terminus of the 18K protein showed similarity with the sequence of beta-lactoglobulin. PMID:7591156

  20. Promoting crystallisation of the Salmonella enteritidis fimbriae 14 pilin SefD using deuterium oxide

    SciTech Connect

    Liu, Bing; Garnett, James A.; Lee, Wei-chao; Lin, Jing; Salgado, Paula; Taylor, Jonathan; Xu, Yingqi; Lambert, Sebastian; Cota, Ernesto; Matthews, Steve

    2012-05-04

    Highlights: Black-Right-Pointing-Pointer The benefits of D{sub 2}O in screening for crystallisation was explored. Black-Right-Pointing-Pointer The crystal structures of the SefD pilin in both H{sub 2}O and D{sub 2}O reveal differences. Black-Right-Pointing-Pointer Crystallisation improvements are explained by altered interactions in D{sub 2}O crystals. Black-Right-Pointing-Pointer D{sub 2}O is useful additive in sparse-matrix screening for crystallisation. -- Abstract: The use of heavy water (D{sub 2}O) as a solvent is commonplace in many spectroscopic techniques for the study of biological macromolecules. A significant deuterium isotope effect exists where hydrogen-bonding is important, such as in protein stability, dynamics and assembly. Here we illustrate the use of D{sub 2}O in additive screening for the production of reproducible diffraction-quality crystals for the Salmonella enteritidis fimbriae 14 (SEF14) putative tip adhesin, SefD.

  1. Role of pili (fimbriae) in attachment of Bradyrhizobium japonicum to soybean roots

    SciTech Connect

    Vesper, S.J.; Bauer, W.D.

    1986-07-01

    Pili (fimbriae) were observed on cells of each of the five strains of Bradyrhizobium japonicum and the one strain of Rhizobium trifolii examined. Pili on B. japonicum were about 4 nm in diameter and polarly expressed. Piliated cells were estimated by transmission electron microscopy and hydrophobic attachment to polystyrene to constitute only a small percentage of the total population. The proportion of piliated cells in these populations was dependent on culture age in some strains. Piliated B. japonicum cells were selectively and quantitatively removed from suspension when cultures were incubated with either soybean roots or hydrophobic plastic surfaces, indicating that pili were involved in the attachment of the bacteria to these surfaces. Pili from B. japonicum 110 ARS were purified and found to have a subunit molecular weight of approximately 21,000. Treatment of B. japonicum suspensions with antiserum against the isolated pili reduced attachment to soybean roots by about 90% and nodulation by about 80%. Pili appear to be important mediators of attachment of B. japonicum to soybean roots under the conditions examined.

  2. Bordetella filamentous hemagglutinin and fimbriae: critical adhesins with unrealized vaccine potential.

    PubMed

    Scheller, Erich V; Cotter, Peggy A

    2015-11-01

    Pertussis, or whooping cough, is a highly contagious respiratory disease that is caused by the Gram-negative bacterium Bordetella pertussis, which is transmitted exclusively from human to human. While vaccination against B. pertussis has been successful, replacement of the whole cell vaccine with an acellular component vaccine has correlated with reemergence of the disease, especially in adolescents and infants. Based on their presumed importance in mediating adherence to host tissues, filamentous hemagglutinin (FHA) and fimbria (FIM) were selected as components of most acellular pertussis vaccines. In this review, we describe the biogenesis of FHA and FIM, recent data that show that these factors do, in fact, play critical roles in adherence to respiratory epithelium, and evidence that they also contribute to persistence in the lower respiratory tract by modulating the host immune response. We also discuss shortcomings of whole cell and acellular pertussis vaccines and the possibility that FHA and FIM could serve as effective protective antigens in next-generation vaccines. PMID:26416077

  3. Development of blood vessel-related radiation damage in the fimbria of the central nervous system

    SciTech Connect

    Reinhold, H.S.; Calvo, W.; Hopewell, J.W.; van der Berg, A.P. )

    1990-01-01

    The identification problem of the dose-limiting tissue component was investigated in the CNS of rats. Moderate single doses of radiation, ranging from 20 to 25 Gy were applied to the brain of adult female rats. The sequence of events was analyzed by scoring a series of morphological changes in one of the white matter structures that appears to represent a sensitive location, that is the fimbria hippocampi. The previously defined Tissue Injury Unit, characterized by a dilation of the blood vessel lumen, a thickening of the blood vessel wall, an enlargement of endothelial cell nuclei, and a hypertrophy of the adjacent astrocytes which represents a combined score of four different, but related histological changes, proved to be slightly more sensitive and responsive than the earliest recognizable changes in the neurological structures, that is demyelination. In addition, the incidence of demyelination could be expressed as a function of the intensity of the Tissue Injury Unit. These findings can be interpreted as an additional indication that blood vessel changes and the hypertrophy of the perivascular astrocytes precede degenerative changes in the white matter of the CNS after moderate doses of X rays.

  4. The Role of Thin Aggregative Fimbriae on Pathogenic Bacterial Transport Through Porous Media

    NASA Astrophysics Data System (ADS)

    Salvucci, A. E.; Fuka, D. R.; Marjerison, R. D.; Hay, A. G.; Zhang, W.; Caballero, L. A.; Zevi, Y.; Richards, B. K.; Steenhuis, T. S.

    2008-05-01

    Pathogenic bacteria, such as Escherichia coli and Salmonella sp., are responsible for many deaths worldwide every year. Their survival in the natural environment is enhanced by the production of biofilms, which provide a resistance to environmental stresses. However, it remains unclear how these biofilms affect the bacterias' ability to move through the soil matrix and potentially contaminate groundwater or water from drainage systems. In this presentation, we discuss the role of thin aggregative fimbriae (curli), a key biofilm component, on transport through porous media. An experiment was performed consisting of 96 sand columns created using a deep-well microtiter plate. We used well-characterized strains of E. coli, one with the ability to form curli and one without. Pulsing the E. coli strains through the sand column, mimicking natural leaching processes, showed less transport, by greater retention, in the strains that produce curli versus those strains that do not. In addition, when cultured in conditions unfavorable to curli production, transport between strains did not differ significantly. These preliminary results indicate that curli, and to a larger extent biofilms, could be an important component influencing the transport of bacterial strains through the soil matrix. This determination of pathogens' ability to move through the environment, as related to how well they form biofilms, will facilitate a better understanding of the fate of pathogenic bacteria in the environment.

  5. Escherichia coli K1 RS218 Interacts with Human Brain Microvascular Endothelial Cells via Type 1 Fimbria Bacteria in the Fimbriated State

    PubMed Central

    Teng, Ching-Hao; Cai, Mian; Shin, Sooan; Xie, Yi; Kim, Kee-Jun; Khan, Naveed Ahmed; Di Cello, Francescopaolo; Kim, Kwang Sik

    2005-01-01

    Escherichia coli K1 is a major gram-negative organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMEC) are a prerequisite for E. coli penetration into the central nervous system in vivo. In the present study, we showed using DNA microarray analysis that E. coli K1 associated with HBMEC expressed significantly higher levels of the fim genes compared to nonassociated bacteria. We also showed that E. coli K1 binding to and invasion of HBMEC were significantly decreased with its fimH deletion mutant and type 1 fimbria locked-off mutant, while they were significantly increased with its type 1 fimbria locked-on mutant. E. coli K1 strains associated with HBMEC were predominantly type 1 fimbria phase-on (i.e., fimbriated) bacteria. Taken together, we showed for the first time that type 1 fimbriae play an important role in E. coli K1 binding to and invasion of HBMEC and that type 1 fimbria phase-on E. coli is the major population interacting with HBMEC. PMID:15845498

  6. Aggregative adherence fimbriae I (AAF/I) mediate colonization of fresh produce and abiotic surface by Shiga toxigenic enteroaggregative Escherichia coli O104:H4.

    PubMed

    Nagy, Attila; Xu, Yunfeng; Bauchan, Gary R; Shelton, Daniel R; Nou, Xiangwu

    2016-07-16

    The Shiga toxigenic Escherichia coli O104:H4 isolated during the 2011 European outbreak expresses Shiga toxin 2a and possess virulence genes associated with the enteroaggregative E. coli (EAEC) pathotype. It produces plasmid encoded aggregative adherence fimbriae I (AAF/I) which mediate cell aggregation and biofilm formation in human intestine and promote Shiga-toxin adsorption, but it is not clear whether the AAF/I fimbriae are involved in the colonization and biofilm formation on food and environmental matrices such as the surface of fresh produce. We deleted the gene encoding for the AAF/I fimbriae main subunit (AggA) from an outbreak associated E. coli O104:H4 strain, and evaluated the role of AAF/I fimbriae in the adherence and colonization of E. coli O104:H4 to spinach and abiotic surfaces. The deletion of aggA did not affect the adherence of E. coli O104:H4 to these surfaces. However, it severely diminished the colonization and biofilm formation of E. coli O104:H4 on these surfaces. Strong aggregation and biofilm formation on spinach and abiotic surfaces were observed with the wild type strain but not the isogenic aggA deletion mutant, suggesting that AAF/I fimbriae play a crucial role in persistence of O104:H4 cells outside of the intestines of host species, such as on the surface of fresh produce. PMID:27099984

  7. Comprehensive analysis of type 1 fimbriae regulation in fimB-null strains from the multidrug resistant Escherichia coli ST131 clone.

    PubMed

    Sarkar, Sohinee; Roberts, Leah W; Phan, Minh-Duy; Tan, Lendl; Lo, Alvin W; Peters, Kate M; Paterson, David L; Upton, Mathew; Ulett, Glen C; Beatson, Scott A; Totsika, Makrina; Schembri, Mark A

    2016-09-01

    Uropathogenic Escherichia coli (UPEC) of sequence type 131 (ST131) are a pandemic multidrug resistant clone associated with urinary tract and bloodstream infections. Type 1 fimbriae, a major UPEC virulence factor, are essential for ST131 bladder colonization. The globally dominant sub-lineage of ST131 strains, clade C/H30-R, possess an ISEc55 insertion in the fimB gene that controls phase-variable type 1 fimbriae expression via the invertible fimS promoter. We report that inactivation of fimB in these strains causes altered regulation of type 1 fimbriae expression. Using a novel read-mapping approach based on Illumina sequencing, we demonstrate that 'off' to 'on' fimS inversion is reduced in these strains and controlled by recombinases encoded by the fimE and fimX genes. Unlike typical UPEC strains, the nucleoid-associated H-NS protein does not strongly repress fimE transcription in clade C ST131 strains. Using a genetic screen to identify novel regulators of fimE and fimX in the clade C ST131 strain EC958, we defined a new role for the guaB gene in the regulation of type 1 fimbriae and in colonisation of the mouse bladder. Our results provide a comprehensive analysis of type 1 fimbriae regulation in ST131, and highlight important differences in its control compared to non-ST131 UPEC. PMID:27309594

  8. Mfa4, an Accessory Protein of Mfa1 Fimbriae, Modulates Fimbrial Biogenesis, Cell Auto-Aggregation, and Biofilm Formation in Porphyromonas gingivalis

    PubMed Central

    Izumigawa, Masashi; Nagano, Keiji; Yoshida, Yasuo; Kitai, Noriyuki; Lamont, Richard J.; Yoshimura, Fuminobu; Murakami, Yukitaka

    2015-01-01

    Porphyromonas gingivalis, a gram-negative obligate anaerobic bacterium, is considered to be a key pathogen in periodontal disease. The bacterium expresses Mfa1 fimbriae, which are composed of polymers of Mfa1. The minor accessory components Mfa3, Mfa4, and Mfa5 are incorporated into these fimbriae. In this study, we characterized Mfa4 using genetically modified strains. Deficiency in the mfa4 gene decreased, but did not eliminate, expression of Mfa1 fimbriae. However, Mfa3 and Mfa5 were not incorporated because of defects in posttranslational processing and leakage into the culture supernatant, respectively. Furthermore, the mfa4-deficient mutant had an increased tendency to auto-aggregate and form biofilms, reminiscent of a mutant completely lacking Mfa1. Notably, complementation of mfa4 restored expression of structurally intact and functional Mfa1 fimbriae. Taken together, these results indicate that the accessory proteins Mfa3, Mfa4, and Mfa5 are necessary for assembly of Mfa1 fimbriae and regulation of auto-aggregation and biofilm formation of P. gingivalis. In addition, we found that Mfa3 and Mfa4 are processed to maturity by the same RgpA/B protease that processes Mfa1 subunits prior to polymerization. PMID:26437277

  9. Structural insight in the inhibition of adherence of F4 fimbriae producing enterotoxigenic Escherichia coli by llama single domain antibodies.

    PubMed

    Moonens, Kristof; Van den Broeck, Imke; Okello, Emmanuel; Pardon, Els; De Kerpel, Maia; Remaut, Han; De Greve, Henri

    2015-01-01

    Enterotoxigenic Escherichia coli that cause neonatal and post-weaning diarrhea in piglets express F4 fimbriae to mediate attachment towards host receptors. Recently we described how llama single domain antibodies (VHHs) fused to IgA, produced in Arabidopsis thaliana seeds and fed to piglets resulted in a progressive decline in shedding of F4 positive ETEC bacteria. Here we present the structures of these inhibiting VHHs in complex with the major adhesive subunit FaeG. A conserved surface, distant from the lactose binding pocket, is targeted by these VHHs, highlighting the possibility of targeting epitopes on single-domain adhesins that are non-involved in receptor binding. PMID:25828907

  10. Diversification of the Salmonella Fimbriae: A Model of Macro- and Microevolution

    PubMed Central

    Yue, Min; Rankin, Shelley C.; Blanchet, Ryan T.; Nulton, James D.; Edwards, Robert A.; Schifferli, Dieter M.

    2012-01-01

    Bacteria of the genus Salmonella comprise a large and evolutionary related population of zoonotic pathogens that can infect mammals, including humans and domestic animals, birds, reptiles and amphibians. Salmonella carries a plethora of virulence genes, including fimbrial adhesins, some of them known to participate in mammalian or avian host colonization. Each type of fimbria has its structural subunit and biogenesis genes encoded by one fimbrial gene cluster (FGC). The accumulation of new genomic information offered a timely opportunity to better evaluate the number and types of FGCs in the Salmonella pangenome, to test the use of current classifications based on phylogeny, and to infer potential correlations between FGC evolution in various Salmonella serovars and host niches. This study focused on the FGCs of the currently deciphered 90 genomes and 60 plasmids of Salmonella. The analysis highlighted a fimbriome consisting of 35 different FGCs, of which 16 were new, each strain carrying between 5 and 14 FGCs. The Salmonella fimbriome was extremely diverse with FGC representatives in 8 out of 9 previously categorized fimbrial clades and subclades. Phylogenetic analysis of Salmonella suggested macroevolutionary shifts detectable by extensive FGC deletion and acquisition. In addition, microevolutionary drifts were best depicted by the high level of allelic variation in predicted or known adhesins, such as the type 1 fimbrial adhesin FimH for which 67 different natural alleles were identified in S. enterica subsp. I. Together with strain-specific collections of FGCs, allelic variation among adhesins attested to the pathoadaptive evolution of Salmonella towards specific hosts and tissues, potentially modulating host range, strain virulence, disease progression, and transmission efficiency. Further understanding of how each Salmonella strain utilizes its panel of FGCs and specific adhesin alleles for survival and infection will support the development of new approaches

  11. Gonadal sex differentiation and effects of dietary methyltestosterone treatment in sablefish (Anoplopoma fimbria).

    PubMed

    Luckenbach, J Adam; Fairgrieve, William T

    2016-02-01

    Methods for sex control are needed to establish monosex aquaculture of sablefish (Anoplopoma fimbria). Here we conducted the first characterization of sex differentiation by histology and hormonal sex reversal experiment in sablefish. Ovarian differentiation was first discernible at ~80 mm fork length (FL) and characterized by development of lamellar structures and onset of meiosis. Testes exhibited a dual-lobe appearance over much of their length and remained non-meiotic until males were ≥520 mm FL (2 years post-fertilization). Juveniles with undifferentiated gonads were provided diets containing 0 (control), 5 or 50 mg 17α-methyltestosterone (MT)/kg for 2 months. Following treatment, controls possessed either ovaries or non-meiotic testes, whereas MT-treated fish exhibited meiotic testes (60% of the fish), intersex gonads (~30%), or gonads that appeared sterile (~10%). A genetic sex marker revealed that all intersex fish were genetic females, although other females appeared to be completely sex reversed (i.e., neomales). One year after treatment, MT-treated fish possessed non-meiotic testes similar to control males or intersex gonads with reduced ovarian features, presumably due to atresia following MT withdrawal. Milt collected from neomales and genetic males 3 years post-treatment permitted sperm motility analyses; however, neomale sperm were virtually immotile. These results demonstrated that sablefish are differentiated gonochorists and that MT treatment from 76 to 196 mm FL induced permanent masculinization of a portion of the genetic females, but acquisition of sperm motility was impaired. Earlier administration of MT may be necessary to sex reverse a higher proportion of genetic females and reduce negative effects on fertility. PMID:26400269

  12. Sialic acid and N-acetylglucosamine Regulate type 1 Fimbriae Synthesis.

    PubMed

    Blomfield, Ian C

    2015-06-01

    Type 1 fimbriae of E. coli, a chaperon-usher bacterial adhesin, are synthesized by the majority of strains of the bacterium. Although frequently produced by commensal strains, the adhesin is nevertheless a virulence factor in Extraintestinal Pathogenic E. coli (ExPEC). The role of the adhesin in pathogenesis is best understood in Uropathogenic E. coli (UPEC). Host attachment and invasion by type 1 fimbriate bacteria activates inflammatory pathways, with TLR4 signaling playing a predominant role. In a mouse model of cystitis, type 1 fimbriation not only enhances UPEC adherence to the surface of superficial umbrella cells of the bladder urothelium, but is both necessary and sufficient for their invasion. Moreover the adhesin plays a role in the formation of transient intracellular bacterial communities (IBCs) within the cytoplasm of urothelial cells as part of UPEC cycles of invasion. The expression of type 1 fimbriation is controlled by phase variation at the transcriptional level, a mode of gene regulation in which bacteria switch reversibly between fimbriate and afimbriate phases. Phase variation has been widely considered to be a mechanism enabling immune evasion. Notwithstanding the apparently random nature of phase variation, switching of type 1 fimbrial expression is nevertheless controlled by a range of environmental signals that include the amino sugars sialic acid and N-acetylglucosamine (GlcNAc). Sialic acid plays a pivotal role in innate immunity, including signaling by the toll-like receptors. Here how sialic acid and GlcNAc control type 1 fimbriation is described and the potential significance of this regulatory response is discussed. PMID:26185091

  13. Reduction of enterotoxin induced fluid accumulation in ileal loops of neonatal calves with anti-F5 fimbriae recombinant antibody.

    PubMed

    Sahagun-Ruiz, Alfredo; Velazquez, Leticia V; Bhaskaran, Shoba; Jay, Chris M; Morales-Salinas, E; Rathore, Keerti; Wagner, Gale G; Waghela, Suryakant D

    2015-12-01

    Neonatal calf colibacillosis caused by enterotoxigenic Escherichia coli (ETEC) is an economically significant problem in most parts of the world. The most common ETEC found in calves express the F5 (K99) fimbriae, which are necessary for the attachment of the bacteria to the ganglioside receptors on enterocytes. It is known that prevention of ETEC F5(+) adhesion to its ganglioside receptors with specific antibodies protects calves from colibacillosis. Previously we have described the development and characterization of a mouse recombinant antibody fragment (moRAb) that prevents F5 fimbrial protein induced agglutination of horse red blood cells (HRBC), which exhibit the same gangloside receptor for F5 fimbriae. Here we demonstrate that this recombinant antibody fragment inhibits in vitro the attachment of ETEC F5(+) bacteria to HRBC as well as isolated calf enterocytes, and in vivo it decreases fluid accumulation in intestinal loops of calves. Thus, correct oral administration of this anti-F5 moRAb may serve as an immunoprophylactic for cost effective control of colibacillosis in calves. PMID:26521056

  14. Biofilm formation by multidrug resistant Escherichia coli ST131 is dependent on type 1 fimbriae and assay conditions.

    PubMed

    Sarkar, Sohinee; Vagenas, Dimitrios; Schembri, Mark A; Totsika, Makrina

    2016-04-01

    Escherichia coli sequence type 131 (ST131) has emerged as a pandemic lineage of important multidrug resistant pathogens worldwide. Despite many studies examining the epidemiology of ST131, only a few studies to date have investigated the capacity of ST131 strains to form biofilms. Some of these studies have reported contrasting findings, with no specific ST131 biofilm-promoting factors identified. Here, we examined a diverse collection of ST131 isolates for in vitro biofilm formation in different media and assay conditions, including urine from healthy adult women. We found significant differences among strains and assay conditions, which offers an explanation for the contrasting findings reported by previous studies using a single condition. Importantly, we showed that expression of type 1 fimbriae is a critical determinant for biofilm formation by ST131 strains and that inhibition of the FimH adhesin significantly reduces biofilm formation. We also offer direct genetic evidence for the contribution of type 1 fimbriae in biofilm formation by the reference ST131 strain EC958, a representative of the clinically dominant H30-Rx ST131 subgroup. This is the first study of ST131 biofilm formation in biologically relevant conditions and paves the way for the application of FimH inhibitors in treating drug resistant ST131 biofilm infections. PMID:26940589

  15. Acetyl-L-carnitine restores choline acetyltransferase activity in the hippocampus of rats with partial unilateral fimbria-fornix transection.

    PubMed

    Piovesan, P; Quatrini, G; Pacifici, L; Taglialatela, G; Angelucci, L

    1995-02-01

    Transection of the fimbria-fornix bundle in adult rats results in degeneration of the septohippocampal cholinergic pathway, reminiscent of that occurring in aging as well as Alzheimer disease. We report here a study of the effect of a treatment with acetyl-L-carnitine (ALCAR) in three-month-old Fischer 344 rats bearing a partial unilateral fimbria-fornix transection. ALCAR is known to ameliorate some morphological and functional disturbances in the aged central nervous system (CNS). We used choline acetyltransferase (ChAT) and acetyl cholinesterase (AChE) as markers of central cholinergic function, and nerve growth factor (NGF) levels as indicative of the trophic regulation of the medio-septal cholinergic system. ChAT and AChE activities were significantly reduced in the hippocampus (HIPP) ipsilateral to the lesion as compared to the contralateral one, while no changes were observed in the septum (SPT), nucleus basalis magnocellularis (NBM) or frontal cortex (FCX). ALCAR treatment restored ChAT activity in the ipsilateral HIPP, while AChE levels were not different from those of untreated animals, and did not affect NGF content in either SPT or HIPP. PMID:7793306

  16. Aggregative adherence fimbriae I (AAF/I) mediate colonization of fresh produce and abiotic surface by Shiga toxigenic enteroaggregative Escherichia coli O104:H4

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Shiga toxigenic Escherichia coli O104:H4 bares the characteristics of both enterohemorrhagic (EHEC) and enteroaggregative (EAEC) E. coli. It produces plasmid encoded aggregative adherence fimbriae I (AAF/I) which mediate cell aggregation and biofilm formation in human intestine and promote Shiga...

