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T F Meyer

Publications and source records attributed to T F Meyer.

At least 127 records · Page 7Linked to original sources
PubMed1994

Highly polarized primary urothelial cells from human ureter grown as spheroid-like vesicles.

Epithelial cells growing in vitro are frequently non-polarized and lack histophysiological characteristics. Furthermore, the quality of two-dimensional cell layers is limited by the physico-chemical properties of the support. Therefore, for an in vitro system to reflect the normal epithelial physiology, it is necessary to maintain the inner and outer geometrical configuration of the cells. In order to avoid the disadvantages of two-dimensional cultures we have established an in vitro model that closely resembles the in vivo situation. Human ureteral epithelial cells (HUEC) were used to prepare multicellular vesicles which maintain a geometrically intact cell organization that is not achieved in conventional cultures. Light and electron microscopy investigations showed the morphology of the cells to be similar to that in situ. HUEC vesicles are more in vivo-like than two-dimensional cultures and therefore represent a suitable model for a variety of research purposes including studies on the pathogenesis of micro-organisms.

Cell Aggregation↗
PubMed1993

Characterization of the Neisseria Iga beta-core. The essential unit for outer membrane targeting and extracellular protein secretion.

Extracellular transport of Neisseria IgA proteases across the bacterial outer membrane is accomplished by the translocation function contained within the C-terminal Iga beta domain of IgA protease precursor proteins. Recently, we reported that Iga beta from N. gonorrhoeae MS11 (Val1097 to Phe1505), fused to a periplasmic passenger protein, facilitated its transport across the outer membrane, leading to surface exposure of the passenger. In the present work we show, by systematic N-terminal truncation of Iga beta, that the functional and structural unit, termed Iga beta-core, corresponds to the C-terminal approximately 274 amino acid residues (Ser1231 to Phe1505). This minimal region retains all the essential features necessary for the translocation of an N-terminally attached passenger across the outer membrane of Escherichia coli, and for its own correct integration into the outer membrane, even in the absence of a passenger protein. The membrane-integrated Iga beta-core constitutes a conserved entity found in the C-terminal regions of Iga beta domains of different N. gonorrhoeae, N. meningitidis and Haemophilus influenzae strains. In contrast, the surface-exposed N termini of the Iga beta domains vary in size and sequence. Based on secondary structure predictions, the key structural feature of the core is a beta-barrel (amphipathic, antiparallel transmembrane beta-strands, interspersed by hairpin turns and loops) which is common to many integral outer membrane proteins of Gram-negative bacteria. We propose that the core has been conserved in evolution, to provide a selective outer membrane export channel for covalently attached polypeptides.

Amino Acid Sequence↗
PubMed1993

TnMax--a versatile mini-transposon for the analysis of cloned genes and shuttle mutagenesis.

A series of Tn1721-based mini-transposons (TnMax) has been developed which are suitable for the targeting of cloned genes, shuttle mutagenesis, identification of protein export signals and the rescue of chromosomal markers. TnMax mini-Tn consist of two 38-bp inverted repeats (IR) flanking a resolution site (res), a suicide replication origin (orifd), and a gene conferring resistance to either chloramphenicol (TnMax1) or erythromycin (TnMax2). Other versions of TnMax (TnMax3 and TnMax4) carry a promoterless alkaline phosphatase-encoding gene (phoA') useful for the identification of protein export signals. The various mini-Tn cartridges are fused on the same plasmid with a common transposition control unit (TCU) comprising the tnpA (transposase) and tnpR (resolvase) genes under control of the inducible Ptrc promoter. This configuration causes a high frequency of transposition in Escherichia coli (approx. 10(-2) events/copy of target plasmid) and a minimum size of the mini-Tn. Like Tn1721, TnMax variants prefer random insertion into plasmids rather than into the E. coli chromosome, thus representing superb tools for the insertion mutagenesis of cloned genes. The TnMax-borne orifd simplifies the identification of targeted plasmids and facilitates shuttle mutagenesis, i.e., suicide delivery of a mutated gene with subsequent allelic replacement of a corresponding resident gene, in a variety of microorganisms. Rescue of such targeted chromosomal genes is easily accomplished by the excision of TnMax plus flanking segments using appropriate restriction endonucleases, ligation, and transformation of an E. coli host permissive for orifd-directed replication.

Base Sequence↗
PubMed1993

A plasmid system for high-level expression and in vitro processing of recombinant proteins.