  17. Both flagella and F4 fimbriae from F4ac+ enterotoxigenic Escherichia coli contribute to attachment to IPEC-J2 cells in vitro.

    PubMed

    Zhou, Mingxu; Duan, Qiangde; Zhu, Xiaofang; Guo, Zhiyan; Li, Yinchau; Hardwidge, Philip R; Zhu, Guoqiang

    2013-01-01

    The role of flagella in the pathogenesis of F4ac+ Enterotoxigenic Escherichia coli (ETEC) mediated neonatal and post-weaning diarrhea (PWD) is not currently understood. We targeted the reference C83902 ETEC strain (O8:H19:F4ac+ LT+ STa+ STb+), to construct isogenic mutants in the fliC (encoding the major flagellin protein), motA (encoding the flagella motor), and faeG (encoding the major subunit of F4 fimbriae) genes. Both the ΔfliC and ΔfaeG mutants had a reduced ability to adhere to porcine intestinal epithelial IPEC-J2 cells. F4 fimbriae expression was significantly down-regulated after deleting fliC, which revealed that co-regulation exists between flagella and F4 fimbriae. However, there was no difference in adhesion between the ΔmotA mutant and its parent strain. These data demonstrate that both flagella and F4 fimbriae are required for efficient F4ac+ ETEC adhesion in vitro. PMID:23668601

  18. Development and Efficacy Assessment of an Enteric Coated Porous Tablet Loaded With F4 Fimbriae for Oral Vaccination of Piglets against F4+ Escherichia coli Infections.

    PubMed

    Srivastava, Atul; Gowda, D V; Madhunapantula, SubbaRao V; Siddaramaiah

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) infection is one of the major causes contributing to the development of diarrhoea and mortality in new born, suckling and newly weaned piglets. To date, no preventive/treatment strategy showed promising results, which could be due to the lack of potent vaccines, and/or due to the development of resistance of ETEC to antibiotics. Therefore, in the present investigation, a novel porous sodium alginate (SA) tablet formulation loaded with F4 fimbriae antigen was developed and tested for efficacy against ETEC infections in piglet models. Precompression parameters of the powder mixes and post compression parameters of tablets have been evaluated and results were found to be satisfactory. Loading of F4 fimbrial antigens into the tablets was achieved by inducing pores in the tablets via the sublimation of camphor followed by incubation with purified F4 fimbriae. The loaded tablets have been coated with Eudragit L100 to protect the F4 fimbriae from (a) highly acidic gastric environment; (b) proteolytic cleavage by pepsin; and (c) to promote subsequent release in the intestine. Evaluation of developed F4 fimbrial tablets in a Pig model demonstrated induction of mucosal immunity, and a significant reduction of F4+ E. coli in faeces. Therefore, F4 fimbriae loaded porous tablets could be a novel oral vaccination candidate to induce mucosal and systemic immunity against ETEC infections. PMID:26212139

  19. Preliminary X-ray diffraction analysis of CfaA, a molecular chaperone essential for the assembly of CFA/I fimbriae of human enterotoxigenic Escherichia coli

    SciTech Connect

    Bao, Rui; Esser, Lothar; Poole, Steven; McVeigh, Annette; Chen, Yu-xing; Savarino, Stephen J.; Xia, Di

    2014-01-21

    The molecular chaperone CfaA plays a critical role in the bioassembly of the surface-adhesive CFA/I fimbriae of enterotoxigenic E. coli. Purified CfaA was crystallized and the phase solution was determined by the multiple isomorphous replacement coupled with anomalous scattering method.

  20. Transcriptional Analysis of the sfa Determinant Revealing Multiple mRNA Processing Events in the Biogenesis of S Fimbriae in Pathogenic Escherichia coli

    PubMed Central

    Balsalobre, Carlos; Morschhäuser, Joachim; Jass, Jana; Hacker, Jörg; Uhlin, Bernt Eric

    2003-01-01

    Among the virulence factors present in pathogenic extraintestinal Escherichia coli strains, expression of fimbrial adhesins is necessary for attachment to the host tissues and subsequent colonization. Occurrence of the sfa determinant coding for the S fimbriae is widespread among the uropathogens and meningitis isolates. The sfa operon consists of nine genes. In the biogenesis of S fimbriae, the proteins encoded by the sfa genes are presumably required in a specific stoichiometry. In the present work we studied how differential expression of the sfa operon genes occurs. Our findings indicate that a number of endoribonucleolytic cleavages occur in the mRNA from the sfa operon, and we detected the presence of different distinct transcriptional products, including sfaBA, sfaA, sfaADE, and sfaGSH. The sfaGSH transcript represents the three distal genes of the sfa operon, which code for the minor subunits of the S fimbriae. Analysis of the proteins in S fimbriae suggested that expression of the sfaGSH transcript provides equimolar amounts of the minor subunits. Furthermore, we showed that in the generation of the major sfaA transcript, the processing included RNase E endoribonuceolytic cleavage of the precursor sfaBA transcript. We suggest that posttranscriptional mRNA processing events result in differential gene expression important to achieve the stoichiometry necessary for fimbrial adhesin biogenesis. PMID:12511509

  1. Live attenuated Salmonella displaying HIV-1 10E8 epitope on fimbriae: systemic and mucosal immune responses in BALB/c mice by mucosal administration

    PubMed Central

    Li, Qing-Hai; Jin, Gang; Wang, Jia-Ye; Li, Hai-Ning; Liu, Huidi; Chang, Xiao-Yun; Wang, Fu-Xiang; Liu, Shu-Lin

    2016-01-01

    The HIV-1 membrane proximal external region (MPER) that is targeted by several broadly neutralizing antibodies (BNAbs) has been considered a potential immunogen for vaccine development. However, to date the immunogenicity of these BNAb epitopes has not been made sufficiently adequate. In the present work, we used live attenuated Salmonella as a platform to present the HIV-1 MPER 10E8 epitope in the fimbriae. The insertion of the 10E8 epitope into the fimbriae had no significant influence on the expression and the absorption capacity of bacterial fimbriae, nor on the virulence and invasiveness of the attenuated Salmonella. After oral administration of the vaccine construct to mice followed by 10E8 epitope peptide boost, specific antibody responses in serum and mucosa as well as memory lymphocytes in spleen and plasma cells in bone marrow were induced. We also found that the live attenuated Salmonella vector directed the immunity toward Th1 bias, induced Th1 and Th2 cytokine responses and stimulated significant B cell differentiation into GC B, memory B and plasma cells. Therefore, we propose that the live attenuated Salmonella constitutively expressing HIV-1 BNAb epitopes on the fimbriae will be an effective approach to improving immune microenvironment and enhancing the immunogenicity of HIV-1 epitope vaccines. PMID:27411313

  2. Conservation of fimbriae and the hemagglutinating adhesin HA-Ag2 among Porphyromonas gingivalis strains and other anaerobic bacteria studied by epitope mapping analysis.

    PubMed Central

    Du, L; Pellen-Mussi, P; Chandad, F; Mouton, C; Bonnaure-Mallet, M

    1997-01-01

    Monoclonal antibodies characterized as antifimbria and anti-HA-Ag2 were used in immunoblotting to examine the antigenic distribution of fimbriae and HA-Ag2 among a collection of human and animal Porphyromonas strains and human Prevotella and Bacteroides strains. The results showed that fimbrial and HA-Ag2 antigenic structures are peculiar to the species Porphyromonas gingivalis. PMID:9384294

  3. Permissive linker insertion sites in the outer membrane protein of 987P fimbriae of Escherichia coli.

    PubMed Central

    Schifferli, D M; Alrutz, M A

    1994-01-01

    The FasD protein is essential for the biogenesis of 987P fimbriae of Escherichia coli. In this study, subcellular fractionation was used to demonstrate that FasD is an outer membrane protein. In addition, the accessibility of FasD to proteases established the presence of surface-exposed FasD domains on both sides of the outer membrane. The fasD gene was sequenced, and the deduced amino acid sequence was shown to share homologous domains with a family of outer membrane proteins from various fimbrial systems. Similar to porins, fimbrial outer membrane proteins are relatively polar, lack typical hydrophobic membrane-spanning domains, and posses secondary structures predicted to be rich in turns and amphipathic beta-sheets. On the basis of the experimental data and structural predictions, FasD is postulated to consist essentially of surface-exposed turns and loops and membrane-spanning interacting amphipathic beta-strands. In an attempt to test this prediction, the fasD gene was submitted to random in-frame linker insertion mutagenesis. Preliminary experiments demonstrated that it was possible to produce fasD mutants, whose products remain functional for fimbrial export and assembly. Subsequently, 11 fasD alleles, containing linker inserts encoding beta-turn-inducing residues, were shown to express functional proteins. The insertion sites were designated permissive sites. The inserts used are expected to be least detrimental to the function of FasD when they are inserted into surface-exposed domains not directly involved in fimbrial export. In contrast, FasD is not expected to accommodate such residues in its amphipathic beta-strands without being destabilized in the membrane and losing function. All permissive sites were sequenced and shown to be located in or one residue away from predicted turns. In contrast, 5 of 10 sequenced nonpermissive sites were mapped to predicted amphipathic beta-strands. These results are consistent with the structural predictions for Fas

  4. The Role of Long Polar Fimbriae in Escherichia coli O104:H4 Adhesion and Colonization

    PubMed Central

    Ross, Brittany N.; Rojas-Lopez, Maricarmen; Cieza, Roberto J.; McWilliams, Brian D.; Torres, Alfredo G.

    2015-01-01

    A renewed interest in Shiga toxin-producing Escherichia coli (STEC) strains was sparked due to the appearance of an outbreak in 2011, causing 3,816 diarrheal cases and some deaths in Europe. The causative strain was classified as enteroaggregative E. coli of serotype O104:H4 that had acquired Shiga toxin genes. The ability of STEC O104:H4 to cause disease relies greatly on the bacteria’s capacity to colonize, persist, and produce Shiga toxin. However, not much is known about the colonization factors of this strain. Because long polar fimbriae (lpf) lpf1 and lpf2 operons encode important colonization factors in other STEC isolates and E. coli O104:H4 possesses both loci, we hypothesized that Lpf is required for adhesion and colonization. In this study, isogenic lpfA1 and lpfA2 major fimbrial subunit mutants were constructed. To determine their role in O104:H4’s virulence, we assessed their ability to adhere to non-polarized and polarized intestinal epithelial cells. The ΔlpfA1 showed decreased adherence in both cell systems, while the ΔlpfA2 only showed a decrease in adherence to polarized Caco-2 cells. We also tested the O104:H4 mutants’ ability to form biofilm and found that the ΔlpfA1 was unable to form a stable biofilm. In an in vivo murine model of intestinal colonization, the ΔlpfA1 had a reduced ability to colonize the cecum and large intestine, consistent with the in vitro data. Further, we tested the lpfA1 mutants’ ability to compete against the wild type. We found that in the in vitro and in vivo models, the presence of the wild type O104:H4 facilitates increased adherence of the ΔlpfA1 to levels exceeding that of the wild type. Overall, our data demonstrated that Lpf1 is one of the factors responsible for O104:H4 intestinal adhesion and colonization. PMID:26517878

  5. Environmental regulation of the long polar fimbriae 2 of enterohemorrhagic Escherichia coli O157:H7

    PubMed Central

    Arenas-Hernández, Margarita M.P.; Rojas-López, Maricarmen; Medrano-López, Abraham; Nuñez-Reza, Karen J.; Puente, José Luis; Martínez-Laguna, Ygnacio; Torres, Alfredo G.

    2014-01-01

    The molecular mechanisms controlling expression of the Long Polar Fimbriae 2 (Lpf2) of enterohemorrhagic E. coli (EHEC) O157:H7 were evaluated. Primer extension was used to locate the lpfA2 transcriptional start site in EHEC strain EDL933 at 171 bp upstream of the lpfA2 start codon. Semi-quantitative RT-PCR demonstrated that the highest lpfA2 expression occurs between an OD600 of 1.0 and 1.2 in DMEM at pH 6.5 and 37°C. The level of lpfA2 transcription at OD600 1.2 and pH 6.5 was 4-times greater than that at pH 7.2. Although lpfA2 expression was decreased under iron-depleted conditions, its expression was increased in a Ferric-uptake-regulator (Fur) mutant strain. The lpfA2 transcript was 0.7 and 2-times more abundant in wt EHEC grown in DMEM pH 6.5 plus iron and MacConkey broth at 25°C, respectively, than in DMEM at pH 6.5. The lpf2 expression in DMEM pH 6.5 plus iron and bile salts was 2.7-times more abundant and similar to MacConkey. Further, transcription in the EDL933Δfur was 0.6 and 0.8-times higher as compared to the wt strain grown in DMEM pH 6.5 plus iron and MacConkey broth, respectively. Electrophoretic mobility shift assays (EMSA) showed that purified Fur interacts with the lpf2 regulatory region, indicating that Fur-repression is exerted by direct binding to the promoter region. In summary, we demonstrated that the EHEC lpf2 operon is regulated in response to temperature, pH, bile salts and iron, during exponential phase of growth, and controlled by Fur. PMID:24966050

  6. Oral Immunization with a Salmonella typhimurium Vaccine Vector Expressing Recombinant Enterotoxigenic Escherichia coli K99 Fimbriae Elicits Elevated Antibody Titers for Protective Immunity

    PubMed Central

    Ascón, Miguel A.; Hone, David M.; Walters, Nancy; Pascual, David W.

    1998-01-01

    Bovine enterotoxigenic Escherichia coli (ETEC) continues to cause mortality in piglets and newborn calves. In an effort to develop a safe and effective vaccine for the prevention of F5+ ETEC infections, a balanced lethal asd+ plasmid carrying the complete K99 operon was constructed and designated pMAK99-asd+. Introduction of this plasmid into an attenuated Salmonella typhimurium Δaro Δasd strain, H683, resulted in strain AP112, which stably expresses E. coli K99 fimbriae. A single oral immunization of BALB/c and CD-1 mice with strain AP112 elicited significant mucosal immunoglobulin A (IgA) titers that remained elevated for >11 weeks. IgA and IgG responses in serum specific for K99 fimbriae were also induced, with a prominent IgG1, as well as IgG2a and IgG2b, titer. To assess the derivation of these antibodies, a K99 isotype-specific B-cell ELISPOT analysis was conducted by using mononuclear cells from the lamina propria of the small intestines (LP), Peyer’s patches (PP), and spleens of vaccinated and control BALB/c mice. This analysis revealed elevated numbers of K99 fimbria-specific IgA-producing cells in the LP, PP, and spleen, whereas elevated K99 fimbria-specific IgG-producing cells were detected only in the PP and spleen. These antibodies were important for protective immunity. One-day-old neonates from dams orally immunized with AP112 were provided passive protection against oral challenge with wild-type ETEC, in contrast to challenged neonates from unvaccinated dams or from dams vaccinated with a control Salmonella vector. These results confirm that oral Salmonella vaccine vectors effectively deliver K99 fimbriae to mucosal inductive sites for sustained elevation of IgA and IgG antibodies and for eliciting protective immunity. PMID:9784559

  7. Micro-evolutionary processes in sablefish Anoplopoma fimbria, based on polymorphism of the two sites of mitochondrial DNA.

    PubMed

    Orlova, S Y; Orlov, A M; Volkov, A A; Novikov, R N

    2014-01-01

    Sablefish Anoplopoma fimbria is a deep-sea fish, endemic to the North Pacific Ocean, with continuous range from southern California to the central part of Honshu Island, including the Bering and Okhotsk Seas. It is an important commercial species and a promising object for aquaculture. Compared to the eastern part of the range the population structure of sablefish in Asian waters is poorly studied. It is believed that sablefish goes to the Bering Sea and Pacific waters of Kamchatka and the Kuril Islands from the northeastern Pacific, and Asian waters are its eviction zone. Other authors suggest that replenishment of sablefish off the eastern Kamchatka and the Kuril Islands is not only due to migration of the adult fish from the Bering Sea along the continental slope, but also due to the drift of yearlings by Aleutian current over the American coast. PMID:25366282

  8. Phylogenetic group-associated differences in regulation of the common colonization factor Mat fimbria in Escherichia coli.

    PubMed

    Lehti, Timo A; Bauchart, Philippe; Kukkonen, Maini; Dobrindt, Ulrich; Korhonen, Timo K; Westerlund-Wikström, Benita

    2013-03-01

    Heterogeneity of cell population is a key component behind the evolutionary success of Escherichia coli. The heterogeneity supports species adaptation and mainly results from lateral gene transfer. Adaptation may also involve genomic alterations that affect regulation of conserved genes. Here we analysed regulation of the mat (or ecp) genes that encode a conserved fimbrial adhesin of E. coli. We found that the differential and temperature-sensitive expression control of the mat operon is dependent on mat promoter polymorphism and closely linked to phylogenetic grouping of E. coli. In the mat promoter lineage favouring fimbriae expression, the mat operon-encoded regulator MatA forms a positive feedback loop that overcomes the repression by H-NS and stabilizes the fimbrillin mRNA under low growth temperature, acidic pH or elevated levels of acetate. The study exemplifies phylogenetic group-associated expression of a highly common surface organelle in E. coli. PMID:23347101

  9. Intestinal receptors for adhesive fimbriae of enterotoxigenic Escherichia coli (ETEC) K88 in swine--a review.

    PubMed

    Jin, L Z; Zhao, X

    2000-09-01

    Determining the structure of the intestinal receptor for enterotoxigenic Escherichia coli (ETEC) K88 fimbriae will make it possible to develop new strategies to prevent K88+ ETEC-induced disease in pigs. Putative K88 adhesin receptors have been identified in both intestinal brush border and mucus preparations as either glycoproteins or glycolipids. Proteins with sizes of 25, 35, 40-42, 60, and 80 kDa in the intestinal mucus and 16, 23, 35, 40-70, 74, 210, and 240 kDa in brush border membranes were reported to bind specifically to K88ab and K88ac fimbriae. The factors accounting for these variable results may include the variants of K88, ages, breeds, and phenotypes of pigs, and even the sampling sites in the small intestine. Of the reported K88 receptors, only three brush border receptors, i.e., a pair of mucin-type sialoglycoproteins (210 kDa or 240 kDa), an intestinal neutral glycosphingolipid (IGLad), and a 74-kDa transferrin glycoprotein (GP74), have fulfilled the criteria as phenotype-specific K88 fimbrial receptors. Inhibiting the attachment of ETEC to intestine by modifying the receptor attachment sites has been the key for developing novel approaches to preventing ETEC-induced diarrhea in pigs. These include: (1) receptor analogs from a variety of biological sources, (2) an enteric protected protease, (3) chicken egg-yolk containing anti-K88 fimbrial antibodies, and (4) some Lactobacillus isolates producing proteinaceous components or carbohydrates interacting with mucus components. Future studies should be directed to further characterize the carbohydrate and protein moieties of receptors recognized by the K88 adhesin variants and to identify the genes responsible for susceptibility to K88+ infections. PMID:11030565

  10. Flagellin and F4 fimbriae have opposite effects on biofilm formation and quorum sensing in F4ac+ enterotoxigenic Escherichia coli.

    PubMed

    Zhou, Mingxu; Guo, Zhiyan; Yang, Yang; Duan, Qiangde; Zhang, Qi; Yao, Fenghua; Zhu, Jun; Zhang, Xinjun; Hardwidge, Philip R; Zhu, Guoqiang

    2014-01-10

    Bacteria that form biofilms are often highly resistant to antibiotics and are capable of evading the host immune system. To evaluate the role of flagellin and F4 fimbriae on biofilm formation by enterotoxigenic Escherichia coli (ETEC), we deleted the fliC (encoding the major flagellin protein) and/or the faeG (encoding the major subunit of F4 fimbriae) genes from ETEC C83902. Biofilm formation was reduced in the fliC mutant but increased in the faeG mutant, as compared with the wild-type strain. The expression of AI-2 quorum sensing associated genes was regulated in the fliC and faeG mutants, consistent with the biofilm formation of these strains. But, deleting fliC and/or faeG also inhibited AI-2 quorum sensing activity. PMID:24238669

  11. Salmonella enterica Serovar Typhimurium Requires the Lpf, Pef, and Tafi Fimbriae for Biofilm Formation on HEp-2 Tissue Culture Cells and Chicken Intestinal Epithelium

    PubMed Central

    Ledeboer, Nathan A.; Frye, Jonathan G.; McClelland, Michael; Jones, Bradley D.

    2006-01-01

    Recent work has demonstrated that Salmonella enterica serovar Typhimurium forms biofilms on HEp-2 tissue culture cells in a type 1 fimbria-dependent manner. To investigate how biofilm growth of HEp-2 tissue culture cells affects gene expression in Salmonella, we compared global gene expression during planktonic growth and biofilm growth. Microarray results indicated that the transcription of ∼100 genes was substantially altered by growth in a biofilm. These genes encode proteins with a wide range of functions, including antibiotic resistance, central metabolism, conjugation, intracellular survival, membrane transport, regulation, and fimbrial biosynthesis. The identification of five fimbrial gene clusters was of particular interest, as we have demonstrated that type 1 fimbriae are required for biofilm formation on HEp-2 cells and murine intestinal epithelium. Mutations in each of these fimbriae were constructed in S. enterica serovar Typhimurium strain BJ2710, and the mutants were found to have various biofilm phenotypes on plastic, HEp-2 cells, and chicken intestinal tissue. The pef and csg mutants were defective for biofilm formation on each of the three surfaces tested, while the lpf mutant exhibited a complete loss of the ability to form a biofilm on chicken intestinal tissue but only an intermediate loss of the ability to form a biofilm on tissue culture cells and plastic surfaces. The bcf mutant displayed increased biofilm formation on both HEp-2 cells and chicken intestinal epithelium, while the sth mutant had no detectable biofilm defects. In all instances, the mutants could be restored to a wild-type phenotype by a plasmid carrying the functional genes. This is the first work to identify the genomic responses of Salmonella to biofilm formation on host cells, and this work highlights the importance of fimbriae in adhering to and adapting to a eukaryotic cell surface. An understanding of these interactions is likely to provide new insights for intervention

  12. Chloroplasts assemble the major subunit FaeG of Escherichia coli F4 (K88) fimbriae to strand-swapped dimers.

    PubMed

    Van Molle, Inge; Joensuu, Jussi J; Buts, Lieven; Panjikar, Santosh; Kotiaho, Mirkka; Bouckaert, Julie; Wyns, Lode; Niklander-Teeri, Viola; De Greve, Henri

    2007-05-01

    F4 fimbriae encoded by the fae operon are the major colonization factors associated with porcine neonatal and postweaning diarrhoea caused by enterotoxigenic Escherichia coli (ETEC). Via the chaperone/usher pathway, the F4 fimbriae are assembled as long polymers of the major subunit FaeG, which also possesses the adhesive properties of the fimbriae. Intrinsically, the incomplete fold of fimbrial subunits renders them unstable and susceptible to aggregation and/or proteolytic degradation in the absence of a specific periplasmic chaperone. In order to test the possibility of producing FaeG in plants, FaeG expression was studied in transgenic tobacco plants. FaeG was directed to different subcellular compartments by specific targeting signals. Targeting of FaeG to the chloroplast results in much higher yields than FaeG targeting to the endoplasmic reticulum or the apoplast. Two chloroplast-targeted FaeG variants were purified from tobacco plants and crystallized. The crystal structures show that chloroplasts circumvent the absence of the fimbrial assembly machinery by assembling FaeG into strand-swapped dimers. Furthermore, the structures reveal how FaeG combines the structural requirements of a major fimbrial subunit with its adhesive role by grafting an additional domain on its Ig-like core. PMID:17368480

  13. Development of a loop-mediated isothermal amplification (LAMP) for the detection of F5 fimbriae gene in enterotoxigenic Escherichia coli (ETEC).

    PubMed

    Jiang, Kuiyu; Zhu, Ying; Liu, Wenxin; Feng, Yufei; He, Lili; Guan, Weikun; Hu, Wenxia; Shi, Dongfang

    2012-11-01

    The objective of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the detection of F5 fimbriae gene in Enterotoxigenic Escherichia coli. A set of four primers were designed based on the conservative sequence of coding F5 fimbriae. Temperature and time condition, specificity test, and sensitivity test were performed with the DNA of Escherichia coli (F5+). The results showed that the optimal reaction condition for LAMP was achieved at 61 °C for 45 min in a water bath. Ladder-like products were produced with those F5-positive samples by LAMP, while no product was generated with other negative samples. The assay of LAMP had a detection limit equivalent to 72 cfu/tube, which was more sensitive than PCR (7.2 × 10(2) cfu/tube). The agreement rate between LAMP and PCR was 100 % in detecting simulation samples. Thus, the LAMP assay may be a new method for rapid detection of F5 fimbriae gene of ETEC. PMID:22890294

  14. IscR Regulates Synthesis of Colonization Factor Antigen I Fimbriae in Response to Iron Starvation in Enterotoxigenic Escherichia coli

    PubMed Central

    Haines, Sara; Arnaud-Barbe, Nadège; Poncet, David; Reverchon, Sylvie; Wawrzyniak, Julien; Nasser, William

    2015-01-01

    ABSTRACT Iron availability functions as an environmental cue for enteropathogenic bacteria, signaling arrival within the human host. As enterotoxigenic Escherichia coli (ETEC) is a major cause of human diarrhea, the effect of iron on ETEC virulence factors was evaluated here. ETEC pathogenicity is directly linked to production of fimbrial colonization factors and secretion of heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (ST). Efficient colonization of the small intestine further requires at least the flagellin binding adhesin EtpA. Under iron starvation, production of the CFA/I fimbriae was increased in the ETEC H10407 prototype strain. In contrast, LT secretion was inhibited. Furthermore, under iron starvation, gene expression of the cfa (CFA/I) and etp (EtpBAC) operons was induced, whereas transcription of toxin genes was either unchanged or repressed. Transcriptional reporter fusion experiments focusing on the cfa operon further showed that iron starvation stimulated cfaA promoter activity in ETEC, indicating that the impact of iron on CFA/I production was mediated by transcriptional regulation. Evaluation of cfaA promoter activity in heterologous E. coli single mutant knockout strains identified IscR as the regulator responsible for inducing cfa fimbrial gene expression in response to iron starvation, and this was confirmed in an ETEC ΔiscR strain. The global iron response regulator, Fur, was not implicated. IscR binding sites were identified in silico within the cfaA promoter and fixation confirmed by DNase I footprinting, indicating that IscR directly binds the promoter region to induce CFA/I. IMPORTANCE Pathogenic enterobacteria modulate expression of virulence genes in response to iron availability. Although the Fur transcription factor represents the global regulator of iron homeostasis in Escherichia coli, we show that several ETEC virulence factors are modulated by iron, with expression of the major fimbriae under the control of the iron

  15. Identification of novel sRNAs involved in biofilm formation, motility, and fimbriae formation in Escherichia coli.

    PubMed

    Bak, Geunu; Lee, Jungmin; Suk, Shinae; Kim, Daun; Young Lee, Ji; Kim, Kwang-Sun; Choi, Byong-Seok; Lee, Younghoon

    2015-01-01

    Bacterial small RNAs (sRNAs) are known regulators in many physiological processes. In Escherichia coli, a large number of sRNAs have been predicted, among which only about a hundred are experimentally validated. Despite considerable research, the majority of their functions remain uncovered. Therefore, collective analysis of the roles of sRNAs in specific cellular processes may provide an effective approach to identify their functions. Here, we constructed a collection of plasmids overexpressing 99 individual sRNAs, and analyzed their effects on biofilm formation and related phenotypes. Thirty-three sRNAs significantly affecting these cellular processes were identified. No consistent correlations were observed, except that all five sRNAs suppressing type I fimbriae inhibited biofilm formation. Interestingly, IS118, yet to be characterized, suppressed all the processes. Our data not only reveal potentially critical functions of individual sRNAs in biofilm formation and other phenotypes but also highlight the unexpected complexity of sRNA-mediated metabolic pathways leading to these processes. PMID:26469694

  16. Identification of novel sRNAs involved in biofilm formation, motility, and fimbriae formation in Escherichia coli

    PubMed Central

    Bak, Geunu; Lee, Jungmin; Suk, Shinae; Kim, Daun; Young Lee, Ji; Kim, Kwang-sun; Choi, Byong-Seok; Lee, Younghoon

    2015-01-01

    Bacterial small RNAs (sRNAs) are known regulators in many physiological processes. In Escherichia coli, a large number of sRNAs have been predicted, among which only about a hundred are experimentally validated. Despite considerable research, the majority of their functions remain uncovered. Therefore, collective analysis of the roles of sRNAs in specific cellular processes may provide an effective approach to identify their functions. Here, we constructed a collection of plasmids overexpressing 99 individual sRNAs, and analyzed their effects on biofilm formation and related phenotypes. Thirty-three sRNAs significantly affecting these cellular processes were identified. No consistent correlations were observed, except that all five sRNAs suppressing type I fimbriae inhibited biofilm formation. Interestingly, IS118, yet to be characterized, suppressed all the processes. Our data not only reveal potentially critical functions of individual sRNAs in biofilm formation and other phenotypes but also highlight the unexpected complexity of sRNA-mediated metabolic pathways leading to these processes. PMID:26469694

  17. Curli fimbriae are conditionally required in Escherichia coli O157:H7 for initial attachment and biofilm formation.

    PubMed

    Carter, Michelle Qiu; Louie, Jacqueline W; Feng, Doris; Zhong, Wayne; Brandl, Maria T

    2016-08-01

    Several species of enteric pathogens produce curli fimbriae, which may affect their interaction with surfaces and other microbes in nonhost environments. Here we used two Escherichia coli O157:H7 outbreak strains with distinct genotypes to understand the role of curli in surface attachment and biofilm formation in several systems relevant to fresh produce production and processing. Curli significantly enhanced the initial attachment of E. coli O157:H7 to spinach leaves and stainless steel surfaces by 5-fold. Curli was also required for E. coli O157:H7 biofilm formation on stainless steel and enhanced biofilm production on glass by 19-27 fold in LB no-salt broth. However, this contribution was not observed when cells were grown in sterile spinach lysates. Furthermore, both strains of E. coli O157:H7 produced minimal biofilms on polypropylene in LB no-salt broth but considerable amounts in spinach lysates. Under the latter conditions, curli appeared to slightly increase biofilm production. Importantly, curli played an essential role in the formation of mixed biofilm by E. coli O157:H7 and plant-associated microorganisms in spinach leaf washes, as revealed by confocal microscopy. Little or no E. coli O157:H7 biofilms were detected at 4 °C, supporting the importance of temperature control in postharvest and produce processing environments. PMID:27052705

  18. Uropathogenic Escherichia coli Modulates Immune Responses and Its Curli Fimbriae Interact with the Antimicrobial Peptide LL-37

    PubMed Central

    Kai-Larsen, Ylva; Lüthje, Petra; Chromek, Milan; Peters, Verena; Wang, Xiaoda; Holm, Åsa; Kádas, Lavinia; Hedlund, Kjell-Olof; Johansson, Jan; Chapman, Matthew R.; Jacobson, Stefan H.; Römling, Ute; Agerberth, Birgitta; Brauner, Annelie

    2010-01-01

    Bacterial growth in multicellular communities, or biofilms, offers many potential advantages over single-cell growth, including resistance to antimicrobial factors. Here we describe the interaction between the biofilm-promoting components curli fimbriae and cellulose of uropathogenic E. coli and the endogenous antimicrobial defense in the urinary tract. We also demonstrate the impact of this interplay on the pathogenesis of urinary tract infections. Our results suggest that curli and cellulose exhibit differential and complementary functions. Both of these biofilm components were expressed by a high proportion of clinical E. coli isolates. Curli promoted adherence to epithelial cells and resistance against the human antimicrobial peptide LL-37, but also increased the induction of the proinflammatory cytokine IL-8. Cellulose production, on the other hand, reduced immune induction and hence delayed bacterial elimination from the kidneys. Interestingly, LL-37 inhibited curli formation by preventing the polymerization of the major curli subunit, CsgA. Thus, even relatively low concentrations of LL-37 inhibited curli-mediated biofilm formation in vitro. Taken together, our data demonstrate that biofilm components are involved in the pathogenesis of urinary tract infections by E. coli and can be a target of local immune defense mechanisms. PMID:20661475

  19. Genes Related to Long Polar Fimbriae of Pathogenic Escherichia coli Strains as Reliable Markers To Identify Virulent Isolates▿ †

    PubMed Central

    Torres, Alfredo G.; Blanco, Miguel; Valenzuela, Patricio; Slater, Terry M.; Patel, Shilpa D.; Dahbi, Ghizlane; López, Cecilia; Barriga, Ximena Fernández; Blanco, Jesús E.; Gomes, Tânia A. T.; Vidal, Roberto; Blanco, Jorge

    2009-01-01

    Lpf (stands for long polar fimbriae) is one of the few adhesive factors of enterohemorrhagic Escherichia coli O157:H7 associated with colonization of the intestine. E. coli O157:H7 strains possess two lpf loci encoding highly regulated fimbrial structures. Database analysis of the genes encoding the major fimbrial subunits demonstrated that they are present in commensal as well as pathogenic (both intestinal and extraintestinal) E. coli strains and in Salmonella strains and that the lpfA1 and lpfA2 genes are highly prevalent among LEE (locus of enterocyte effacement)-positive E. coli strains associated with severe and/or epidemic disease. Further DNA sequence analysis of the lpfA1 and lpfA2 genes from different attaching-and-effacing E. coli strains has led us to the identification of several polymorphisms and the classification of the major fimbrial subunits into distinct variants. Using collections of pathogenic E. coli isolates from Europe and Latin America, we demonstrated that the different lpfA types are associated with the presence of specific intimin (eae) adhesin variants and, most importantly, that they are found in specific E. coli pathotypes. Our results showed that the use of these fimbrial genes as markers, in combination with the different intimin types, resulted in a specific test for the identification of E. coli O157:H7, distinguishing it from other pathogenic E. coli strains. PMID:19494071

  20. Immunohistochemical analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity on the developmental dentate gyrus and hippocampal fimbria in fetal mice.

    PubMed

    Kobayashi, Yoshihiro; Hirano, Tetsushi; Omotehara, Takuya; Hashimoto, Rie; Umemura, Yuria; Yuasa, Hideto; Masuda, Natsumi; Kubota, Naoto; Minami, Kiichi; Yanai, Shogo; Ishihara-Sugano, Mitsuko; Mantani, Youhei; Yokoyama, Toshifumi; Kitagawa, Hiroshi; Hoshi, Nobuhiko

    2015-11-01

    Dioxins are widespread persistent environmental contaminants with adverse impacts on humans and experimental animals. Behavioral and cognitive functions are impaired by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. TCDD exerts its toxicity via the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. The hippocampus, which plays important roles in episodic memory and spatial function, is considered vulnerable to TCDD-induced neurotoxicity, because it contains the AhR. We herein investigated the effects of TCDD toxicity on hippocampal development in embryonic mice. TCDD was administered to dams at 8.5 days postcoitum with a single dose of 20, 200, 2,000 and 5,000 ng/kg body weight (groups T20, T200, T2000 and T5000, respectively), and the brains were dissected from their pups at embryonic day 18.5. Immunohistochemical analysis demonstrated that the Glial Fibrillary Acidic Protein (GFAP) immunoreactivities in the dentate gyrus (DG) were reduced in the T5000 group. Granular GFAP immunoreactivity was observed in the hippocampal fimbria, and the number of immunoreactive fimbria was significantly decreased in the T5000 group. The number of Proliferating Cell Nuclear Antigen (PCNA)-positive cells was decreased in all TCDD-exposed groups and significantly reduced in the T20, T200 and T5000 groups. Together, these results demonstrate that maternal TCDD exposure has adverse impacts on neural stem cells (NSCs), neural precursor cells (NPCs) and granular cells in the DG and disrupts the NSC maintenance and timing of differentiation in the hippocampal fimbria, which in turn interrupt neuronal development in future generations of mice. PMID:26096965

  1. Immunohistochemical analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity on the developmental dentate gyrus and hippocampal fimbria in fetal mice

    PubMed Central

    KOBAYASHI, Yoshihiro; HIRANO, Tetsushi; OMOTEHARA, Takuya; HASHIMOTO, Rie; UMEMURA, Yuria; YUASA, Hideto; MASUDA, Natsumi; KUBOTA, Naoto; MINAMI, Kiichi; YANAI, Shogo; ISHIHARA-SUGANO, Mitsuko; MANTANI, Youhei; YOKOYAMA, Toshifumi; KITAGAWA, Hiroshi; HOSHI, Nobuhiko

    2015-01-01

    Dioxins are widespread persistent environmental contaminants with adverse impacts on humans and experimental animals. Behavioral and cognitive functions are impaired by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. TCDD exerts its toxicity via the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. The hippocampus, which plays important roles in episodic memory and spatial function, is considered vulnerable to TCDD-induced neurotoxicity, because it contains the AhR. We herein investigated the effects of TCDD toxicity on hippocampal development in embryonic mice. TCDD was administered to dams at 8.5 days postcoitum with a single dose of 20, 200, 2,000 and 5,000 ng/kg body weight (groups T20, T200, T2000 and T5000, respectively), and the brains were dissected from their pups at embryonic day 18.5. Immunohistochemical analysis demonstrated that the Glial Fibrillary Acidic Protein (GFAP) immunoreactivities in the dentate gyrus (DG) were reduced in the T5000 group. Granular GFAP immunoreactivity was observed in the hippocampal fimbria, and the number of immunoreactive fimbria was significantly decreased in the T5000 group. The number of Proliferating Cell Nuclear Antigen (PCNA)-positive cells was decreased in all TCDD-exposed groups and significantly reduced in the T20, T200 and T5000 groups. Together, these results demonstrate that maternal TCDD exposure has adverse impacts on neural stem cells (NSCs), neural precursor cells (NPCs) and granular cells in the DG and disrupts the NSC maintenance and timing of differentiation in the hippocampal fimbria, which in turn interrupt neuronal development in future generations of mice. PMID:26096965

  2. Preliminary X-ray diffraction analysis of CfaA, a molecular chaperone essential for the assembly of CFA/I fimbriae of human enterotoxigenic Escherichia coli

    PubMed Central

    Bao, Rui; Esser, Lothar; Poole, Steven; McVeigh, Annette; Chen, Yu-xing; Savarino, Stephen J.; Xia, Di

    2014-01-01

    Understanding of pilus bioassembly in Gram-negative bacteria stems mainly from studies of P pili and type 1 fimbriae of uropathogenic Escherichia coli, which are mediated by the classic chaperone–usher pathway (CUP). However, CFA/I fimbriae, a class 5 fimbria and intestinal colonization factor for enterotoxigenic E. coli (ETEC), are proposed to assemble via the alternate chaperone pathway (ACP). Both CUP and ACP fimbrial bioassembly pathways require the function of a periplasmic chaperone, but their corresponding proteins share very low similarity in primary sequence. Here, the crystallization of the CFA/I periplasmic chaperone CfaA by the hanging-drop vapor-diffusion method is reported. X-ray diffraction data sets were collected from a native CfaA crystal to 2 Å resolution and to 1.8 and 2.8 Å resolution, respectively, from a lead and a platinum derivative. These crystals displayed the symmetry of space group C2, with unit-cell parameters a = 103.6, b = 28.68, c = 90.60 Å, β = 119.7°. Initial phases were derived from multiple isomorphous replacement with anomalous scattering experiments using the data from the platinum and lead derivatives. This resulted in an interpretable electron-density map showing one CfaA molecule in an asymmetric unit. Sequence assignments were aided by anomalous signals from the heavy-atom derivatives. Refinement of the atomic model of CfaA is ongoing, which is expected to further understanding of the essential aspects and allowable variations in tertiary structure of the greater family of chaperones involved in chaperone–usher mediated bioassembly. PMID:24637755

  3. Preliminary X-ray diffraction analysis of CfaA, a molecular chaperone essential for the assembly of CFA/I fimbriae of human enterotoxigenic Escherichia coli.

    PubMed

    Bao, Rui; Esser, Lothar; Poole, Steven; McVeigh, Annette; Chen, Yu Xing; Savarino, Stephen J; Xia, Di

    2014-02-01

    Understanding of pilus bioassembly in Gram-negative bacteria stems mainly from studies of P pili and type 1 fimbriae of uropathogenic Escherichia coli, which are mediated by the classic chaperone-usher pathway (CUP). However, CFA/I fimbriae, a class 5 fimbria and intestinal colonization factor for enterotoxigenic E. coli (ETEC), are proposed to assemble via the alternate chaperone pathway (ACP). Both CUP and ACP fimbrial bioassembly pathways require the function of a periplasmic chaperone, but their corresponding proteins share very low similarity in primary sequence. Here, the crystallization of the CFA/I periplasmic chaperone CfaA by the hanging-drop vapor-diffusion method is reported. X-ray diffraction data sets were collected from a native CfaA crystal to 2 Å resolution and to 1.8 and 2.8 Å resolution, respectively, from a lead and a platinum derivative. These crystals displayed the symmetry of space group C2, with unit-cell parameters a = 103.6, b = 28.68, c = 90.60 Å, β = 119.7°. Initial phases were derived from multiple isomorphous replacement with anomalous scattering experiments using the data from the platinum and lead derivatives. This resulted in an interpretable electron-density map showing one CfaA molecule in an asymmetric unit. Sequence assignments were aided by anomalous signals from the heavy-atom derivatives. Refinement of the atomic model of CfaA is ongoing, which is expected to further understanding of the essential aspects and allowable variations in tertiary structure of the greater family of chaperones involved in chaperone-usher mediated bioassembly. PMID:24637755

  4. Equal effects of typical environmental and specific social enrichment on posttraumatic cognitive functioning after fimbria-fornix transection in rats.

    PubMed

    Gajhede Gram, Marie; Gade, Louise; Wogensen, Elise; Mogensen, Jesper; Malá, Hana

    2015-12-10

    Enriched environment (EE) has been shown to have beneficial effects on cognitive recovery after brain injury. Typical EE comprises three components: (i) enlarged living area providing physical activation, (ii) sensory stimulation, and (iii) social stimulation. The present study assessed the specific contribution of the social stimulation. Animals were randomly divided into groups of (1) a typical EE, (2) pure social enrichment (SE), or (3) standard housing (SH) and subjected to either a sham operation or transection of the fimbria-fornix (FF). The effect of these conditions on acquisition of a delayed alternation task in a T-maze was assessed. The sham control groups were not affected by housing conditions. In the lesioned groups, both typical EE and SE improved the task acquisition, compared to SH. A baseline one-hour activity measurement confirmed an equal level of physical activity in the EE and SE groups. After delayed alternation testing, pharmacological challenges (muscarinergic antagonist scopolamine and dopaminergic antagonist SKF-83566) were used to assess cholinergic and dopaminergic contributions to task solution. Scopolamine led to a marked impairment in all groups. SKF-83566 significantly enhanced the performance of the lesioned group subjected to SE. The results demonstrate that housing in a typical as well as atypical EE can enhance cognitive recovery after mechanical injury to the hippocampus. The scopolamine challenge revealed a cholinergic dependency during task performance in all groups, regardless of lesion and housing conditions. The dopaminergic challenge revealed a difference in the neural substrates mediating recovery in the lesioned groups exposed to different types of housing. PMID:26499260

  5. Molecular characterization of the gonadal kisspeptin system: Cloning, tissue distribution, gene expression analysis and localization in sablefish (Anoplopoma fimbria).

    PubMed

    Fairgrieve, Marian R; Shibata, Yasushi; Smith, Elizabeth K; Hayman, Edward S; Luckenbach, J Adam

    2016-01-01

    The kisspeptin system plays pivotal roles in the regulation of vertebrate reproduction. Classically, kisspeptin produced in the brain stimulates brain gonadotropin-releasing hormone signaling, which in turn activates the pituitary-gonad axis. Expression of the kisspeptin system has also been documented in peripheral tissues, including gonads of mammals and fishes. However, the fish gonadal kisspeptin system remained uncharacterized. Herein we report identification and characterization of four kisspeptin system mRNAs (kisspeptin 1 (kiss1), kiss2, and G protein-coupled receptor 54-1 (gpr54-1) and gpr54-2) in sablefish, Anoplopoma fimbria. Sablefish predicted protein sequences were highly similar to those of other marine teleosts, but less so to freshwater teleosts. Tissue distribution analyses revealed that all four kisspeptin-system transcripts were expressed in both brain and gonad. However, kiss2 was the predominant transcript in the gonads and the only transcript detected in ovulated eggs. Ontogenetic analysis of kiss2 expression in juvenile sablefish gonads demonstrated that levels were low during sex differentiation but increased with fish size and gonadal development. Dramatic increases in kiss2 mRNA occurred during primary oocyte growth, while levels remained relatively low in testes. In situ hybridization revealed that kiss2 mRNA was localized to cytoplasm of perinucleolus stage oocytes, suggesting it could play a local role in oogenesis or could be synthesized and stored within oocytes as maternal mRNA. This represents the first study to focus on the gonadal kisspeptin system in fishes and provides important tools for further investigation of both the gonadal and brain kisspeptin systems in sablefish. PMID:26386183

  6. Delayed intensive acquisition training alleviates the lesion-induced place learning deficits after fimbria-fornix transection in the rat.