A novel plasmid expression system has been constructed that combines two useful functions: it facilitates single-step affinity purification of cytoplasmically overproduced fusion proteins and the in vitro processing of fusions with IgA protease (Igase). The significant features directing the high expression rate of pEV41-based gene fusions in Escherichia coli are the lambda pL promoter for temperature-regulated transcription and the translation initiation region of the bacteriophage MS2 polymerase gene including a downstream box (db) within the first few codons of the open reading frame. Fusion proteins generated with this system contain a short N-terminal carrier peptide allowing convenient affinity purification by means of the His6 peptide. As exemplified by the production of the variable heavy (VH) and light (VL)-chain domains of a monoclonal antibody, the fusion proteins can be specifically processed with Igase either in purified form or simply by incubation with the culture medium of recombinant E. coli [pJP10] cells. Chemical cross-linking of processed VH and VL domains resulted in a recombinant antibody Fv fragment that can specifically bind to its antigen.

Amino Acid Sequence↗
PubMed1993

The secretion pathway of IgA protease-type proteins in gram-negative bacteria.

The pathogenic, Gram-negative bacteria, Neisseria gonorrhoeae, Neisseria meningitidis and Haemophilus influenzae, secrete immunoglobulin A1 proteases into their extracellular surroundings. An extraordinary feature in the secretory pathway of these putative virulence factors is a self-directed outer membrane transport step allowing the proteins to be secreted autonomously, even from foreign Gram-negative host cells like Escherichia coli. Here we summarize recent achievements in the understanding of IgA protease outer membrane translocation.

Amino Acid Sequence↗
PubMed1993

Variable opacity (Opa) outer membrane proteins account for the cell tropisms displayed by Neisseria gonorrhoeae for human leukocytes and epithelial cells.

Opacity proteins (Opa) of Neisseria gonorrhoeae, a family of variant outer membrane proteins implicated in pathogenesis, are subject to phase variation. In strain MS11, 11 different opa gene alleles have been identified, the expression of which can be turned on and off independently. Using a reverse genetic approach, we demonstrate that a single Opa protein variant of strain MS11, Opa50, enables gonococci to invade epithelial cells. The remaining variant Opa proteins show no, or very little, specificity for epithelial cells but instead confer interaction with human polymorphonuclear neutrophils (PMNs). Thus, depending on the opa allele expressed, gonococci are capable of invading epithelial cells or of interacting with human leukocytes. The respective properties of Opa proteins are maintained independent of the gonococcal strain; thus, the specificity for epithelial cells or leukocytes is intrinsic to Opa proteins. Significant homology exists in the surface exposed variable regions of two invasion supporting Opa proteins from independent strains. Efficient epithelial cell invasion is favoured by high level Opa production, however, a 10-fold reduction still allows significant invasion by gonococci. In contrast, recombinant Escherichia coli expressing Opa proteins adhered or invaded poorly under similar experimental conditions, thus indicating that additional factors besides Opa are required in the Opa-mediated interaction with human cells.

Amino Acid Sequence↗
PubMed1993

A novel determinant (comA) essential for natural transformation competence in Neisseria gonorrhoeae and the effect of a comA defect on pilin variation.

A novel genetic determinant (comA) has been identified and found to be required for the transformation of piliated Neisseria gonorrhoeae. Mutants in comA of strain MS11 grow normally and are DNA-uptake proficient but blocked in the translocation of DNA into the cytoplasm. Here we show by site-specific mutagenesis and genetic complementation that only one of two open reading frames identified in comA is essential for competence: it encodes a protein (ComA) with a predicted size of 74 kDa. The comA gene maps upstream of the iga locus and is transcribed in the opposite orientation, probably under the control of a putative sigma 54-type promoter. While DNA probes specific for the N. gonorrhoeae iga locus reveal only a little cross-reactivity with commensal Neisseria species, the neighbouring comA gene appears to be present in most of them. ComA fusion proteins were obtained by in vitro translation. The synthesized gene products migrated atypically in SDS gels indicating its strong hydrophobicity. Several transmembrane alpha-helices were predicted from the amino acid sequence of ComA which, in the context of an observed sequence similarity with other inner membrane proteins, suggests a location for the protein in the inner membrane. Using piliated and non-piliated comA mutants the consequences of transformation deficiency on pilin phase variation were assessed. We show that the comA defect affects some but not all types of DNA rearrangements associated with pilE variation. The results are in agreement with previous observations supporting the notion that multiple recombination pathways contribute to the variability of pilE.

Amino Acid Sequence↗
PubMed1993

Aflagellated mutants of Helicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis.