    PubMed

    Malá, Hana; Rodríguez Castro, María; Pearce, Hadley; Kingod, Siff Camilla; Nedergaard, Signe Kjær; Scharff, Zakaryiah; Zandersen, Maja; Mogensen, Jesper

    2012-03-22

    This study evaluates the effects of two learning paradigms, intensive vs. baseline, on the posttraumatic acquisition of a water maze based place learning task. Rats were subjected either to a control operation (Sham) or to a fimbria-fornix (FF) transection, which renders the hippocampus dysfunctional and disrupts the acquisition of allocentric place learning. All animals were administered 30 post-lesion acquisition sessions, which spanned either 10 or 30days. The acquisition period was followed by a 7day pause after which a retention probe was administered. The lesioned animals were divided into 3 groups: i) Baseline Acquisition Paradigm (BAP) once daily for 30days starting 1week post-surgery; ii) Early Intensive Acquisition Paradigm (EIAP) 3 times daily for 10days starting 1week post-surgery; and iii) Late Intensive Acquisition Paradigm (LIAP) 3 times daily for 10days starting 3weeks post-surgery. Within the control animals, one group followed the schedule of BAP, and one group followed the schedule of Intensive Acquisition Paradigm (IAP). All lesioned animals showed an impaired task acquisition. LIAP was beneficial in FF animals, in that it led to a better acquisition of the place learning task than the two other acquisition paradigms. The FF/EIAP group did not show improved acquisition compared to the FF/BAP group. The control animals were not differentially affected by the two learning schedules. The findings have implications for cognitive rehabilitation after brain injury and support the assumption that intensive treatment can lead to an improved learning, even when the neural structures underlying such a process are compromised. However, the timing of intensive treatment needs to be considered further. PMID:22322151

  7. Immunization with the Recombinant Cholera Toxin B Fused to Fimbria 2 Protein Protects against Bordetella pertussis Infection

    PubMed Central

    Castuma, Celina E.; Hozbor, Daniela; Gaillard, María E.; Rumbo, Martín; Gómez, Ricardo M.

    2014-01-01

    This study examined the immunogenic properties of the fusion protein fimbria 2 of Bordetella pertussis (Fim2)—cholera toxin B subunit (CTB) in the intranasal murine model of infection. To this end B. pertussis Fim2 coding sequence was cloned downstream of the cholera toxin B subunit coding sequence. The expression and assembly of the fusion protein into pentameric structures (CTB-Fim2) were evaluated by SDS-PAGE and monosialotetrahexosylgaglioside (GM1-ganglioside) enzyme-linked immunosorbent assay (ELISA). To evaluate the protective capacity of CTB-Fim2, an intraperitoneal or intranasal mouse immunization schedule was performed with 50 μg of CTB-Fim2. Recombinant (rFim2) or purified (BpFim2) Fim2, CTB, and phosphate-buffered saline (PBS) were used as controls. The results showed that mice immunized with BpFim2 or CTB-Fim2 intraperitoneally or intranasally presented a significant reduction in bacterial lung counts compared to control groups (P < 0.01 or P < 0.001 , resp.). Moreover, intranasal immunization with CTB-Fim2 induced significant levels of Fim2-specific IgG in serum and bronchoalveolar lavage (BAL) and Fim2-specific IgA in BAL. Analysis of IgG isotypes and cytokines mRNA levels showed that CTB-Fim2 results in a mixed Th1/Th2 (T-helper) response. The data presented here provide support for CTB-Fim2 as a promising recombinant antigen against Bordetella pertussis infection. PMID:24982881

  8. Role of Proteus mirabilis MR/P fimbriae and flagella in adhesion, cytotoxicity and genotoxicity induction in T24 and Vero cells.

    PubMed

    Scavone, Paola; Villar, Silvia; Umpiérrez, Ana; Zunino, Pablo

    2015-06-01

    Proteus mirabilis is frequently associated with complicated urinary tract infections (UTI). It is proposed that several virulence factors are associated with P. mirabilis uropathogenicity. The aim of this work was to elucidate genotoxic and cytotoxic effects mediated by MR/P fimbriae and flagella in eukaryotic cells in vitro. Two cell lines (kidney- and bladder-derived) were infected with a clinical wild-type P. mirabilis strain and an MR/P and a flagellar mutant. We evaluated adhesion, genotoxicity and cytotoxicity by microscopy, comet assay and triple staining technique, respectively. Mutant strains displayed lower adhesion rates than the P. mirabilis wild-type strain and were significantly less effective to induce genotoxic and cytotoxic effects compared to the wild type. We report for the first time that P. mirabilis MR/P fimbriae and flagella mediate genotoxic and cytotoxic effects on eukaryotic cells, at least in in vitro conditions. These results could contribute to design new strategies for the control of UTI. PMID:25724892

  9. Crystallization and preliminary X-ray diffraction analyses of several forms of the CfaB major subunit of enterotoxigenic Escherichia coli CFA/I fimbriae

    PubMed Central

    Li, Yong-Fu; Poole, Steven; Rasulova, Fatima; McVeigh, Annette L.; Savarino, Stephen J.; Xia, Di

    2009-01-01

    Enterotoxigenic Escherichia coli (ETEC), a major global cause of diarrhea, initiates the pathogenic process via fimbriae-mediated attachment to the small intestinal epithelium. A common prototypic ETEC fimbria, colo­nization factor antigen I (CFA/I), consists of a tip-localized minor adhesive subunit CfaE and the stalk-forming major subunit CfaB, both of which are necessary for fimbrial assembly. To elucidate the structure of CFA/I at atomic resolution, three recombinant proteins were generated consisting of fusions of the minor and major subunits (CfaEB) and of two (CfaBB) and three (CfaBBB) repeats of the major subunit. Crystals of CfaEB diffracted X-rays to 2.1 Å resolution and displayed the symmetry of space group P21. CfaBB exhibited a crystal diffraction limit of 2.3 Å resolution and had the symmetry of space group P21212. CfaBBB crystallized in the monoclinic space group C2 and diffracted X-­rays to 2.3 Å resolution. These structures were determined using the molecular-replacement method. PMID:19255474

  10. Crystallization and preliminary X-ray diffraction analysis of CfaE, the adhesive subunit of the CFA/I fimbriae from human enterotoxigenic Escherichia coli

    SciTech Connect

    Li, Yong-Fu; Poole, Steven; Rasulova, Fatima; Esser, Lothar; Savarino, Stephen J.; Xia, Di

    2006-02-01

    The adhesin CfaE of the CFA/I fimbriae from human enterotoxigenic E. coli has been crystallized. CfaE crystals diffracted X-rays to better than 2.4 Å and phasing was solved by the SIRAS method. Enterotoxigenic Escherichia coli (ETEC) represents a formidable food and waterborne diarrheal disease threat of global importance. The first step in ETEC pathogenesis is bacterial attachment to small-intestine epithelial cells via adhesive fimbriae, many of which are genetically related to the prototype colonization factor antigen I (CFA/I). The minor fimbrial subunit CfaE is required for initiation of CFA/I fimbrial assembly and mediates bacterial attachment to host cell-surface receptors. A donor-strand complemented variant of CfaE (dscCfaE) was expressed with a hexahistidine tag, purified to homogeneity and crystallized using the hanging-drop vapor-diffusion method. X-ray diffraction data sets were collected to 2.4 Å resolution for both native and derivatized crystals and showed the symmetry of space group P6{sub 2}22, with unit-cell parameters a = b = 142.9, c = 231.9 Å. Initial phases were derived from the SIRAS approach and electron density showed two molecules in the crystallographic asymmetric unit. Sequence assignments were aided by anomalous signals from the selenium of an SeMet-derivatized crystal and from S atoms of a native crystal.

  11. Escherichia coli K88ac fimbriae expressing heat-labile and heat-stable (STa) toxin epitopes elicit antibodies that neutralize cholera toxin and STa toxin and inhibit adherence of K88ac fimbrial E. coli.

    PubMed

    Zhang, Chengxian; Zhang, Weiping

    2010-12-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Bacterial adhesins and heat-labile (LT) and heat-stable (ST) enterotoxins are the virulence determinants in ETEC diarrhea. It is believed that vaccines inducing anti-adhesin immunity to inhibit bacterial adherence and anti-toxin immunity to eliminate toxin activity would provide broad-spectrum protection against ETEC. In this study, an ETEC fimbrial adhesin was used as a platform to express LT and STa for adhesin-toxin fusion antigens to induce anti-toxin and anti-adhesin immunity. An epitope from the B subunit of LT toxin (LTP1, (8)LCSEYRNTQIYTIN(21)) and an STa toxoid epitope ((5)CCELCCNPQCAGCY(18)) were embedded in the FaeG major subunit of E. coli K88ac fimbriae. Constructed K88ac-toxin chimeric fimbriae were harvested and used for rabbit immunization. Immunized rabbits developed anti-K88ac, anti-LT, and anti-STa antibodies. Moreover, induced antibodies not only inhibited adherence of K88ac fimbrial E. coli to porcine small intestinal enterocytes but also neutralized cholera toxin and STa toxin. Data from this study demonstrated that K88ac fimbriae expressing LT and STa epitope antigens elicited neutralizing anti-toxin antibodies and anti-adhesin antibodies and suggested that E. coli fimbriae could serve as a platform for the development of broad-spectrum vaccines against ETEC. PMID:20980482

  12. Escherichia coli K88ac Fimbriae Expressing Heat-Labile and Heat-Stable (STa) Toxin Epitopes Elicit Antibodies That Neutralize Cholera Toxin and STa Toxin and Inhibit Adherence of K88ac Fimbrial E. coli▿

    PubMed Central

    Zhang, Chengxian; Zhang, Weiping

    2010-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Bacterial adhesins and heat-labile (LT) and heat-stable (ST) enterotoxins are the virulence determinants in ETEC diarrhea. It is believed that vaccines inducing anti-adhesin immunity to inhibit bacterial adherence and anti-toxin immunity to eliminate toxin activity would provide broad-spectrum protection against ETEC. In this study, an ETEC fimbrial adhesin was used as a platform to express LT and STa for adhesin-toxin fusion antigens to induce anti-toxin and anti-adhesin immunity. An epitope from the B subunit of LT toxin (LTP1, 8LCSEYRNTQIYTIN21) and an STa toxoid epitope (5CCELCCNPQCAGCY18) were embedded in the FaeG major subunit of E. coli K88ac fimbriae. Constructed K88ac-toxin chimeric fimbriae were harvested and used for rabbit immunization. Immunized rabbits developed anti-K88ac, anti-LT, and anti-STa antibodies. Moreover, induced antibodies not only inhibited adherence of K88ac fimbrial E. coli to porcine small intestinal enterocytes but also neutralized cholera toxin and STa toxin. Data from this study demonstrated that K88ac fimbriae expressing LT and STa epitope antigens elicited neutralizing anti-toxin antibodies and anti-adhesin antibodies and suggested that E. coli fimbriae could serve as a platform for the development of broad-spectrum vaccines against ETEC. PMID:20980482

  13. F1C Fimbriae Play an Important Role in Biofilm Formation and Intestinal Colonization by the Escherichia coli Commensal Strain Nissle 1917▿

    PubMed Central

    Lasaro, Melissa A.; Salinger, Nina; Zhang, Jing; Wang, Yantao; Zhong, Zhengtao; Goulian, Mark; Zhu, Jun

    2009-01-01

    Bacterial biofilm formation is thought to enhance survival in natural environments and during interaction with hosts. A robust colonizer of the human gastrointestinal tract, Escherichia coli Nissle 1917, is widely employed in probiotic therapy. In this study, we performed a genetic screen to identify genes that are involved in Nissle biofilm formation. We found that F1C fimbriae are required for biofilm formation on an inert surface. In addition, these structures are also important for adherence to epithelial cells and persistence in infant mouse colonization. The data suggest a possible connection between Nissle biofilm formation and the survival of this commensal within the host. Further study of the requirements for robust biofilm formation may improve the therapeutic efficacy of Nissle 1917. PMID:18997018

  14. Development and application of pathovar-specific monoclonal antibodies that recognize the lipopolysaccharide O antigen and the type IV fimbriae of Xanthomonas hyacinthi

    SciTech Connect

    Doorn, J. van; Ojanen-Reuhs, T.; Hollinger, T.C.; Reuhs, B.L.; Schots, A.; Boonekamp, P.M.; Oudega, B.

    1999-09-01

    The objective of this study was to develop a specific immunological diagnostic assay for yellow disease in hyacinths, using monoclonal antibodies (MAbs). Mice were immunized with a crude cell wall preparation (shear fraction) from Xanthomonas hyacinthi and with purified type IV fimbriae. Hybridomas were screened for a positive reaction with X. hyacinthi cells or fimbriae and for a negative reaction with X. translucens pv. graminis or Erwinia carotovora subsp. carotovora. Nine MAbs recognized fimbrial epitopes, as shown by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy; however, three of these MAbs had weak cross-reactions with two X. translucens pathovars in immunoblotting experiments. Seven MAbs reacted with lipopolysaccharides and yielded a low-mobility ladder pattern on immunoblots. Subsequent analysis of MAb 2E5 showed that it specifically recognized an epitope on the O antigen, which was found to consist of rhamnose and fucose in a 2:1 molar ratio. The cross-reaction of MAb 2E5 with all X. hyacinthi strains tested showed that this O antigen is highly conserved within this species. MAb 1B10 also reacted with lipopolysaccharides. MAbs 2E5 and 1B10 were further tested in ELISA and immunoblotting experiments with cells and extracts from other pathogens. No cross-reaction was found with 27 other Xanthomonas pathovars tested or with 14 other bacterial species from other genera, such as Erwinia and Pseudomonas, indicating the high specificity of these antibodies. MAbs 2E5 and 1B10 were shown to be useful in ELISA for the detection of X. hyacinthi in infected hyacinths.

  15. Development and Application of Pathovar-Specific Monoclonal Antibodies That Recognize the Lipopolysaccharide O Antigen and the Type IV Fimbriae of Xanthomonas hyacinthi

    PubMed Central

    van Doorn, J.; Ojanen-Reuhs, T.; Hollinger, T. C.; Reuhs, B. L.; Schots, A.; Boonekamp, P. M.; Oudega, B.

    1999-01-01

    The objective of this study was to develop a specific immunological diagnostic assay for yellow disease in hyacinths, using monoclonal antibodies (MAbs). Mice were immunized with a crude cell wall preparation (shear fraction) from Xanthomonas hyacinthi and with purified type IV fimbriae. Hybridomas were screened for a positive reaction with X. hyacinthi cells or fimbriae and for a negative reaction with X. translucens pv. graminis or Erwinia carotovora subsp. carotovora. Nine MAbs recognized fimbrial epitopes, as shown by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy; however, three of these MAbs had weak cross-reactions with two X. translucens pathovars in immunoblotting experiments. Seven MAbs reacted with lipopolysaccharides and yielded a low-mobility ladder pattern on immunoblots. Subsequent analysis of MAb 2E5 showed that it specifically recognized an epitope on the O antigen, which was found to consist of rhamnose and fucose in a 2:1 molar ratio. The cross-reaction of MAb 2E5 with all X. hyacinthi strains tested showed that this O antigen is highly conserved within this species. MAb 1B10 also reacted with lipopolysaccharides. MAbs 2E5 and 1B10 were further tested in ELISA and immunoblotting experiments with cells and extracts from other pathogens. No cross-reaction was found with 27 other Xanthomonas pathovars tested or with 14 other bacterial species from other genera, such as Erwinia and Pseudomonas, indicating the high specificity of these antibodies. MAbs 2E5 and 1B10 were shown to be useful in ELISA for the detection of X. hyacinthi in infected hyacinths. PMID:10473431

  16. Implementation of a Functional Observation Battery for the Assessment of Postoperative Well-being in Rats Subjected to Fimbria-Fornix Transection.

    PubMed

    Marschner, Linda; Wogensen, Elise; Mogensen, Jesper; Abelson, Klas

    2016-01-01

    The postoperative well-being of Wistar rats subjected to fimbria-fornix transections was assessed using a functional observational battery (FOB), including observations of relative body weight change, general condition, fur quality, body posture and movement, appetite, and pica behavior. Fimbria-fornix transected animals (FF), sham-operated animals (Sham), and two non-operated control groups with and without administration of buprenorphine (+BUP and -BUP, respectively) were observed twice daily for seven days after surgery. Buprenorphine (0.4 mg/kg) mixed in a nut paste for voluntary ingestion was supplied twice daily for 84 h to all groups except the -BUP control group starting on the day of surgery. Body weight was slightly decreased postoperatively in both surgical groups (FF and Sham) compared to control groups. The +BUP control group lost weight starting at day four after discontinuation of buprenorphine. Furthermore, the FF group exhibited significantly reduced general condition one day after surgery, with significantly affected body posture and movement for two days after surgery. In addition, mild pica behavior was observed in the FF group during the first postsurgical day. In conclusion, the FOB implemented in the present study appears to be a sensitive and accurate protocol for assessing animal well-being in the experimental setup applied. It is apparent that the FF transection is an invasive procedure that causes mildly adverse postoperative effects on the rats' well-being. We therefore recommend that this FOB is applied as a routine welfare monitoring protocol in experiments using mechanical central nervous system injury models, such as FF transection. PMID:26912816

  17. The Klebsiella pneumoniae YfgL (BamB) lipoprotein contributes to outer membrane protein biogenesis, type-1 fimbriae expression, anti-phagocytosis, and in vivo virulence.

    PubMed

    Hsieh, Pei-Fang; Hsu, Chun-Ru; Chen, Chun-Tang; Lin, Tzu-Lung; Wang, Jin-Town

    2016-07-01

    Klebsiella pneumoniae is an opportunistic pathogen that causes several kinds of infections, including pneumonia, bacteremia, urinary tract infection and community-acquired pyogenic liver abscess (PLA). Adhesion is the critical first step in the infection process. Our previous work demonstrated that the transcellular translocation is exploited by K. pneumoniae strains to migrate from the gut flora into other tissues, resulting in systemic infections. However, the initial stages of K. pneumoniae infection remain unclear. In this study, we demonstrated that a K. pneumoniae strain deleted for yfgL (bamB) exhibited reduced adherence to and invasion of host cells; changed biogenesis of major β-barrel outer membrane proteins; decreased transcriptional expression of type-1 fimbriae; and increased susceptibility to vancomycin and erythromycin. The yfgL deletion mutant also had reduced ability to against neutrophil phagocytosis; exhibited decreased induction of host IL-6 production; and was profoundly attenuated for virulence in a K. pneumoniae model of bacteremia. Thus, the K. pneumoniae YfgL lipoprotein mediates in outer membrane proteins biogenesis and is crucial for anti-phagocytosis and survival in vivo. These data provide a new insight for K. pneumoniae attachment and such knowledge could facilitate preventive therapies or alternative therapies against K. pneumoniae. PMID:27029012

  18. Crystallization and preliminary X-ray diffraction analysis of CfaE, the adhesive subunit of the CFA/I fimbriae from human enterotoxigenic Escherichia coli

    PubMed Central

    Li, Yong-Fu; Poole, Steven; Rasulova, Fatima; Esser, Lothar; Savarino, Stephen J.; Xia, Di

    2006-01-01

    Enterotoxigenic Escherichia coli (ETEC) represents a formidable food and waterborne diarrheal disease threat of global importance. The first step in ETEC pathogenesis is bacterial attachment to small-intestine epithelial cells via adhesive fimbriae, many of which are genetically related to the prototype colonization factor antigen I (CFA/I). The minor fimbrial subunit CfaE is required for initiation of CFA/I fimbrial assembly and mediates bacterial attachment to host cell-surface receptors. A donor-strand complemented variant of CfaE (dscCfaE) was expressed with a hexahistidine tag, purified to homogeneity and crystallized using the hanging-drop vapor-diffusion method. X-ray diffraction data sets were collected to 2.4 Å resolution for both native and derivatized crystals and showed the symmetry of space group P6222, with unit-cell parameters a = b = 142.9, c = 231.9 Å. Initial phases were derived from the SIRAS approach and electron density showed two molecules in the crystallographic asymmetric unit. Sequence assignments were aided by anomalous signals from the selenium of an SeMet-derivatized crystal and from S atoms of a native crystal. PMID:16511280

  19. Crohn disease–associated adherent-invasive E. coli bacteria target mouse and human Peyer’s patches via long polar fimbriae

    PubMed Central

    Chassaing, Benoit; Rolhion, Nathalie; de Vallée, Amélie; Salim, Sa’ad Y.; Prorok-Hamon, Maelle; Neut, Christel; Campbell, Barry J.; Söderholm, Johan D.; Hugot, Jean-Pierre; Colombel, Jean-Frédéric; Darfeuille-Michaud, Arlette

    2011-01-01

    Crohn disease (CD) is a multifactorial disease in which an abnormal immune response in the gastrointestinal (GI) tract leads to chronic inflammation. The small intestine, particularly the ileum, of patients with CD is colonized by adherent-invasive E. coli (AIEC) — a pathogenic group of E. coli able to adhere to and invade intestinal epithelial cells. As the earliest inflammatory lesions are microscopic erosions of the epithelium lining the Peyer’s patches (PPs), we investigated the ability of AIEC bacteria to interact with PPs and the virulence factors involved. We found that AIEC bacteria could interact with mouse and human PPs via long polar fimbriae (LPF). An LPF-negative AIEC mutant was highly impaired in its ability to interact with mouse and human PPs and to translocate across monolayers of M cells, specialized epithelial cells at the surface of PPs. The prevalence of AIEC strains harboring the lpf operon was markedly higher in CD patients compared with controls. In addition, increased numbers of AIEC, but not LPF-deficient AIEC, bacteria were found interacting with PPs from Nod2–/– mice compared with WT mice. In conclusion, we have identified LPF as a key factor for AIEC to target PPs. This could be the missing link between AIEC colonization and the presence of early lesions in the PPs of CD patients. PMID:21339647

  20. Role of type 1 and S fimbriae in the pathogenesis of Escherichia coli O18:K1 bacteremia and meningitis in the infant rat.