Three out of 10 Helicobacter pylori clinical isolates were found to be naturally competent for genetic transformation to streptomycin resistance by chromosomal DNA extracted from a spontaneous streptomycin-resistant H. pylori mutant. The frequency of transformation varied between 5 x 10(-4) and 4 x 10(-6), depending on the H. pylori isolate used. Transposon shuttle mutagenesis based on this natural competence was established using the flagellin gene flaA as the target. The cloned flaA gene was interrupted by insertion of TnMax1, a mini-Tn1721 transposon carrying a modified chloramphenicol-acetyltransferase gene, the catGC cassette. Natural transformation of competent H. pylori strains with plasmid constructs harbouring a catGC-inactivated flaA gene resulted in chloramphenicol-resistant transformants at an average frequency of 4 x 10(-5). Southern hybridization experiments confirmed the replacement of the chromosomal H. pylori flaA gene by the cat-inactivated cloned gene copy via homologous recombination resulting in allelic exchange. Phenotypic characterization of the mutants demonstrated the absence of flagella under the electron microscope and the loss of bacterial motility. Immunoblots of cell lysates of the H. pylori mutants with an antiserum raised against the C-terminal portion of recombinant H. pylori major flagellin (FlaA) confirmed the absence of the 54 kDa FlaA protein. This efficient transposon shuttle mutagenesis procedure for H. pylori based on natural competence opens up new possibilities for the genetic assessment of putative H. pylori virulence determinants.

Bacterial Proteins↗
PubMed1993

Highly polarized primary epithelial cells from human nasopharynx grown as spheroid-like vesicles.

The value of experimental culture models using epithelial cells often depends on the degree of polarization and other critical features observed in natural tissues, including the formation of tight junctions, desmosomes and membrane interdigitations. However, growth of normal epithelial cells as monolayers on artificial supports also leads to partial loss of the original characteristics of epithelial cells, and the quality of the monolayer is strongly influenced by the physicochemical properties of the support. In addition, not all normal epithelial cell types are able to adhere and to grow well on artificial substrata. In order to circumvent the drawbacks of two-dimensional cultures we established an in vitro model that closely resembles the in vivo situation of the intact epithelium. Human epithelial cells from nasopharynx (HNPEC) were used to prepare multicellular epithelial vesicles consisting of both non-ciliated and ciliated mucosal cells. Electron microscopy investigations showed that the morphological appearance of the epithelial cells was similar to that in situ. HNPEC vesicle cultures maintain a geometrically intact organization of individual cells that is not achieved using conventional culture conditions. HNPEC vesicles are more in vivo-like than two-dimensional cultures and therefore represent a suitable model for a variety of research purposes including studies on the pathogenesis of invasive microorganisms.

Cell Adhesion↗
PubMed1992

Transformation-mediated exchange of virulence determinants by co-cultivation of pathogenic Neisseriae.

The horizontal flow of genetic material between microbes utilizes three principal routes: conjugation, transduction and transformation. While the significance in nature of the first two pathways is generally accepted, the in vivo role of transformation remains uncertain, despite the early observations by Griffith in 1928 on the transformation of streptococci from an avirulent to a virulent state [1]. Recently, circumstantial evidence was collected suggesting a role for transformation-mediated horizontal exchange in the modulation of virulence determinants of pathogenic Neisseriae and the variation of surface structures. In order to further assess the significance of transformation-mediated exchange we performed simple co-cultivation experiments of different Neisseria strains. We observed an efficient intra- and interspecies transfer of essential virulence determinants; the process was sensitive to the presence of DNaseI in the culture and was blocked in transformation-deficient recipients.

Genes, Bacterial↗
PubMed1992

Selective extracellular release of cholera toxin B subunit by Escherichia coli: dissection of Neisseria Iga beta-mediated outer membrane transport.

The C-terminal domain (Iga beta) of the Neisseria IgA protease precursor is involved in the transport of covalently attached proteins across the outer membrane of Gram-negative bacteria. We investigated outer membrane transport in Escherichia coli using fusion proteins consisting of an N-terminal signal sequence for inner membrane transport, the Vibrio cholerae toxin B subunit (CtxB) as a passenger and Iga beta. The process probably involves two distinct steps: (i) integration of Iga beta into the outer membrane and (ii) translocation of the passenger across the membrane. The outer membrane integrated part of Iga beta is the C-terminal 30 kDa core, which serves as a translocator for both the passenger and the linking region situated between the passenger and Iga beta core. The completeness of the translocation is demonstrated by the extracellular release of the passenger protein owing to the action of the E. coli outer membrane OmpT protease. Translocation of the CtxB moiety occurs efficiently under conditions preventing intramolecular disulphide bond formation. In contrast, if disulphide bond formation in the periplasm proceeds, then translocation halts after the export of the linking region. In this situation transmembrane intermediates are generated which give rise to characteristic fragments resulting from rapid proteolytic degradation of the periplasmically trapped portion. Based on the identification of translocation intermediates we propose that the polypeptide chain of the passenger passes in a linear fashion across the bacterial outer membrane.