    PubMed Central

    Saukkonen, K M; Nowicki, B; Leinonen, M

    1988-01-01

    The role of fimbriae in the pathogenesis of Escherichia coli infection was studied in the infant rat model. Rat pups were challenged intraperitoneally at the age of 5 days with E. coli K1 (strain IH3080, O18:K1:H7) and three different subpopulations (type 1, type S, or nonfimbriated) of it. All bacterial subpopulations were able to produce peritonitis, bacteremia, and meningitis. However, the type 1 fraction was the least virulent and the type S fraction was the most virulent, as judged by the bacterial counts in body fluids and by the mortality rates of the pups. Fimbrial phase variation to mainly the type-S-fimbriated forms was observed in all body fluids. An initially type-S-fimbriated inoculum remained predominantly type S fimbriated in the peritoneal fluid and blood. In the cerebrospinal fluid, however, about 50% of the bacteria were type S fimbriated and 50% were nonfimbriated 1 h after challenge with the type-S-fimbriated subpopulation; at later times the share of type-S-fimbriated bacteria also increased in the cerebrospinal fluid. PMID:2894363

  1. Characterization of F107 fimbriae of Escherichia coli 107/86, which causes edema disease in pigs, and nucleotide sequence of the F107 major fimbrial subunit gene, fedA.

    PubMed Central

    Imberechts, H; De Greve, H; Schlicker, C; Bouchet, H; Pohl, P; Charlier, G; Bertschinger, H; Wild, P; Vandekerckhove, J; Van Damme, J

    1992-01-01

    F107 fimbriae were isolated and purified from edema disease strain 107/86 of Escherichia coli. Plasmid pIH120 was constructed, which contains the gene cluster that codes for adhesive F107 fimbriae. The major fimbrial subunit gene, fedA, was sequenced. An open reading frame that codes for a protein with 170 amino acids, including a 21-amino-acid signal peptide, was found. The protein without the signal sequence has a calculated molecular mass of 15,099 Da. Construction of a nonsense mutation in the open reading frame of fedA abolished both fimbrial expression and the capacity to adhere to isolated porcine intestinal villi. In a screening of 28 reference edema disease strains and isolates from clinically ill piglets, fedA was detected in 24 cases (85.7%). In 20 (83.3%) of these 24 strains, fedA was found in association with Shiga-like toxin II variant genes, coding for the toxin that is characteristic for edema disease strains of E. coli. The fimbrial subunit gene was not detected in enterotoxigenic E. coli strains. Because of the capacity of E. coli HB101(pIH120) transformants to adhere to isolated porcine intestinal villi, the high prevalence of fedA in edema disease strains, and the high correlation with the Shiga-like toxin II variant toxin-encoding genes, we suggest that F107 fimbriae are an important virulence factor in edema disease strains of E. coli. Images PMID:1348723

  2. Role of F1C Fimbriae, Flagella, and Secreted Bacterial Components in the Inhibitory Effect of Probiotic Escherichia coli Nissle 1917 on Atypical Enteropathogenic E. coli Infection

    PubMed Central

    Kleta, Sylvia; Nordhoff, Marcel; Tedin, Karsten; Wieler, Lothar H.; Kolenda, Rafal; Oswald, Sibylle; Oelschlaeger, Tobias A.; Bleiß, Wilfried

    2014-01-01

    Enteropathogenic Escherichia coli (EPEC) is recognized as an important intestinal pathogen that frequently causes acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probiotic E. coli strain Nissle 1917 (EcN) is known to be effective in the treatment of several gastrointestinal disorders. While both in vitro and in vivo studies have described strong inhibitory effects of EcN on enteropathogenic bacteria, including pathogenic E. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenic E. coli (aEPEC) with respect to single infection steps, including adhesion, microcolony formation, and the attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagella, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific to EcN, but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of the inhibitory factors. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence-associated proteins of EPEC, but not their expression. PMID:24549324

  3. Protection of piglets against enteric colibacillosis by intranasal immunization with K88ac (F4ac) fimbriae and heat labile enterotoxin of Escherichia coli.

    PubMed

    Lin, Jun; Mateo, Kristina S; Zhao, Mojun; Erickson, Alan K; Garcia, Nuria; He, Dong; Moxley, Rodney A; Francis, David H

    2013-03-23

    Enterotoxigenic Escherichia coli (ETEC) is an important diarrheal agent of young domestic animals. Currently, there are no commercially available non-living vaccines to protect weaned pigs from the disease and no major veterinary biologics company markets a postweaning ETEC vaccine of any kind. While efforts have been made to develop a non-living postweaning ETEC vaccine for pigs, studies have been limited to the assessment of immune responses to experimental immunogens. In the present study, we describe a reproducible gnotobiotic piglet model of post-weaning ETEC diarrhea and efficacy tests in that model of subunit vaccines consisting of K88 (F4) fimbriae and/or heat labile enterotoxin (LT) delivered by the intranasal route. We also report antibody responses to the vaccine antigens. Piglets vaccinated with both antigens mounted a substantial immune response with serum and cecal antibody titers to K88 antigen significantly greater than those of controls. Serum anti-LT antibody titers were also significantly greater than those of controls. Piglets vaccinated with both antigens remained healthy following challenge with ETEC. At least some pigs vaccinated with either antigen alone, and most of the control piglets developed dehydrating diarrhea and suffered significant weight loss. The results of this study suggest that an intranasal vaccine consisting of both antigens is highly protective against a vigorous experimental challenge of pigs with K88+ ETEC, while that against either antigen alone is not. The current study provides a system whereby various ETEC antigens and/or combinations of antigens can be tested in exploring strategies for the development of vaccines for ETEC. PMID:23089483

  4. Fimbria-Encoding Gene yadC Has a Pleiotropic Effect on Several Biological Characteristics and Plays a Role in Avian Pathogenic Escherichia coli Pathogenicity

    PubMed Central

    Rojas, Thaís Cabrera Galvão; Maluta, Renato Pariz; Leite, Janaína Luisa; da Silva, Livia Pilatti Mendes; Nakazato, Gerson

    2015-01-01

    The extraintestinal pathogen termed avian pathogenic Escherichia coli (APEC) is known to cause colibacillosis in chickens. The molecular basis of APEC pathogenesis is not fully elucidated yet. In this work, we deleted a component of the Yad gene cluster (yadC) in order to understand the role of Yad in the pathogenicity of the APEC strain SCI-07. In vitro, the transcription level of yadC was upregulated at 41°C and downregulated at 22°C. The yadC expression in vivo was more pronounced in lungs than in spleen, suggesting a role in the early steps of the infection. Chicks infected with the wild-type and mutant strains presented, respectively, 80% and 50% mortality rates. The ΔyadC strain presented a slightly decreased ability to adhere to HeLa cells with or without the d-mannose analog compared with the wild type. Real-time PCR (RT-PCR) assays showed that fimH was downregulated (P < 0.05) and csgA and ecpA were slightly upregulated in the mutant strain, showing that yadC modulates expression of other fimbriae. Bacterial internalization studies showed that the ΔyadC strain had a lower number of intracellular bacteria recovered from Hep-2 cells and HD11 cells than the wild-type strain (P < 0.05). Motility assays in soft agar demonstrated that the ΔyadC strain was less motile than the wild type (P < 0.01). Curiously, flagellum-associated genes were not dramatically downregulated in the ΔyadC strain. Taken together, the results show that the fimbrial adhesin Yad contributes to the pathogenicity and modulates different biological characteristics of the APEC strain SCI-07. PMID:26502907

  5. Fimbria-Encoding Gene yadC Has a Pleiotropic Effect on Several Biological Characteristics and Plays a Role in Avian Pathogenic Escherichia coli Pathogenicity.

    PubMed

    Verma, Renu; Rojas, Thaís Cabrera Galvão; Maluta, Renato Pariz; Leite, Janaína Luisa; da Silva, Livia Pilatti Mendes; Nakazato, Gerson; Dias da Silveira, Wanderley

    2016-01-01

    The extraintestinal pathogen termed avian pathogenic Escherichia coli (APEC) is known to cause colibacillosis in chickens. The molecular basis of APEC pathogenesis is not fully elucidated yet. In this work, we deleted a component of the Yad gene cluster (yadC) in order to understand the role of Yad in the pathogenicity of the APEC strain SCI-07. In vitro, the transcription level of yadC was upregulated at 41°C and downregulated at 22°C. The yadC expression in vivo was more pronounced in lungs than in spleen, suggesting a role in the early steps of the infection. Chicks infected with the wild-type and mutant strains presented, respectively, 80% and 50% mortality rates. The ΔyadC strain presented a slightly decreased ability to adhere to HeLa cells with or without the d-mannose analog compared with the wild type. Real-time PCR (RT-PCR) assays showed that fimH was downregulated (P < 0.05) and csgA and ecpA were slightly upregulated in the mutant strain, showing that yadC modulates expression of other fimbriae. Bacterial internalization studies showed that the ΔyadC strain had a lower number of intracellular bacteria recovered from Hep-2 cells and HD11 cells than the wild-type strain (P < 0.05). Motility assays in soft agar demonstrated that the ΔyadC strain was less motile than the wild type (P < 0.01). Curiously, flagellum-associated genes were not dramatically downregulated in the ΔyadC strain. Taken together, the results show that the fimbrial adhesin Yad contributes to the pathogenicity and modulates different biological characteristics of the APEC strain SCI-07. PMID:26502907

  6. The study of adhesive forces between the type-3 fimbriae of Klebsiella pneumoniae and collagen-coated surfaces by using optical tweezers

    NASA Astrophysics Data System (ADS)

    Chan, Chiahan; Fan, Chia-chieh; Huang, Ying-Jung; Peng, Hwei-Ling; Long, Hsu

    2004-10-01

    Adherence to host cells by a bacterial pathogen is a critical step for establishment of infection. It will contribute greatly to the understanding of bacterial pathogenesis by studying the biological force between a single pair of pathogen and host cell. In our experiment, we use a calibrated optical tweezers system to detach a single Klebsiella pneumoniae, the pathogen, from collagen, the host. By gradually increasing the laser power of the optical tweezers until the Klebsiella pneumoniae is detached from the collagen, we obtain the magnitude of the adhesive force between them. This happens when the adhesive force is barely equal to the trapping force provided by the optical tweezers at that specific laser power. This study is important because Klebsiella pneumoniae is an opportunistic pathogen which causes suppurative lesions, urinary and respiratory tract infections. It has been proved that type 3 fimbrial adhesin (mrkD) is strongly associated with the adherence of Klebsiella pneumoniae. Besides, four polymorphic mrkD alleles: namely, mrkDv1, v2, v3, and v4, are typed by using RFLP. In order to investigate the relationship between the structure and the function for each of these variants, DNA fragments encoding the major fimbrial proteins mrkA, mrkB, mrkC are expressed together with any of the four mrkD adhesins in E. coli JM109. Our study shows that the E. coli strain carrying the mrkDv3 fimbriae has the strongest binding activity. This suggests that mrkDv3 is a key factor that enhances the adherence of Klebsiella Pneumoniae to human body.

  7. Contributions of EspA Filaments and Curli Fimbriae in Cellular Adherence and Biofilm Formation of Enterohemorrhagic Escherichia coli O157:H7.

    PubMed

    Sharma, Vijay K; Kudva, Indira T; Bearson, Bradley L; Stasko, Judith A

    2016-01-01

    In Escherichia coli O157:H7 (O157), the filamentous structure of the type III secretion system is produced from the polymerization of the EspA protein. EspA filaments are essential for O157 adherence to epithelial cells. In previous studies, we demonstrated that O157 hha deletion mutants showed increased adherence to HEp-2 cells and produced abundant biofilms. Transcriptional analysis revealed increased expression of espA as well as the csgA gene, which encodes curli fimbriae that are essential for biofilm formation. In the present study, we constructed hha espA, hha csgA, and hha csgA espA deletion mutants to determine the relative importance of EspA and CsgA in O157 adherence to HEp-2 cells and biofilm formation. In vitro adherence assays, conducted at 37°C in a tissue culture medium containing 0.1% glucose, showed that HEp-2 cell adherence required EspA because hha espA and hha csgA espA mutants adhered to HEp-2 cells at higher levels only when complemented with an espA-expressing plasmid. Biofilm assays performed at 28°C in a medium lacking glucose showed dependency of biofilm formation on CsgA; however EspA was not produced under these conditions. Despite production of detectable levels of EspA at 37°C in media supplemented with 0.1% glucose, the biofilm formation occurred independent of EspA. These results indicate dependency of O157 adherence to epithelial cells on EspA filaments, while CsgA promoted biofilm formation under conditions mimicking those found in the environment (low temperature with nutrient limitations) and in the digestive tract of an host animal (higher temperature and low levels of glucose). PMID:26900701

  8. Contributions of EspA Filaments and Curli Fimbriae in Cellular Adherence and Biofilm Formation of Enterohemorrhagic Escherichia coli O157:H7

    PubMed Central

    Sharma, Vijay K.; Kudva, Indira T.; Bearson, Bradley L.; Stasko, Judith A.

    2016-01-01

    In Escherichia coli O157:H7 (O157), the filamentous structure of the type III secretion system is produced from the polymerization of the EspA protein. EspA filaments are essential for O157 adherence to epithelial cells. In previous studies, we demonstrated that O157 hha deletion mutants showed increased adherence to HEp-2 cells and produced abundant biofilms. Transcriptional analysis revealed increased expression of espA as well as the csgA gene, which encodes curli fimbriae that are essential for biofilm formation. In the present study, we constructed hha espA, hha csgA, and hha csgA espA deletion mutants to determine the relative importance of EspA and CsgA in O157 adherence to HEp-2 cells and biofilm formation. In vitro adherence assays, conducted at 37°C in a tissue culture medium containing 0.1% glucose, showed that HEp-2 cell adherence required EspA because hha espA and hha csgA espA mutants adhered to HEp-2 cells at higher levels only when complemented with an espA-expressing plasmid. Biofilm assays performed at 28°C in a medium lacking glucose showed dependency of biofilm formation on CsgA; however EspA was not produced under these conditions. Despite production of detectable levels of EspA at 37°C in media supplemented with 0.1% glucose, the biofilm formation occurred independent of EspA. These results indicate dependency of O157 adherence to epithelial cells on EspA filaments, while CsgA promoted biofilm formation under conditions mimicking those found in the environment (low temperature with nutrient limitations) and in the digestive tract of an host animal (higher temperature and low levels of glucose). PMID:26900701

  9. Role of F1C fimbriae, flagella, and secreted bacterial components in the inhibitory effect of probiotic Escherichia coli Nissle 1917 on atypical enteropathogenic E. coli infection.

    PubMed

    Kleta, Sylvia; Nordhoff, Marcel; Tedin, Karsten; Wieler, Lothar H; Kolenda, Rafal; Oswald, Sibylle; Oelschlaeger, Tobias A; Bleiss, Wilfried; Schierack, Peter

    2014-05-01

    Enteropathogenic Escherichia coli (EPEC) is recognized as an important intestinal pathogen that frequently causes acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probiotic E. coli strain Nissle 1917 (EcN) is known to be effective in the treatment of several gastrointestinal disorders. While both in vitro and in vivo studies have described strong inhibitory effects of EcN on enteropathogenic bacteria, including pathogenic E. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenic E. coli (aEPEC) with respect to single infection steps, including adhesion, microcolony formation, and the attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagella, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific to EcN, but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of the inhibitory factors. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence-associated proteins of EPEC, but not their expression. PMID:24549324

  10. Altered neuronal responses and regulation of neurotrophic proteins in the medial septum following fimbria-fornix transection in CNTF- and leukaemia inhibitory factor-deficient mice.

    PubMed

    Naumann, Thomas; Steup, Andreas; Schnell, Oliver; Schubert, Klaus Oliver; Zhi, Qixia; Guijarro, Christian; Kirsch, Matthias; Hofmann, Hans-Dieter

    2006-10-01

    Degeneration of axotomized GABAergic septohippocampal neurones has been shown to be enhanced in ciliary neurotrophic factor (CNTF)-deficient mice following fimbria-fornix transection (FFT), indicating a neuroprotective function of endogenous CNTF. Paradoxically, however, the cholinergic population of septohippocampal neurones was more resistant to axotomy in these mutants. As leukaemia inhibitory factor (LIF) has been identified as a potential neuroprotective factor for the cholinergic medial septum (MS) neurones, FFT-induced responses were compared in CNTF(-/-), LIF(-/-) and CNTF/LIF double knockout mice. In CNTF(-/-) mice, FFT-induced cholinergic degeneration was confirmed to be attenuated as compared with wildtype mice. The expression of both LIF and LIF receptor beta was increased in the MS providing a possible explanation for the enhanced neuronal resistance to FFT in these animals. However, ablation of the LIF gene also produced paradoxical effects; following FFT in LIF(-/-) mice no loss of GABAergic or cholinergic MS neurones was detectable during the first postlesional week, suggesting that other efficient neuroprotective mechanisms are activated in these animals. In fact, enhanced activation of astrocytes, a source of neurotrophic proteins, was indicated by increased up-regulation of glial fibrillary acidic protein and vimentin expression. In addition, mRNA levels for neurotrophin signalling components (e.g. nerve growth factor, p75(NTR)) were differentially regulated. The positive effect on axotomized cholinergic neurones seen in CNTF(-/-) and LIF(-/-) mice as well as the increased up-regulation of astrogliose markers was abolished in CNTF/LIF double knockout animals. Our results indicate that endogenous CNTF and LIF are involved in the regulation of neuronal survival following central nervous system lesion and are integrated into a network of neurotrophic signals that mutually influence their expression and function. PMID:17074046

  11. The secondary Müllerian system, field effect, BRCA, and tubal fimbria: our evolving understanding of the origin of tubo-ovarian high-grade serous carcinoma and why assignment of primary site matters.

    PubMed

    Singh, Naveena; Gilks, C Blake; Wilkinson, Nafisa; McCluggage, W Glenn

    2015-08-01

    It has long been held that most epithelial ovarian carcinomas arise from the ovarian surface epithelium. Theories on origin were based on the assumption that there was a common cell of origin for all ovarian carcinoma histotypes, and that these histotypes were closely related and frequently admixed. It is now recognised that the histotypes are distinct diseases. Recent studies on early, organ-confined, non-uterine high-grade serous carcinoma (HGSC) have led to a change in our understanding of their anatomical site of origin. These studies were initially on patients at high risk of developing HGSC but more recently have been extended to cases without family history or genetic markers of increased risk. These have shown that incidental HGSC, when detected before dissemination, is most commonly identified in the tubal fimbria. As a result, we have had to revisit theories on the cell and site of origin of HGSC. This progress in our understanding has necessitated a change in how we handle cases in clinical practice, as it impacts on primary site assignment, which in turn has implications for staging. In this review we will discuss the evolution of our understanding of the cell of origin of HGSC, the evidence for the tubal fimbria as the anatomical site of origin of most non-uterine HGSC, and the clinical implications of these recent developments. PMID:26126051

  12. Inheritance of porcine receptors for enterotoxigenic Escherichia coli with fimbriae F4ad and their relation to other F4 receptors.

    PubMed

    Rampoldi, A; Bertschinger, H U; Bürgi, E; Dolf, G; Sidler, X; Bratus, A; Vögeli, P; Neuenschwander, S

    2014-06-01

    Enteric Escherichia coli infections are a highly relevant cause of disease and death in young pigs. Breeding genetically resistant pigs is an economical and sustainable method of prevention. Resistant pigs are protected against colonization of the intestine through the absence of receptors for the bacterial fimbriae, which mediate adhesion to the intestinal surface. The present work aimed at elucidation of the mode of inheritance of the F4ad receptor which according to former investigations appeared quite confusing. Intestines of 489 pigs of an experimental herd were examined by a microscopic adhesion test modified in such a manner that four small intestinal sites instead of one were tested for adhesion of the fimbrial variant F4ad. Segregation analysis revealed that the mixed inheritance model explained our data best. The heritability of the F4ad phenotype was estimated to be 0.7±0.1. There are no relations to the strong receptors for variants F4ab and F4ac. Targeted matings allowed the discrimination between two F4ad receptors, that is, a fully adhesive receptor (F4adRFA) expressed on all enterocytes and at all small intestinal sites, and a partially adhesive receptor (F4adRPA) variably expressed at different sites and often leading to partial bacterial adhesion. In pigs with both F4ad receptors, the F4adRPA receptor is masked by the F4adRFA. The hypothesis that F4adRFA must be encoded by at least two complementary or epistatic dominant genes is supported by the Hardy-Weinberg equilibrium statistics. The F4adRPA receptor is inherited as a monogenetic dominant trait. A comparable partially adhesive receptor for variant F4ab (F4abRPA) was also observed but the limited data did not allow a prediction of the mode of inheritance. Pigs were therefore classified into one of eight receptor phenotypes: A1 (F4abRFA/F4acR+/F4adRFA); A2 (F4abRFA/F4acR+/F4adRPA); B (F4abRFA/F4acR+/F4adR-); C1 (F4abRPA/F4acR-/F4adRFA); C2 (F4abRPA/F4acR-/F4adRPA); D1 (F4abR-/F4acR-/F4ad

  13. Proteins with molecular masses of 50 and 80 kilodaltons encoded by genes downstream from the fimbrilin gene (fimA) are components associated with fimbriae in the oral anaerobe Porphyromonas gingivalis.

    PubMed Central

    Yoshimura, F; Takahashi, Y; Hibi, E; Takasawa, T; Kato, H; Dickinson, D P

    1993-01-01

    Flanking DNA regions of the fimbrilin gene (designated fimA), which encodes the major subunit protein of Porphyromonas (Bacteroides) gingivalis fimbriae, were cloned in several manners from the P. gingivalis chromosome into Escherichia coli by screening with probes derived from a 2.5-kb SacI DNA fragment previously cloned. A total of 10.4 kb of DNA fragments from the P. gingivalis genome was cloned in the pUC plasmid. Expression of the fimA gene and possible flanking genes in the fragments cloned was examined in a pUC plasmid vector system and in a bacteriophage T7 RNA polymerase-promoter expression vector system. The results show that in the pUC plasmid system, a 45-kDa protein, a product of fimA, was only poorly expressed as a precursor of the fimbrilin protein (FimA) and could be detected from cell extracts in Western blotting (immunoblotting) analysis as a sharp band but not in colony immunoblotting analysis. On the other hand, in the T7 RNA polymerase-promoter system, the product of fimA and products of the possible flanking genes responsible for fimbriation were overproduced as thick bands of the 45-kDa protein and as 63-, 50-, and 80-kDa proteins, respectively, in stained electrophoresis gels. All of the recombinant proteins were insoluble and seemed to be expressed as precursors with leader peptides. The 63-kDa, 45-k*Da (a truncated protein of the 50-kDa protein), and 80-kDa proteins were purified after solubilization with sodium dodecyl sulfate. N-terminal amino acid sequences of the 45-k*Da and 80-kDa proteins were analyzed up to the first 35 residues with a gas-phase sequencer. Monospecific antibodies directed to the recombinant proteins, i.e., the 63-kDa, 45-k*Da, and 80-kDa proteins, were raised in rabbits. By using the antibodies, localization of their matured proteins in P. gingivalis was investigated by Western blotting analysis. Immunoblotting analysis suggests that at least the 50- and 80-kDa proteins, encoded by genes downstream from the fim

  14. Hemagglutinin, urease, and hemolysin production by Proteus mirabilis from clinical sources.

    PubMed

    Mobley, H L; Chippendale, G R

    1990-03-01

    Proteus mirabilis, a common cause of urinary tract infection, can lead to serious complications including pyelonephritis. Adherence factors, urease, and hemolysin may be virulence determinants. These factors were compared for bacteria cultured from 16 patients with acute pyelonephritis and 35 with catheter-associated bacteriuria and for 20 fecal isolates. Pyelonephritis isolates were more likely (P less than .05) to express the mannose-resistant/Proteus-like (MR/P) hemagglutinin in the absence of mannose-resistant/Klebsiella-like (MR/K) hemagglutinin than were catheter-associated or fecal isolates. Pyelonephritis isolates produced urease activity of 63 +/- 27 (mean +/- SD) mumol of NH3/min/mg of protein, not significantly different from catheter-associated or fecal isolates. Hybridization of Southern blots of P. mirabilis chromosomal DNA with two urease gene probes demonstrated that urease gene sequences were conserved in all isolates. Geometric mean of reciprocal hemolytic titers for pyelonephritis isolates was 27.9; for urinary catheter isolates, 18.0; and for fecal isolates, 55.7 (not significantly different, P greater than .1). Although in vivo expression of urease and hemolysin may not be reliable indexes of virulence, MR/P hemagglutination in the absence of MR/K hemagglutination may be necessary for development of pyelonephritis. PMID:2179424

  15. “Kinin danger signals proteolytically released by gingipain induce fimbriae-specific IFN-γ and IL-17-producing T cells in mice infected intramucosally with Porphyromonas gingivalis”

    PubMed Central

    Monteiro, Ana Carolina; Scovino, Aline; Raposo, Susane; Gaze, Vinicius Mussa; Cruz, Catia; Svensjö, Erik; Narciso, Marcelo Sampaio; Colombo, Ana Paula; Pesquero, João B.; Feres-Filho, Eduardo; Nguyen, Ky-Anh; Sroka, Aneta; Potempa, Jan; Scharfstein, Julio

    2009-01-01

    Porphyromonas gingivalis, a gram-negative bacterium that causes periodontitis, activates the kinin system via the cysteine protease R-gingipain. Using a model of buccal infection based on P. gingivalis inoculation in the anterior mandibular vestibule, here we studied whether kinins released by gingipain may link mucosal inflammation to T cell-dependent immunity through the activation of bradykinin B2 receptors (B2R). Our data show that P. gingivalis W83 (WT), but not gingipain deficient mutant or WT bacteria pretreated with gingipain inhibitors, elicited buccal edema and gingivitis in Balb/C or C57BL/6 mice. Studies in TLR2−/−, B2R−/− and neutrophil-depleted C57Bl/6 mice revealed that P. gingivalis induced edema through the sequential activation of TLR2/neutrophils, with the initial plasma leakage being amplified by gingipain-dependent release of vasoactive kinins from plasma-borne kininogens. We then used fimbriae (Fim) Ag as a read-out to verify if activation of the TLR2>PMN>B2R axis at early-stages of mucosal infection had impact on adaptive immunity. Analyzes of T cell recall responses indicated that gingipain drives B2R-dependent generation of IFN-γ-producing Fim T cells in submandibular draining LNs of Balb/C and C57BL/6 mice while IL-17-producing Fim T cells were generated only in Balb/C mice. In summary, our studies suggest that two virulence factors, LPS (an atypical TLR2 ligand) and gingipain, forges a trans-cellular cross-talk between TLR2/B2R, thus forming an innate axis that guides the development of Fim-specific T cells in mice challenged intrabuccally by P. gingivalis. Ongoing research may clarify if kinin-driven modulation of T cell responses may also influence the severity of chronic periodontitis. PMID:19687097

  16. A tripartite fusion, FaeG-FedF-LT(192)A2:B, of enterotoxigenic Escherichia coli (ETEC) elicits antibodies that neutralize cholera toxin, inhibit adherence of K88 (F4) and F18 fimbriae, and protect pigs against K88ac/heat-labile toxin infection.

    PubMed

    Ruan, Xiaosai; Liu, Mei; Casey, Thomas A; Zhang, Weiping

    2011-10-01

    Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) or F18 fimbriae and heat-labile (LT) and/or heat-stable (ST) toxins are the major cause of diarrhea in young pigs. Effective vaccines inducing antiadhesin (anti-K88 and anti-F18) and antitoxin (anti-LT and anti-ST) immunity would provide broad protection to young pigs against ETEC. In this study, we genetically fused nucleotides coding for peptides from K88ac major subunit FaeG, F18 minor subunit FedF, and LT toxoid (LT(192)) A2 and B subunits for a tripartite adhesin-adhesin-toxoid fusion (FaeG-FedF-LT(192)A2:B). This fusion was used for immunizations in mice and pigs to assess the induction of antiadhesin and antitoxin antibodies. In addition, protection by the elicited antiadhesin and antitoxin antibodies against a porcine ETEC strain was evaluated in a gnotobiotic piglet challenge model. The data showed that this FaeG-FedF-LT(192)A2:B fusion elicited anti-K88, anti-F18, and anti-LT antibodies in immunized mice and pigs. In addition, the anti-porcine antibodies elicited neutralized cholera toxin and inhibited adherence against both K88 and F18 fimbriae. Moreover, immunized piglets were protected when challenged with ETEC strain 30302 (K88ac/LT/STb) and did not develop clinical disease. In contrast, all control nonvaccinated piglets developed severe diarrhea and dehydration after being challenged with the same ETEC strain. This study clearly demonstrated that this FaeG-FedF-LT(192)A2:B fusion antigen elicited antibodies that neutralized LT toxin and inhibited the adherence of K88 and F18 fimbrial E. coli strains and that this fusion could serve as an antigen for vaccines against porcine ETEC diarrhea. In addition, the adhesin-toxoid fusion approach used in this study may provide important information for developing effective vaccines against human ETEC diarrhea. PMID:21813665

  17. Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa.

    PubMed Central

    Hoyne, P A; Haas, R; Meyer, T F; Davies, J K; Elleman, T C

    1992-01-01

    Pseudomonas aeruginosa K/2PfS, when transformed with an expression plasmid harboring the pilin gene (pilE1) of Neisseria gonorrhoeae MS11, was able to express and assemble gonococcal pilin monomers into surface-associated pili, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoelectron microscopy. Concomitant with the expression of gonococcal pili in P. aeruginosa was the virtual loss of production of P. aeruginosa K/2PfS pili normally associated with the host cell. Images PMID:1358873

  18. Correlation between virulence factors and in vitro biofilm formation by Escherichia coli strains.

    PubMed

    Naves, Plínio; del Prado, Gema; Huelves, Lorena; Gracia, Matilde; Ruiz, Vicente; Blanco, Jorge; Dahbi, Ghizlane; Blanco, Miguel; Ponte, María del Carmen; Soriano, Francisco

    2008-08-01

    The ability of 15 Escherichia coli strains to form biofilms on polystirene plates was studied. The strains were serotyped, and their phenotypic expression of surface virulence factors (VFs), and antibiotic susceptibility was also determined. Moreover, 30 VFs-associated genes were analysed, including 15 adhesins (papC, papG and its three alleles, sfa/focDE, sfaS, focG, afa/draBC, iha, bmaE, gafD, nfaE, fimH, fimAvMT78, agn43, F9 fimbriae and type 3 fimbriae-encoding gene clusters), four toxins (hlyA, cnf1, sat and tsh), four siderophore (iron, fyuA, iutA and iucD), five proctetins/invasion-encoding genes (kpsM II, kpsMT III, K1 kps variant- neuC, traT and ibeA), and the pathogenicity island malX and cvaC. Morphological appearance and thickness of biofilms of two strong and three weak biofilm producers were also studied by confocal laser scanning microscopy (CLSM). Seven strains were classified as strong biofilm producers and the remaining eight strains were regarded as weak biofilm producers. Mannose-resistant haemagglutination was the only phenotypically expressed surface virulence factor more frequently found in the strong biofilm group. Five virulence-associated genes were more common (p<0.05) in strong biofilm producers: papC and papG alleles, sfa/focDE, focG, hlyA and cnf1. CLSM images showed irregular biofilms with projections at the top mainly in strong biofilm. PMID:18486439

  19. Virulence factors in Escherichia coli urinary tract infection.

    PubMed Central

    Johnson, J R

    1991-01-01

    Uropathogenic strains of Escherichia coli are characterized by the expression of distinctive bacterial properties, products, or structures referred to as virulence factors because they help the organism overcome host defenses and colonize or invade the urinary tract. Virulence factors of recognized importance in the pathogenesis of urinary tract infection (UTI) include adhesins (P fimbriae, certain other mannose-resistant adhesins, and type 1 fimbriae), the aerobactin system, hemolysin, K capsule, and resistance to serum killing. This review summarizes the virtual explosion of information regarding the epidemiology, biochemistry, mechanisms of action, and genetic basis of these urovirulence factors that has occurred in the past decade and identifies areas in need of further study. Virulence factor expression is more common among certain genetically related groups of E. coli which constitute virulent clones within the larger E. coli population. In general, the more virulence factors a strain expresses, the more severe an infection it is able to cause. Certain virulence factors specifically favor the development of pyelonephritis, others favor cystitis, and others favor asymptomatic bacteriuria. The currently defined virulence factors clearly contribute to the virulence of wild-type strains but are usually insufficient in themselves to transform an avirulent organism into a pathogen, demonstrating that other as-yet-undefined virulence properties await discovery. Virulence factor testing is a useful epidemiological and research tool but as yet has no defined clinical role. Immunological and biochemical anti-virulence factor interventions are effective in animal models of UTI and hold promise for the prevention of UTI in humans. Images PMID:1672263

  20. Phenotypic Assays to Determine Virulence Factors of Uropathogenic Escherichia coli (UPEC) Isolates and their Correlation with Antibiotic Resistance Pattern

    PubMed Central

    Tabasi, Mohsen; Asadi Karam, Mohammad Reza; Habibi, Mehri; Yekaninejad, Mir Saeed; Bouzari, Saeid

    2015-01-01

    Objectives Urinary tract infection caused by uropathogenic Escherichia coli (UPEC) strains is one of the most important infections in the world. UPEC encode widespread virulence factors closely related with pathogenesis of the bacteria. The purpose of this study was to evaluate the presence of different phenotypic virulence markers in UPEC isolates and determine their correlation with antibiotic resistance pattern. Methods UPEC isolates from patients with different clinical symptoms of UTI were collected and screened for biofilm and hemolysin production, mannose resistant, and mannose sensitive hemagglutination (MRHA and MSHA, respectively). In addition, antimicrobial resistance pattern and ESBL-producing isolates were recorded. Results Of the 156 UPEC isolates, biofilm and hemolysin formation was seen in 133 (85.3%) and 53 (34%) isolates, respectively. Moreover, 98 (62.8%) and 58 (37.2%) isolates showed the presence of Types 1 fimbriae (MSHA) and P fimbriae (MRHA), respectively. Our results also showed a relationship between biofilm formation in UPEC isolated from acute cystitis patients and recurrent UTI cases. Occurrence of UTI was dramatically correlated with the patients' profiles. We observed that the difference in antimicrobial susceptibilities of the biofilm and nonbiofilm former isolates was statistically significant. The UPEC isolates showed the highest resistance to ampicillin, tetracycline, amoxicillin, and cotrimoxazole. Moreover, 26.9% of isolates were ESBL producers. Conclusion This study indicated that there is a relationship between the phenotypic virulence traits of the UPEC isolates, patients' profiles, and antibiotic resistance. Detection of the phenotypic virulence factors could help to improve understanding of pathogenesis of UPEC isolates and better medical intervention. PMID:26473094

  1. Discovery and phylogenetic analysis of novel members of class b enterotoxigenic Escherichia coli adhesive fimbriae.

    PubMed

    Nada, Rania A; Shaheen, Hind I; Khalil, Sami B; Mansour, Adel; El-Sayed, Nasr; Touni, Iman; Weiner, Matthew; Armstrong, Adam W; Klena, John D

    2011-04-01

    Enterotoxigenic Escherichia coli (ETEC) is recognized to be a common cause of acute watery diarrhea in children from developing countries. Colonization factors (CFAs) have been identified predominantly in ETEC isolates secreting heat-stable enterotoxin (ST) or cosecreting ST with a heat-labile toxin (LT). We hypothesized that LT-only-secreting ETEC produces unique colonization factors not previously described in ST and LTST-secreting ETEC. A set of degenerate primers based on nucleotide sequence similarities between the major structural genes of CS20 (csnA), CS18 (fotA), CS12 (cswA), and porcine antigen 987 (fasA) was developed and used to screen a collection of 266 LT-secreting ETEC isolates in which no known CFA was detected. PCR-amplified products of different molecular masses were obtained from 49 (18.4%) isolates. Nucleotide sequence analysis of the PCR amplicons followed by GenBank nucleotide BLASTn analysis revealed five novel DNA sequences; translated amino acid BLASTx analysis confirmed sequence similarity to class 1b major structural proteins encoded by csnA, fotA, and fasA. Strains expressing the novel CFAs were phylotyped and analyzed using multilocus sequence typing (MLST; Achtman scheme), and the types detected were compared to those of a collection of archived global E. coli strains. In conclusion, application of the degenerate primer sets to ETEC isolates from surveillance studies increased the total number of ETEC isolates with detectable CFAs by almost 20%. Additionally, MLST analysis suggests that for many CFAs, there may be a requirement for certain genetic backgrounds to acquire and maintain plasmids carrying genes encoding CFAs. PMID:21289147

  2. Epigenetic reprogramming of fallopian tube fimbriae in BRCA mutation carriers defines early ovarian cancer evolution

    PubMed Central

    Bartlett, Thomas E.; Chindera, Kantaraja; McDermott, Jacqueline; Breeze, Charles E.; Cooke, William R.; Jones, Allison; Reisel, Daniel; Karegodar, Smita T.; Arora, Rupali; Beck, Stephan; Menon, Usha; Dubeau, Louis; Widschwendter, Martin

    2016-01-01

    The exact timing and contribution of epigenetic reprogramming to carcinogenesis are unclear. Women harbouring BRCA1/2 mutations demonstrate a 30–40-fold increased risk of high-grade serous extra-uterine Müllerian cancers (HGSEMC), otherwise referred to as ‘ovarian carcinomas', which frequently develop from fimbrial cells but not from the proximal portion of the fallopian tube. Here we compare the DNA methylome of the fimbrial and proximal ends of the fallopian tube in BRCA1/2 mutation carriers and non-carriers. We show that the number of CpGs displaying significant differences in methylation levels between fimbrial and proximal fallopian tube segments are threefold higher in BRCA mutation carriers than in controls, correlating with overexpression of activation-induced deaminase in their fimbrial epithelium. The differentially methylated CpGs accurately discriminate HGSEMCs from non-serous subtypes. Epigenetic reprogramming is an early pre-malignant event integral to BRCA1/2 mutation-driven carcinogenesis. Our findings may provide a basis for cancer-preventative strategies. PMID:27216078

  3. Tubal Ligation Induces Quiescence in the Epithelia of the Fallopian Tube Fimbria.

    PubMed

    Tiourin, Ekaterina; Velasco, Victor S; Rosales, Miguel A; Sullivan, Peggy S; Janzen, Deanna M; Memarzadeh, Sanaz

    2015-10-01

    Tubal ligation keeps the fimbriated end of the fallopian tube intact while interrupting the conduit for sperm and egg between the uterus and ovary. Tubal ligation is associated with an approximately 20% decreased risk of high-grade serous ovarian cancers, which mounting evidence suggests arise from the distal fallopian tube epithelium. We postulated that biological changes at the epithelial cellular level of the distal fallopian tube may account for the surgical procedure's observed risk reduction. We compared the histology, presence of epithelial progenitors (basally located CD44-positive cells), and degree of epithelial proliferation (Ki67-positive cells) of distal fallopian tube from 10 patients with previous tubal ligation and 10 age-matched patients with uncut fallopian tubes. A significantly reduced population of proliferating epithelial progenitors (basally located CD44/Ki67 dual-positive cells) was detected in the tubal ligated specimens (P = .0002). To functionally assess the effect of tubal ligation, a murine model was utilized to compare the growth capacity of distal fallopian tube epithelial cells isolated from either ligated or sham-operated tubal epithelia. Murine fallopian tube epithelial cells isolated after tubal ligation showed a significantly reduced capacity to grow organoids in culture compared to sham-operated controls (P = .002). The findings of this study show that tubal ligation is associated with a reduced presence and decreased proliferation of progenitor cells in the distal fallopian tube epithelium. These compositional and functional changes suggest that tubal ligation induces quiescence of distal fallopian tube epithelial cells. PMID:25736327

  4. Production and regulation of functional amyloid curli fimbriae by Shiga toxin-producing Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Functional amyloid, in the form of adhesive fimbrial proteins termed curli, was first described in Salmonella and Escherichia coli. Curli fibers adhere to various host cells and structural proteins, interact with components of the host immune system, and participate in biofilm formation. Shiga toxin...

  5. Epigenetic reprogramming of fallopian tube fimbriae in BRCA mutation carriers defines early ovarian cancer evolution.

    PubMed

    Bartlett, Thomas E; Chindera, Kantaraja; McDermott, Jacqueline; Breeze, Charles E; Cooke, William R; Jones, Allison; Reisel, Daniel; Karegodar, Smita T; Arora, Rupali; Beck, Stephan; Menon, Usha; Dubeau, Louis; Widschwendter, Martin

    2016-01-01

    The exact timing and contribution of epigenetic reprogramming to carcinogenesis are unclear. Women harbouring BRCA1/2 mutations demonstrate a 30-40-fold increased risk of high-grade serous extra-uterine Müllerian cancers (HGSEMC), otherwise referred to as 'ovarian carcinomas', which frequently develop from fimbrial cells but not from the proximal portion of the fallopian tube. Here we compare the DNA methylome of the fimbrial and proximal ends of the fallopian tube in BRCA1/2 mutation carriers and non-carriers. We show that the number of CpGs displaying significant differences in methylation levels between fimbrial and proximal fallopian tube segments are threefold higher in BRCA mutation carriers than in controls, correlating with overexpression of activation-induced deaminase in their fimbrial epithelium. The differentially methylated CpGs accurately discriminate HGSEMCs from non-serous subtypes. Epigenetic reprogramming is an early pre-malignant event integral to BRCA1/2 mutation-driven carcinogenesis. Our findings may provide a basis for cancer-preventative strategies. PMID:27216078

  6. Nanobody Mediated Inhibition of Attachment of F18 Fimbriae Expressing Escherichia coli

    PubMed Central

    Moonens, Kristof; De Kerpel, Maia; Coddens, Annelies; Cox, Eric; Pardon, Els; Remaut, Han; De Greve, Henri

    2014-01-01

    Post-weaning diarrhea and edema disease caused by F18 fimbriated E. coli are important diseases in newly weaned piglets and lead to severe production losses in farming industry. Protective treatments against these infections have thus far limited efficacy. In this study we generated nanobodies directed against the lectin domain of the F18 fimbrial adhesin FedF and showed in an in vitro adherence assay that four unique nanobodies inhibit the attachment of F18 fimbriated E. coli bacteria to piglet enterocytes. Crystallization of the FedF lectin domain with the most potent inhibitory nanobodies revealed their mechanism of action. These either competed with the binding of the blood group antigen receptor on the FedF surface or induced a conformational change in which the CDR3 region of the nanobody displaces the D″-E loop adjacent to the binding site. This D″-E loop was previously shown to be required for the interaction between F18 fimbriated bacteria and blood group antigen receptors in a membrane context. This work demonstrates the feasibility of inhibiting the attachment of fimbriated pathogens by employing nanobodies directed against the adhesin domain. PMID:25502211

  7. Genomic Subtractive Hybridization and Selective Capture of Transcribed Sequences Identify a Novel Salmonella typhimurium Fimbrial Operon and Putative Transcriptional Regulator That Are Absent from the Salmonella typhi Genome

    PubMed Central

    Morrow, Brian J.; Graham, James E.; Curtiss, Roy

    1999-01-01

    Salmonella typhi, the etiologic agent of typhoid fever, is adapted to the human host and unable to infect nonprimate species. The genetic basis for host specificity in S. typhi is unknown. The avirulence of S. typhi in animal hosts may result from a lack of genes present in the broad-host-range pathogen Salmonella typhimurium. Genomic subtractive hybridization was successfully employed to isolate S. typhimurium genomic sequences which are absent from the S. typhi genome. These genomic subtracted sequences mapped to 17 regions distributed throughout the S. typhimurium chromosome. A positive cDNA selection method was then used to identify subtracted sequences which were transcribed by S. typhimurium following macrophage phagocytosis. A novel putative transcriptional regulator of the LysR family was identified as transcribed by intramacrophage S. typhimurium. This putative transcriptional regulator was absent from the genomes of the human-adapted serovars S. typhi and Salmonella paratyphi A. Mutations within this gene did not alter the level of S. typhimurium survival within macrophages or virulence within mice. A subtracted genomic fragment derived from the ferrichrome operon also hybridized to the intramacrophage cDNA. Nucleotide sequence analysis of S. typhimurium and S. typhi chromosomal sequences flanking the ferrichrome operon identified a novel S. typhimurium fimbrial operon with a high level of similarity to sequences encoding Proteus mirabilis mannose-resistant fimbriae. The novel fimbrial operon was absent from the S. typhi genome. The absence of specific genes may have allowed S. typhi to evolve as a highly invasive, systemic human pathogen. PMID:10496884

  8. Leucine-responsive regulatory protein Lrp and PapI homologues influence phase variation of CS31A fimbriae.

    PubMed

    Graveline, Richard; Garneau, Philippe; Martin, Christine; Mourez, Michaël; Hancock, Mark A; Lavoie, Rémi; Harel, Josée

    2014-08-15

    CS31A, a K88-related surface antigen specified by the clp operon, is a member of the type P family of adhesive factors and plays a key role in the establishment of disease caused by septicemic and enterotoxigenic Escherichia coli strains. Its expression is under the control of methylation-dependent transcriptional regulation, for which the leucine-responsive regulatory protein (Lrp) is essential. CS31A is preferentially in the OFF state and exhibits distinct regulatory features compared to the regulation of other P family members. In the present study, surface plasmon resonance and DNase I protection assays showed that Lrp binds to the distal moiety of the clp regulatory region with low micromolar affinity compared to its binding to the proximal moiety, which exhibits stronger, nanomolar affinity. The complex formation was also influenced by the addition of PapI or FooI, which increased the affinity of Lrp for the clp distal and proximal regions and was required to induce phase variation. The influence of PapI or FooI, however, was predominantly associated with a more complete shutdown of clp expression, in contrast to what has previously been observed with AfaF (a PapI ortholog). Taken together, these results suggest that the preferential OFF state observed in CS31A cells is mainly due to the weak interaction of the leucine-responsive regulatory protein with the clp distal region and that the PapI homolog favors the OFF phase. Within the large repertoire of fimbrial variants in the P family, our study illustrates that having a fimbrial operon that lacks its own PapI ortholog allows it to be more flexibly regulated by other orthologs in the cell. PMID:24914179

  9. Effect of dietary taurine supplementation on growth, feed efficiency, and nutrient composition of juvenile sablefish (Anoplopoma fimbria)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Juvenile sablefish were fed a low taurine, basal feed with seven graded levels of supplemental taurine to determine taurine requirements for growth and feed efficiency. The basal feed was plant based, formulated primarily with soy and corn proteins with a minimal (9%) amount of fishmeal. The unsuppl...

  10. Structure of CfaA Suggests a New Family of Chaperones Essential for Assembly of Class 5 Fimbriae

    PubMed Central

    Bao, Rui; Fordyce, April; Chen, Yu-Xing; McVeigh, Annette; Savarino, Stephen J.; Xia, Di

    2014-01-01

    Adhesive pili on the surface of pathogenic bacteria comprise polymerized pilin subunits and are essential for initiation of infections. Pili assembled by the chaperone-usher pathway (CUP) require periplasmic chaperones that assist subunit folding, maintain their stability, and escort them to the site of bioassembly. Until now, CUP chaperones have been classified into two families, FGS and FGL, based on the short and long length of the subunit-interacting loops between its F1 and G1 β-strands, respectively. CfaA is the chaperone for assembly of colonization factor antigen I (CFA/I) pili of enterotoxigenic E. coli (ETEC), a cause of diarrhea in travelers and young children. Here, the crystal structure of CfaA along with sequence analyses reveals some unique structural and functional features, leading us to propose a separate family for CfaA and closely related chaperones. Phenotypic changes resulting from mutations in regions unique to this chaperone family provide insight into their function, consistent with involvement of these regions in interactions with cognate subunits and usher proteins during pilus assembly. PMID:25122114

  11. Structure of CfaA suggests a new family of chaperones essential for assembly of class 5 fimbriae.

    PubMed

    Bao, Rui; Fordyce, April; Chen, Yu-Xing; McVeigh, Annette; Savarino, Stephen J; Xia, Di

    2014-08-01

    Adhesive pili on the surface of pathogenic bacteria comprise polymerized pilin subunits and are essential for initiation of infections. Pili assembled by the chaperone-usher pathway (CUP) require periplasmic chaperones that assist subunit folding, maintain their stability, and escort them to the site of bioassembly. Until now, CUP chaperones have been classified into two families, FGS and FGL, based on the short and long length of the subunit-interacting loops between its F1 and G1 β-strands, respectively. CfaA is the chaperone for assembly of colonization factor antigen I (CFA/I) pili of enterotoxigenic E. coli (ETEC), a cause of diarrhea in travelers and young children. Here, the crystal structure of CfaA along with sequence analyses reveals some unique structural and functional features, leading us to propose a separate family for CfaA and closely related chaperones. Phenotypic changes resulting from mutations in regions unique to this chaperone family provide insight into their function, consistent with involvement of these regions in interactions with cognate subunits and usher proteins during pilus assembly. PMID:25122114

  12. The role of Type 1, P and S fimbriae in binding of Escherichia coli to the canine endometrium.

    PubMed

    Krekeler, N; Marenda, M S; Browning, G F; Holden, K M; Charles, J A; Wright, P J

    2013-06-28

    Escherichia coli (E. coli) is the most commonly isolated infectious agent causing pyometra in bitches. Many E. coli strains isolated from the uteri of infected dogs carry several adhesin genes (fimH, papGIII and sfa). The objective of this study was to investigate the role of each adhesin gene product, acting alone or expressed in combination, in the bacterial binding to canine endometrium. E. coli strain P3, which was isolated from a uterus of a bitch naturally affected with pyometra, was shown by PCR to carry all three known fimbrial adhesin genes fimH, papGIII and sfa. Knockout (KO) mutants of this wildtype (P3-wt) strain were generated using insertional inactivation. Adhesion assays on anoestrous uteri of three post-pubertal bitches were undertaken. Overall, the number of bacteria adhering to canine endometrial biopsies was comparable between strains and no significant difference in the number of bound bacteria was found between the P3-wt strain and the single or double KO-strains. However, the triple knockout strain displayed less binding to the canine endometrium compared with the P3-wt strain. This study shows that a pathogenic E. coli strain (P3) isolated from the uterus of a bitch with pyometra was able to fully compensate for the loss of two of its three known adhesin genes. It was necessary to inactivate all three known adhesin genes in order to see a significant decrease in binding to canine endometrium. PMID:23523172

  13. Untangling a Repetitive Amyloid Sequence: Correlating Biofilm-Derived and Segmentally Labeled Curli Fimbriae by Solid-State NMR Spectroscopy.

    PubMed

    Schubeis, Tobias; Yuan, Puwei; Ahmed, Mumdooh; Nagaraj, Madhu; van Rossum, Barth-Jan; Ritter, Christiane

    2015-12-01

    Curli are functional bacterial amyloids produced by an intricate biogenesis machinery. Insights into their folding and regulation can advance our understanding of amyloidogenesis. However, gaining detailed structural information of amyloids, and their tendency for structural polymorphisms, remains challenging. Herein we compare high-quality solid-state NMR spectra from biofilm-derived and recombinantly produced curli and provide evidence that they adopt a similar, well-defined β-solenoid arrangement. Curli subunits consist of five sequence repeats, resulting in severe spectral overlap. Using segmental isotope labeling, we obtained the unambiguous sequence-specific resonance assignments and secondary structure of one repeat, and demonstrate that all repeats are most likely structurally equivalent. PMID:26474178

  14. Nucleotide sequence of the gene encoding the major subunit of CS3 fimbriae of enterotoxigenic Escherichia coli.

    PubMed Central

    Boylan, M; Smyth, C J; Scott, J R

    1988-01-01

    The complete nucleotide sequence of a 612-base-pair DNA fragment containing the gene for the major fimbrial subunit of CS3 of enterotoxigenic Escherichia coli is presented. A possible promoter region, a ribosome-binding site, and two potential signal peptidase cleavage sites are indicated. Unlike the best-studied fimbrial proteins, the predicted CS3 sequence has no Cys residues. PMID:2903130

  15. Transcriptional analysis and regulation of the sfa determinant coding for S fimbriae of pathogenic Escherichia coli strains.

    PubMed

    Morschhäuser, J; Uhlin, B E; Hacker, J

    1993-04-01

    The sfa determinant codes for S fimbrial adhesins which constitute adherence factors of pathogenic Escherichia coli strains. We have recently shown that the sfa determinant is transcribed from three promoters, pA, pB, and pC. In comparison with the promoters pB and pC, promoter pA, which is located in front of the structural gene sfaA, showed very weak activity. Here we have determined the exact positions of the mRNA start points by primer extension studies. We have also shown that mRNAs of 500, 700 and 1400 bases can be detected using oligonucleotide probes specific for the genes sfaB, sfaC and sfaA. SfaB and SfaC are positive regulators influencing fimbriation and the production of the S-specific adhesin which is encoded by the gene sfaS located in the distal half of the determinant. In addition, it is demonstrated that SfaB and SfaC interfere with the regulatory effect of the histone-like protein H-NS, encoded by a locus termed drdX or osmZ. In a drdX+ strain the regulators are necessary for transcription of the sfa determinant. In contrast, sfa expression is activator-independent in a drdX- strain. In this latter genetic background, a substantial fraction of the sfa transcripts is initiated from promoter pA. On the basis of these data we discuss a model for the regulation of this adhesin-specific determinant. PMID:8097559

  16. Hha Represses Biofilm Formation in Escherichia coli O157:H7 by Affecting the Expression of Flagella and Curli Fimbriae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen that produces a broad-spectrum of diarrheal illnesses in infected humans. Although the genetic and molecular mechanisms enabling EHEC O157:H7 to produce characteristic adherence on epithelial cells are well characterized, the g...

  17. Effects of erythropoietin on posttraumatic place learning in fimbria-fornix transected rats after a 30-day postoperative pause.

    PubMed

    Malá, Hana; Rodriguez Castro, Maria; Dall Jørgensen, Katrine; Mogensen, Jesper

    2007-10-01

    Human recombinant erythropoietin (EPO) has been shown to exert neuroprotective effects following both vascular and mechanical brain injury. Previously, we showed that behavioral symptoms associated with mechanical lesions of the hippocampus are nearly abolished due to EPO treatment. In these studies, the EPO administration took place simultaneously with the infliction of brain injury and the rehabilitation training started 6-7 days postoperatively. In the present study, we tested whether the therapeutic effect of EPO on the acquisition of an allocentric eight-arm radial maze spatial task also manifests itself if the rehabilitative training is postponed. Postoperatively, the animals were left without any specific stimulation for 30 days. The current results show an improved behavioral performance of the EPO-treated lesioned group relative to the saline-treated lesioned group, and confirm EPO's therapeutic effect even in case of postponed rehabilitation. However, compared to the control group, the EPO-treated lesioned group demonstrated an impaired task acquisition. All subjects eventually recovered functionally. Subsequently, the animals were given behavioral challenges during which the cue constellation in the room was changed. The challenges revealed that, although the EPO-treated lesion group had achieved the same level of task proficiency as the control group, the cognitive mechanisms mediating the task performance in the EPO-treated lesion group (as well as in the saline-treated lesion group) were dissimilar from those mediating the task in the control group. Both the EPO-treated and the saline-treated lesion group demonstrated an increased dependency on the original cue configuration. PMID:17970627

  18. Monitoring F1651 P-Like Fimbria Expression at the Single-Cell Level Reveals a Highly Heterogeneous Phenotype

    PubMed Central

    Graveline, Richard; Lavoie, Rémi; Garneau, Philippe; Daigle, France; Sénéchal, Serge; Martin, Christine

    2015-01-01

    F1651 and the pyelonephritis-associated pili (Pap) are two members of the type P family of adhesive factors. They play a key role in establishing disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) strains in animals and humans. Both F1651 and Pap are under the control of an epigenetic and reversible switch that defines the number of fimbriated (ON) and afimbriated (OFF) cells within a clonal population. Using the Gfp reporter system, we monitored in vitro the level of fluorescence intensity corresponding to the F1651 and Pap fimbrial synthesis. Monitoring individual Escherichia coli cells by flow cytometry and by real-time fluorescence microscopy, we identified cells associated with a low or high level of fluorescence intensity and a large amount of cells with partial levels of fluorescence, mostly present in the F1651 system. This mixed population identified through fluorescence intensity could be attributed to the high switching rate previously observed in F1651-positive bacteria. The fimbrial heterogeneous phenotype for these ExPEC could represent increased fitness in unpredictable environments. Our study illustrates that within the large repertoire of fimbrial variants such as the well-characterized Pap, F1651 is an exquisite example of regulatory expression that arms the bacterium with strategies for surviving in more than one particular environment. PMID:25712930

  19. Inhibition of EGFR pathway signaling and the metastatic potential of breast cancer cells by PA-MSHA mediated by type 1 fimbriae via a mannose-dependent manner.

    PubMed

    Liu, Z-B; Hou, Y-F; Zhu, J; Hu, D-L; Jin, W; Ou, Z-L; Di, G-H; Wu, J; Shen, Z-Z; Shao, Z-M

    2010-05-20

    To identify more therapeutic targets and clarify the detailed mechanisms of Pseudomonas aeruginosa-mannose-sensitive hemagglutinin (PA-MSHA) on breast cancer cells both in vitro and in vivo. PA-MSHA was administered to epidermal growth factor receptor (EGFR)-positive human breast cancer cell lines MDA-MB-231HM and MDA-MB-468 in vitro and to mice bearing tumor xenografts. The mannose cocultured test was used to detect the effect of mannose on PA-MSHA-induced cell proliferation, cell cycle arrest, apoptosis, and EGFR pathway signaling. We found that cells stimulated with PA-MSHA exhibited a downregulation of EGFR signaling. The addition of mannose partially inhibited the PA-MSHA-stimulated cell anti-proliferative effect, cell apoptosis, cell cycle arrest, activation of apoptosis-associated caspases, and even downregulation of the EGFR signaling pathway. In vivo, PA-MSHA treatment significantly suppressed mammary tumorigenesis in xenografts in mice and decreased lung metastasis in MDA-MB-231HM cell-transplanted mice. Tumor sample analyses confirmed inhibition of the EGFR pathway in the PA-MSHA-treated mice. In conclusion, this study showed that the involvement of the mannose-mediated EGFR pathway has a critical function in the preclinical rationale for the development of PA-MSHA for the treatment of human breast cancer. It also suggests the potentially beneficial use of PA-MSHA in adjuvant therapy for breast tumors with EGFR overexpression. PMID:20228837

  20. Contributions of EspA filaments and curli fimbriae in cellular adherence and biofilm formation of enterohemorrhagic Escherichia coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In Escherichia coli O157:H7 (O157), the filamentous structure of the type III secretion system is produced from the polymerization of the EspA protein. EspA filaments are essential for O157 adherence to epithelial cells. In previous studies, we demonstrated that O157 hha deletion mutants showed incr...

  1. Pathogenicity of an Escherichia coli O115:K"V165" mutant negative for F165(1) fimbriae in septicemia of gnotobiotic pigs.

    PubMed

    Ngeleka, M; Jacques, M; Martineau-Doizé, B; Daigle, F; Harel, J; Fairbrother, J M

    1993-03-01

    To evaluate the role of the F165(1) fimbrial system in the pathogenesis of septicemia, 2-day-old germfree pigs were inoculated intragastrically with Escherichia coli O115:K"V165":F165 wild-type strain 5131, its F165(1)-negative TnphoA mutant M48, or E. coli O115:K(-):F165(-) wild-type strain 862B. Pigs were sacrificed at different times (3, 6, 12, 24, 48, and 96 h) postinfection (p.i.). Pigs inoculated with strain 5131 developed clinical signs (anorexia, lameness, reluctance to move, or lack of motor coordination) and were moribund within 48 h p.i., and, at necropsy, infecting bacteria were isolated in various extraintestinal organs. Strain 5131 was isolated as early as 6 h p.i. from the blood of inoculated pigs. Pigs inoculated with mutant M48 developed only mild clinical signs at 96 h p.i. Mutant M48 colonized extraintestinal organs of pigs but to a lesser extent than the parent strain did. In contrast to the parent strain, this mutant was not isolated in the blood of inoculated pigs. Pigs inoculated with strain 862B remained normal during the experiment. All of the strains colonized the mucus layer of the intestine, but no histological changes of intestinal mucosa were observed by either light or electron microscopy. The parent strain, but not the mutant M48, expressed F165(1) in vivo. In a competitive study in which the parent strain and its afimbrial mutant were inoculated simultaneously, clinical signs of septicemia developed 24 h after inoculation, and only the parent strain 5131 was isolated from the blood of inoculated pigs. Our results suggest that the F165(1) fimbrial system of E. coli O115:K"V165" strains may play an important role in the ability of the bacteria to survive in the blood and spread systemically through the porcine host. PMID:8094383

  2. A cryptic fimbrial gene in Serratia marcescens.

    PubMed Central

    Moriya, T; Kawabata, S; Mizunoe, Y; Amako, K

    1989-01-01

    The gene coding for the mannose-sensitive hemagglutinating fimbriae in Serratia marcescens US5 was cloned into Escherichia coli K4 with a cosmid vector system. One of the transformants, US5-1, expressed two morphologically distinct fimbriae, one that was 5-nm wide and one that was 3-nm wide. The latter fimbria was morphologically and serologically indistinguishable from that of strain US5. Genetic analysis of transformant US5-1 showed that the gene responsible for the 5-nm-wide fimbriae was located more than 10 kilobases away from the gene responsible for the 3-nm-wide fimbriae. The molecular sizes of the subunits of these two fimbriae, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 19 kilodaltons for the 3-nm-wide fimbriae and 20 kilodaltons for the 5-nm-wide fimbriae. Serologically, the 5-nm-wide fimbriae did not cross-react with monoclonal or polyclonal antibodies raised against the mannose-sensitive hemagglutinating fimbriae of strain US5. Strain EL101, which expressed only the 5-nm-wide fimbriae, did not agglutinate chicken or human erythrocytes. These experimental results suggest that the gene for the 5-nm-wide fimbriae is cryptic in strain US5 and is expressed in E. coli K4 only after it is moved by transformation. Images PMID:2574174

  3. Inverse relationship between heat stable enterotoxin-b induced fluid accumulation and adherence of F4ac-positive enterotoxigenic Escherichia coli in ligated jejunal loops of F4ab/ac fimbria receptor-positive swine.

    PubMed

    Erume, Joseph; Wijemanne, Prageeth; Berberov, Emil M; Kachman, Stephen D; Oestmann, Daniel J; Francis, David H; Moxley, Rodney A

    2013-01-25

    Heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) increases bacterial adherence to porcine enterocytes in vitro and enhances small intestinal colonization in swine. Heat-stable enterotoxin-b (STb) is not known to affect colonization; however, through an induction of net fluid accumulation it might reduce bacterial adherence. The relationship between fluid accumulation and bacterial adherence in jejunal loops inoculated with ETEC strains that produce LT, STb, both, or neither toxin was studied. Ligated jejunal loops were constructed in weaned Yorkshire pigs in two independent experiments (Exp. 1, n=5, 8-week-old; Exp. 2, n=6, 6-8-week-old). Each pig was inoculated with six F4ac(+)E. coli strains: (1) LT(+), STb(+) parent (WAM2317); (2) STb(-) (ΔestB) mutant (MUN297); (3) MUN297 complemented with STb (MUN298); (4) LT(-) STb(-) (ΔeltAB ΔestB) mutant (MUN300); (5) MUN300 complemented with LT (MUN301); and (6) 1836-2 (non-enterotoxigenic, wild-type). Pigs were confirmed to be K88 (F4)ab/ac receptor-positive in Exp. 2 by testing for intestinal mucin-type glycoproteins and inferred to be receptor-positive in both Exp. 1 and 2 based on histopathologic evidence of bacterial adherence. Strains that produced STb induced marked fluid accumulation with the response (ml/cm) to WAM2317 and MUN298 significantly greater than that to the other strains (P<0.0001). Conversely, bacterial adherence scores based on immunohistochemistry and CFU/g of washed mucosa were both lowest in the strains that expressed STb and highest in those that did not. For the two experiments combined, the Pearson correlation coefficient (R) between fluid volume (ml/cm) and log CFU per gram was -0.57021 (P<0.0001); R(2)=0.3521 (n=197). These results support the hypothesis that enterotoxin-induced fluid accumulation flushes progeny organisms into the lumen of the bowel, thereby increasing the likelihood of fecal shedding and transmission of the pathogen to new hosts. PMID:22901529

  4. Purification, morphology, and genetics of a new fimbrial putative colonization factor of enterotoxigenic Escherichia coli O159:H4.

    PubMed Central

    Tacket, C O; Maneval, D R; Levine, M M

    1987-01-01

    The ability to colonize the small intestine is essential for the pathogenesis of diarrhea caused by enterotoxigenic Escherichia coli (ETEC). Colonization is mediated by fimbriae (pili), of which there are several antigenically distinct types, including colonization factor antigen I, colonization factor antigen II (CS1, CS2, and CS3), and PCF8775 (CS4, CS5, and CS6). These fimbriae are associated with certain ETEC O serogroups. Serogroup O159 has had no known colonization factor. We found a distinct plasmid-encoded fimbria composed of 19-kilodalton protein subunits associated with ETEC serotype O159:H4. Rabbit antibody against this purified fimbria reacted with a single 19-kilodalton protein band as seen by Western immunoblot of sheared-cell preparations. The rabbit antibody, treated with colloidal-gold-labeled goat anti-rabbit immunoglobulin G, bound specifically to fimbriae when cells were examined with an electron microscope. Of 10 available ETEC O159:H4 strains from Europe, Bangladesh, and Kenya, 6 expressed this type of fimbria; its true prevalence among ETEC strains is unknown. This putative colonization factor of O159:H4 joins other ETEC fimbriae as potentially useful immunogens against human diarrhea. Images PMID:2883122

  5. The major fimbrial subunit of Bordetella pertussis binds to sulfated sugars.

    PubMed Central

    Geuijen, C A; Willems, R J; Mooi, F R

    1996-01-01

    Bordetella pertussis fimbriae are composed of major and minor subunits, and recently it was shown that the minor fimbrial subunit binds to Vla-5, a receptor located on monocytes (W. Hazenbos, C. Geuijen, B. van den Berg, F. Mooi, and R. van Furth, J. Infect. Dis. 171:924-929, 1995). Here we present evidence that the major subunits bind to sulfated sugars, which are ubiquitous in the respiratory tract. Binding was observed to chondroitin sulfate, heparan sulfate, and dextran sulfate but not to dextran. Removal of the minor subunit from fimbriae did not significantly affect binding to sulfated sugars, indicating that the major subunit alone is sufficient for this binding. Fimbriae were also able to bind HEp-2 cells, which are known to display glycoconjugates on their surface. This binding was not dependent on the presence of the minor subunit. However, binding was dependent on the sulfation state of the glycoconjugates, since inhibition of the sulfation resulted in a significant reduction of fimbria binding. The specificity of fimbria binding was further characterized by using heparan sulfate-derived disaccharides in inhibition assays. Two disaccharides were highly effective inhibitors, and it was observed that both the degree of sulfation and the arrangement of the sulfate groups on the disaccharides were important for binding to fimbriae. B. pertussis bacteria also bound to sulfated sugars and HEp-2 cells, and analysis of B. pertussis mutants indicated that both filamentous hemagglutinin and fimbriae were required for this binding. A host protein present in the extracellular matrix, fibronectin, has binding activities similar to those of B. pertussis fimbriae, binding to both Vla-5 and sulfated sugars. Two regions in the major fimbrial subunit were identified which showed similarity with fibronectin peptides which bind to sulfated sugars. Thus, B. pertussis fimbriae exemplify molecular mimicry and may co-opt host processes by mimicking natural ligand

  6. The Non-Fimbriate Phenotype Is Predominant among Salmonella enterica Serovar Choleraesuis from Swine and Those Non-Fimbriate Strains Possess Distinct Amino Acid Variations in FimH

    PubMed Central

    Lee, Chien-An; Yeh, Kuang-Sheng

    2016-01-01

    Although most Salmonella serovars are able to infect a range of animal hosts, some have acquired the ability to cause systemic infections of specific hosts. For example, Salmonella enterica serovar Choleraesuis is primarily associated with systemic infection in swine. Adherence to host epithelial cells is considered a prerequisite for initial infection, and fimbrial appendages on the outer membrane of the bacteria are implicated in this process. Although type 1 fimbriae encoded by the fim gene cluster are commonly found in Salmonella serovars, it is not known whether S. Choleraesuis produces this fimbrial type and if and how fimbriae are involved in pathogenesis. In the present study, we demonstrated that only four out of 120 S. Choleraesuis isolates from pigs with salmonellosis produced type 1 fimbriae as assayed by the yeast agglutination test and electron microscopy. One of the 116 non-type 1 fimbria-producing isolates was transformed with plasmids carrying different fim genes from S. Typhimurium LB5010, a type 1 fimbria-producing strain. Our results indicate that non-type 1 fimbria-producing S. Choleraesuis required only an intact fimH to regain the ability to produce fimbrial appendages. Sequence comparison revealed six amino acid variations between the FimH of the non-type 1 fimbria-producing S. Choleraesuis isolates and those of the type 1 fimbria-producing S. Choleraesuis isolates. S. Choleraesuis that produced type 1 fimbriae contained FimH with an amino acid sequence identical to that of S. Typhimurium LB5010. Site-directed mutagenesis leading to the replacement of the non-conserved residues revealed that a change from glycine to valine at position of 63 (G63V) resulted in a non-type 1 fimbria-producing S. Choleraesuis being able to express type 1 fimbriae on its outer membrane. It is possible that this particular amino acid change prevents this polypeptide from proper interaction with other Fim subunits required for assembly of an intact type 1 fimbrial

  7. New Surface-Associated Heat-Labile Colonization Factor Antigen (CFA/II) Produced by Enterotoxigenic Escherichia coli of Serogroups O6 and O8

    PubMed Central

    Evans, Dolores G.; Evans, Doyle J.

    1978-01-01

    Enterotoxigenic Escherichia coli (ETEC) belonging to serogroups O6 and O8 do not possess the H-10407-type colonization factor antigen (CFA/I). However, these frequently isolated ETEC were found to possess a second and distinct heat-labile surface-associated colonization factor antigen, termed CFA/II. Whereas CFA/I mediates mannose-resistant hemagglutination of human group A erythrocytes, CFA/II does not. CFA/II mediates mannose-resistant hemagglutination of bovine erythrocytes, and mannose-resistant hemagglutination is rapid only at reduced temperature (4°C). Because CFA/II, like CFA/I, is spontaneously lost by many ETEC isolates in the laboratory, it was possible to produce specific anti-CFA/II serum by preparing antiserum against living cells of a prototype strain (PB-176) and adsorbing this serum with living and heat-treated cells of its CFA/II-negative derivative strain PB-176-P. This serum, which neutralized the colonization factor activity of CFA/II-positive strains in infant rabbits, was employed to confirm the presence of CFA/II on ETEC which exhibited mannose-resistant hemagglutination of bovine but not human erythrocytes. CFA/II, like CFA/I, mediates adherence of the bacteria to the mucosal surface of the small intestine, as demonstrated by indirect immunofluorescence. CFA/II appears to be an important virulence factor for humans since CFA/II-positive ETEC are frequently isolated from diarrhea cases, particularly travelers' diarrhea, in Mexico; these ETEC were not uncommon in a collection of isolates from Bangladesh. The O6:H16 strain of ETEC responsible for an outbreak of diarrhea in the United States was also shown to be CFA/II positive. CFA/I and CFA/II were never found on the same serotypes of ETEC, but 98% of the heat-stable and heat-labile enterotoxin-producing ETEC belonging to the frequently isolated serogroups O6, O8, O15, O25, O63, and O78 were positive for either CFA/I or CFA/II. Images PMID:80383

  8. The role of cellulose and O-antigen capsule in the colonization of plants by Salmonella

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Numerous salmonellosis outbreaks have been associated with vegetables, in particular sprouted seeds. Thin aggregative fimbriae (Tafi), a component of the extracellular matrix responsible for multicellular behavior, are important for Salmonella enterica attachment and colonization of plants. Here we ...

  9. 77 FR 52333 - International Workshop on Alternatives to the Murine Histamine Sensitization Test (HIST) for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-29

    ... Pertussis, also known as whooping cough, is a highly contagious disease caused by the bacterium Bordetella.... pertussis bacteria (e.g., inactivated pertussis toxin , pertactin, and fimbriae) and are less...

  10. Antigenicity and immunogenicity of fused B-subunit of heat labile toxin of Escherichia coli and colonization factor antigen I polyepitopes.

    PubMed

    Savar, Nastaran Sadat; Dashti, Amir; Darzi Eslam, Elham; Jahanian-Najafabadi, Ali; Jafari, Anis

    2014-11-01

    Linear B-cell epitopes ((93)AKEFEAAAL(101) and (66)PQLTDVLN(73)) of CfaB were genetically fused to ltb-(gly)5-cfaB(1-25). Sera of rabbits immunized with fusion proteins reacted strongly with solid-phase bound ETEC bacteria bearing CFA/I fimbriae. Sera failed to agglutinate or inhibit hemagglutination promoted by CFA/I-positive strain which may be due to solvent inaccessibility of epitope residues on intact fimbriae. PMID:25108290

  11. Pathogenic properties of Edwardsiella species.

    PubMed Central

    Janda, J M; Abbott, S L; Kroske-Bystrom, S; Cheung, W K; Powers, C; Kokka, R P; Tamura, K

    1991-01-01

    The pathogenic characteristics of 35 Edwardsiella strains from clinical and environmental sources were investigated. Overall, most Edwardsiella tarda strains were invasive in HEp-2 cell monolayers, produced a cell-associated hemolysin and siderophores, and bound Congo red; many strains also expressed mannose-resistant hemagglutination against guinea pig erythrocytes. Edwardsiella hoshinae strains bound Congo red and were variable in their invasive and hemolytic capabilities while Edwardsiella ictaluri strains did not produce either factor; neither E. hoshinae nor E. ictaluri expressed mannose-resistant hemagglutination nor elaborated siderophores under the tested conditions. Selected strains of each species tested for mouse lethality indicated strain variability in pathogenic potential, with E. tarda strains being the most virulent; 50% lethal doses in individual strains did not correlate with plasmid content, chemotactic motility, serum resistance, or expression of selected enzyme activities. The results suggest some potential important differences in pathogenic properties that may help explain their environmental distribution and ability to cause disease in humans. Images PMID:1774326

  12. Porphyromonas (Bacteroides) gingivalis fimbrillin: size, amino-terminal sequence, and antigenic heterogeneity.

    PubMed Central

    Lee, J Y; Sojar, H T; Bedi, G S; Genco, R J

    1991-01-01

    Bacterial fimbriae mediate cell adhesion and are important in colonization. Fimbrial proteins from strains of Porphyromonas (Bacteroides) gingivalis isolated from different individuals were compared for their size, amino-terminal sequence, and antigenic diversity. Two major protein components of the crude fimbrial preparations differed in apparent molecular mass, ranging from 41 to 49 kDa for the fimbrillin monomer and from 61 to 78 kDa for the other major protein. The amino-terminal sequence of the antigenically related group of proteins of the fimbrillin monomer in the 41- to 49-kDa range showed significant homology; however, minor sequence heterogeneity was observed, mainly in residues 4 to 6. One of the observed amino-terminal sequences, AFGVGDDESKVAKLTVMVYNG, resembled the deduced sequence of P. gingivalis 381 (D.P. Dickinson, M. K. Kubiniec, F. Yoshimura, and R.J. Genco, J. Bacteriol. 170:1658-1665, 1988). Fimbriae from all the strains of P. gingivalis showing this sequence contained a fimbrillin monomer of 43 kDa and showed a strong reaction with both polyclonal and monoclonal antibodies directed to the fimbriae from P. gingivalis 2561 (381). Fimbriae from strains showing amino-terminal sequence variations in residues 4 to 6 (i.e., substitution of VGD with either E or NAG) were more diverse in their molecular sizes. Most of these variant fimbriae showed weak reactions with the polyclonal antibodies and no reaction with the monoclonal antibodies induced to the fimbriae of strain 2561. No correlation could be established between the molecular size and immunological reactivity of the fimbrillin monomer of P. gingivalis strains. Strains 9-14K-1 and HG 564 not only showed markedly different sequences from the other three amino-terminal sequences but also did not react with either polyclonal or monoclonal antibodies to the fimbriae of strain 2561. Strains W50, W83, and AJW 5 failed to show any immunological reactivity with the antibodies to fimbrillin or fimbriae

  13. Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species

    PubMed Central

    2010-01-01

    Background Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii. Results Phylogenetic analysis of the type 3 fimbrial genes (mrkABCD) from 39 strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae MGH78578. The E. coli and K. pneumoniae mrkABCD gene sequences clustered together in two distinct clades, supporting previous evidence for the occurrence of inter-genera lateral gene transfer. All of the strains examined caused type 3 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkD-encoding adhesin gene. Type 3 fimbriae deletion mutants were constructed in 13 representative strains and were used to demonstrate a direct role for type 3 fimbriae in biofilm formation. Conclusions The expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI and is strongly associated with biofilm growth. Our data provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer. Further work is now required to substantiate the clade structure reported here by examining more strains as well as other bacterial genera that make type 3 fimbriae and cause CAUTI. PMID:20576143

  14. The location of surface antigens of Bordetella pertussis by immuno-electron microscopy.

    PubMed

    Ashworth, L A; Dowsett, A B; Irons, L I; Robinson, A

    1985-01-01

    Immuno-electron microscopy using colloidal gold-tagged monoclonal antibodies (McAbs) has been used to detect antigens on the surface of Bordetella pertussis cells. McAbs to serotype-specific agglutinogens 2 and 3 labelled fimbriae in a serotype-specific manner. An attempt was made to determine whether individual fimbriae of serotype 1.2.3 cells bear both antigens 2 and 3. In double labelling experiments, individual cells were found to label with McAbs to antigen 2 (3 nm gold) and to antigen 3 (15 nm gold). Many fimbriae labelled with only one of these reagents, but there were instances where both labels could have been attached to the same fimbria. No fimbrial labelling was obtained with McAb to agglutinogen 1. Gold-tagged McAbs to filamentous haemagglutinin (FHA) labelled neither the fimbriae nor the surface of B. pertussis cells. Unfixed cells on electron microscope grids appeared to shed FHA readily. After fixation, a small proportion of cells bore aggregates of FHA which labelled specifically with anti-FHA McAb-gold. Results with one McAb to lymphocytosis promoting factor indicated that this antigen may be more intimately associated with the surface of B. pertussis than is FHA. PMID:2872100

  15. Structure of the fimbrial protein Mfa4 from Porphyromonas gingivalis in its precursor form: implications for a donor-strand complementation mechanism

    PubMed Central

    Kloppsteck, Patrik; Hall, Michael; Hasegawa, Yoshiaki; Persson, Karina

    2016-01-01

    Gingivitis and periodontitis are chronic inflammatory diseases that can lead to tooth loss. One of the causes of these diseases is the Gram-negative Porphyromonas gingivalis. This periodontal pathogen is dependent on two fimbriae, FimA and Mfa1, for binding to dental biofilm, salivary proteins, and host cells. These fimbriae are composed of five proteins each, but the fimbriae assembly mechanism and ligands are unknown. Here we reveal the crystal structure of the precursor form of Mfa4, one of the accessory proteins of the Mfa1 fimbria. Mfa4 consists of two β-sandwich domains and the first part of the structure forms two well-defined β-strands that run over both domains. This N-terminal region is cleaved by gingipains, a family of proteolytic enzymes that encompass arginine- and lysine-specific proteases. Cleavage of the N-terminal region generates the mature form of the protein. Our structural data allow us to propose that the new N-terminus of the mature protein may function as a donor strand in the polymerization of P. gingivalis fimbriae. PMID:26972441

  16. Structure of the fimbrial protein Mfa4 from Porphyromonas gingivalis in its precursor form: implications for a donor-strand complementation mechanism.

    PubMed

    Kloppsteck, Patrik; Hall, Michael; Hasegawa, Yoshiaki; Persson, Karina

    2016-01-01

    Gingivitis and periodontitis are chronic inflammatory diseases that can lead to tooth loss. One of the causes of these diseases is the Gram-negative Porphyromonas gingivalis. This periodontal pathogen is dependent on two fimbriae, FimA and Mfa1, for binding to dental biofilm, salivary proteins, and host cells. These fimbriae are composed of five proteins each, but the fimbriae assembly mechanism and ligands are unknown. Here we reveal the crystal structure of the precursor form of Mfa4, one of the accessory proteins of the Mfa1 fimbria. Mfa4 consists of two β-sandwich domains and the first part of the structure forms two well-defined β-strands that run over both domains. This N-terminal region is cleaved by gingipains, a family of proteolytic enzymes that encompass arginine- and lysine-specific proteases. Cleavage of the N-terminal region generates the mature form of the protein. Our structural data allow us to propose that the new N-terminus of the mature protein may function as a donor strand in the polymerization of P. gingivalis fimbriae. PMID:26972441

  17. Mannanoligosaccharide agglutination by Salmonella enterica strains isolated from carrier pigs

    PubMed Central

    Borowsky, Luciane; Corção, Gertrudes; Cardoso, Marisa

    2009-01-01

    Type-1 fimbriae are associated with most Salmonella enterica serovars and are an essential factor for host colonization. Mannanoligosaccharides (MOS), a prebiotic that is agglutinated by type-1 fimbriae, are proposed for the control of enterobacteria colonization and may be an alternative to Salmonella control in pigs. The aim of this study was to evaluate the capability of porcine Salmonella strains to adhere to MOS in vitro. A total of 108 strains of Salmonella sp. isolated from carrier pigs were evaluated for the amplification of fimA and fimH genes, agglutination of MOS and hemagglutination. In all tested strains, amplicons of expected size were detected for both fimA and fimH gene. In the hemagglutination assays, 31 (28.7%) strains presented mannose–sensitive agglutination of erythrocytes, indicating that the strains were expressing type-1 fimbriae. Considering only strains expressing the type-1 fimbriae, 23 (74.2%) presented a strong agglutination of MOS, 3 (9.6%) a weak reaction and 5 (16.2%) none. The results indicate that Salmonella enterica strains expressing type-1 fimbriae can agglutinate effectively in vitro to MOS. PMID:24031388

  18. Purification, and biochemical and structural characterization of a fimbrial haemagglutinin of Renibacterium salmoninarum.

    PubMed

    Dubreuil, J D; Jacques, M; Graham, L; Lallier, R

    1990-12-01

    Renibacterium salmoninarum was shown to possess peritrichous fimbriae. Electron microscopy of strains FMV 84-01 and ATCC 33209T revealed short, flexible fimbriae less than 2 nm in diameter. These surface appendages were isolated from the bacteria by a procedure involving water extraction and urea solubilization. The fimbrin was purified to homogeneity by Fast Pressure Liquid Chromatography, and shown by SDS-PAGE to be a protein of 57 kDa. Isoelectric focusing under non-denaturing conditions indicated a pI of 4.8. The protein had an amino acid composition rich in glycine, Asx (aspartic acid and asparagine), valine and alanine; methionine was absent. Approximately 33% of the amino acid residues were hydrophobic. Immunoblotting using a polyclonal antiserum raised against whole cells showed that the 57 kDa protein was the immunodominant antigen on the cell surface. Immunogold labelling using polyclonal antibodies raised against the fimbrin revealed an alignment of gold particles along the fimbriae. Purified fimbriae caused agglutination of rabbit erythrocytes and antifimbrial serum inhibited this haemagglutination. Altogether the results indicate that the fimbriae on the surface of R. salmoninarum are responsible for the haemagglutinating activity. PMID:1981894

  19. Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium.

    PubMed Central

    Bäumler, A J; Heffron, F

    1995-01-01

    A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences. PMID:7721701

  20. Virulence and metabolic characteristics of Salmonella Enteritidis sefD variants in hens.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Salmonella Enteritidis is one of a few pathogenic Salmonella enterica serotypes that have SEF14 fimbriae encoded by the sef operon, which consists of 4 co-transcribed genes sefABCD that are regulated by sefR. To explore the function of sefD within the infection pathway resulting in egg contamination...

  1. How methylglyoxal kills bacteria: An ultrastructural study.

    PubMed

    Rabie, Erika; Serem, June Cheptoo; Oberholzer, Hester Magdalena; Gaspar, Anabella Regina Marques; Bester, Megan Jean

    2016-01-01

    Antibacterial activity of honey is due to the presence of methylglyoxal (MGO), H2O2, bee defensin as well as polyphenols. High MGO levels in manuka honey are the main source of antibacterial activity. Manuka honey has been reported to reduce the swarming and swimming motility of Pseudomonas aeruginosa due to de-flagellation. Due to the complexity of honey it is unknown if this effect is directly due to MGO. In this ultrastructural investigation the effects of MGO on the morphology of bacteria and specifically the structure of fimbriae and flagella were investigated. MGO effectively inhibited Gram positive (Bacillus subtilis; MIC 0.8 mM and Staphylococcus aureus; MIC 1.2 mM) and Gram negative (P. aeruginosa; MIC 1.0 mM and Escherichia coli; MIC 1.2 mM) bacteria growth. The ultrastructural effects of 0.5, 1.0 and 2 mM MGO on B. substilis and E. coli morphology was then evaluated. At 0.5 mM MGO, bacteria structure was unaltered. For both bacteria at 1 mM MGO fewer fimbriae were present and the flagella were less or absent. Identified structures appeared stunted and fragile. At 2 mM MGO fimbriae and flagella were absent while the bacteria were rounded with shrinkage and loss of membrane integrity. Antibacterial MGO causes alterations in the structure of bacterial fimbriae and flagella which would limit bacteria adherence and motility. PMID:26986806

  2. EVALUATING THE ROLE OF SDIA AND HHA IN ENHANCED ADHERENCE OF A SDIA HHA DOUBLE MUTANT OF ENTEROHEMORRHAGIC ESCHERICHIA COLI O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Adherence of Enterohemorrhagic Escherichia coli (EHEC) O157:H7 to biotic (epithelial cells) and abiotic surfaces (biofilm formation) proceeds from an initial reversible adherence to an irreversible stage of intimate adherence. While flagella and fimbriae facilitate initial stage of adherence in both...

  3. Bordetella pertussis infection: pathogenesis, diagnosis, management, and the role of protective immunity.

    PubMed

    Kerr, J R; Matthews, R C

    2000-02-01

    Whooping cough is presently one of the ten most common causes of death from infectious disease worldwide. Despite a high vaccine uptake, resurgences of this disease have been observed in several countries. Virulence factors of Bordetella pertussis include agglutinogens, fimbriae, P.69/pertactin, pertussis toxin, filamentous haemagglutinin, adenylate cyclase, tracheal cytotoxin, dermonecrotic toxin, lipopolysaccharide, tracheal colonisation factor, serum resistance factor, and type III secretion. Virulence factor expression is regulated by the bvgAS locus, a two-component signal transduction system. The pathophysiologic sequence consists of attachment (fimbriae, P.69/pertactin, tracheal colonisation factor, pertussis toxin, filamentous haemagglutinin), evasion of host defence (adenylate cyclase, pertussis toxin, serum resistance factor), local effects (tracheal cytotoxin), and systemic effects (pertussis toxin). Bordetella pertussis is transmitted by respiratory droplets and causes disease only in humans. Various diagnostic methods are available, including culture, serological methods, and the polymerase chain reaction. Serotyping of isolates to detect agglutinogens 2 and 3 is useful because serotype 1,2 may be associated with higher mortality, and antibodies to these antigens (agglutinins) may be protective in both animals and humans. Immunisation using whole-cell vaccine is effective but is reactogenic. Acellular vaccines containing one to five components are being used increasingly in various countries. Protective immunity to pertussis correlates with high levels of antibody to each of pertactin, fimbriae, and pertussis toxin; however, doubt remains as to the relationship between agglutinogen 3 and fimbria 3, making results of trials investigating these virulence factors difficult to interpret. PMID:10746492

  4. Stx1 prophage excision in Escherichia coli strain PA20 confers strong curli and biofilm formation by restoring native mlrA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prophage insertions in Escherichia coli O157:H7 mlrA contribute to the low expression of curli fimbriae and biofilm observed in many clinical isolates. Varying levels of CsgD-dependent curli/biofilm expression are restored to strains bearing prophage insertions in mlrA by mutation of regulatory gene...

  5. ESCHERICHIA COLI O147: AN EMERGING SEROGROUP OF EDEMA DISEASE OUTBREAKS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Edema disease is a systemic disease of weanling pigs caused by E. coli strains that produce a variant of Shiga toxin, Stx2e. These strains usually produce F18ab fimbriae and heat stable enterotoxin b (STb). Historically, edema disease strains from U.S. outbreaks have not produced heat stable enter...

  6. Reduction of Salmonella Enteritidis in the spleens of hens by bacterins that vary in fimbrial protein SefD

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gene sefD is part of operon sefABCD, and it is required for production of the SEF14 fimbria by Salmonella Enteritidis. We compared strains that varied in SefD content for their ability to reduce recovery of Salmonella Enteritidis from the spleens of hens infected by parenteral challenge. The two bac...

  7. RcsB contributes to the distinct stress fitness between Escherichia coli O157:H7 curli variants of 1993 hamburger-associated outbreak strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Curli are adhesive fimbriae of Enterobactericaeae and are involved in surface attachment, cell aggregation and biofilm formation. We previously reported that natural curli variants of E. coli O157:H7 (EcO157) displayed distinct acid resistance; however, this difference was not linked to the curli fi...

  8. Draft Genome Sequence and Gene Annotation of the Uropathogenic Bacterium Proteus mirabilis Pr2921

    PubMed Central

    Giorello, F. M.; Romero, V.; Farias, J.; Scavone, P.; Umpiérrez, A.; Zunino, P.

    2016-01-01

    Here, we report the genome sequence of Proteus mirabilis Pr2921, a uropathogenic bacterium that can cause severe complicated urinary tract infections. After gene annotation, we identified two additional copies of ucaA, one of the most studied fimbrial protein genes, and other fimbriae related-proteins that are not present in P. mirabilis HI4320. PMID:27340058

  9. Growth media and temperature effects on biofilm formation by serotype O157:H7 and non-O157 Shiga toxin-producing Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofilm formation in most Escherichia coli strains is dependent on curli fimbriae and cellulose, and the expression of both varies widely among pathogenic strains. Curli and cellulose expression are often identified by their affinity for Congo red dye (CR). However, media composition and incubation ...

  10. Phenotypic Divergence in Curli Variants of Enterohemorrhagic Escherichia coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Curli are adhesive fimbriae of many Enterobactericeae and are involved in surface attachment, cell aggregation, and biofilm formation. They also mediate host cell invasion and are potent inducers of the host inflammatory response. Variations in curli expression were reported in enterohemorrhagic E. ...