Base Sequence↗
PubMed1992

Changes in the surface composition after transmission of Acanthocheilonema viteae third stage larvae into the jird.

This study describes the dynamics and the biochemical nature of changes in the surface of the filarial nematode Acanthocheilonema viteae after its transmission into the vertebrate host. Vector-derived third-stage larvae (mL3) were inoculated into naive Meriones unguiculatus and recovered from the tissues at different times post-infection until their moult to fourth-stage larvae (L4). Surface-specific labelling with fluoresceinated lectins revealed that the larvae are covered by a carbohydrate envelope. Although the mL3 envelope was strongly reduced one day after transmission, new surface carbohydrates appeared until the onset of moulting, some of which could also be identified on the surface of L4. In general, surface carbohydrates were partially shed by moving larvae, suggesting a loose association of these components in the epicuticle. The fate of cuticular lipids and proteins of L3 and L4 was monitored by external 125I-labelling and differential extraction of the components. Thin-layer chromatography of surface-labelled lipids revealed only minor changes 1 day after parasite transmission. Afterwards the number of lipids accessible to label decreased further until moulting was complete. Two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of surface-labelled proteins showed a consistent surface exposure of mL3 specific proteins until 1 day post-infection. Thereafter, the composition of surface-labelled proteins changed rapidly, resembling that of the L4 as early as several days before moulting. During this period individual differences in the composition of surface proteins were evident.

Animals↗
PubMed1992

Genetic variation in pathogenic bacteria.

In contrast to textbook ideas of pure cultures and defined strains, genetic variation is a fact of life in the microbial world. It not only allows pathogens to establish themselves in their chosen host, but also allows them to resist that host's subsequent attempts to evict them. Here we review some of the mechanisms that bring about this variation, and some of the functional consequences that result from it.

Adaptation, Biological↗
PubMed1992

Silent pilin genes of Neisseria gonorrhoeae MS11 and the occurrence of related hypervariant sequences among other gonococcal isolates.

Pilin variation in Neisseria gonorrhoeae depends on a family of variant genes that undergo homologous, intragenic recombination. This work focuses on the repertoire of silent variant pilin genes in strain MS11, which contribute to the extensive variation of the expressed gene copy. A total of 17 silent copies were identified, which are, to varying degrees, truncated at their 5' coding region and grouped in seven distinct pil loci. Most silent copies belong to loci pilS1, pilS2 and pilS6, which contain six, two and three silent copies, respectively, tandemly arranged. The pilS5 and pilS7 loci each contain only a single copy. In addition, two silent copies are associated with each of the two pilE loci. By comparison with sequences present in the expressed gene of other variants of the same strain, it is suggested that each silent locus is capable of donating variant sequences into the expression locus and, thus, each silent copy can contribute to the variability of pilin expression. Often, concomitant with changes in the expressed copy, the silent copies of the pilE1 locus undergo recombinations as well. Analyses of unrelated clinical isolates of N. gonorrhoeae reveal homologies of hypervariant pilin sequences with those present in strain MS11, suggesting a limited diversity of such sequences within the gonococcal population and the existence of substantial functional constraints on the variability of pilin and pili. The data further indicate that hypervariant pilin sequences are subject to horizontal exchange and interstrain recombination.

Bacterial Outer Membrane Proteins↗
PubMed1992

Interaction of two variable proteins (PilE and PilC) required for pilus-mediated adherence of Neisseria gonorrhoeae to human epithelial cells.

Pili confer the initial ability of Neisseria gonorrhoeae to bind to epithelial cells. Pilin (PilE), the major pilus subunit, and a minor protein termed PilC, reportedly essential for pilus biogenesis, undergo intra-strain phase and structural variation. We demonstrate here that at least two different adherence properties are associated with the gonococcal pili: one is specific for erythrocytes, which is virtually unaffected by PilE variation, and another is specific for epithelial cells, and is modulated in response to the variation of PilE. Based on this finding, mutants of a recA- strain were selected that had lost the ability to bind to human cornea epithelial cells (A-) but retained the ability to form pili (P+) and to agglutinate human erythrocytes (H+). The adherence-negative mutants failed to produce detectable levels of PilC1 or PilC2 proteins, representing piIC phase variants generated in the absence of RecA. The A- pilC phase variants were indistinguishable from their A+ parents and spontaneous A+ revertants with regard to the amount of PilE produced and its electrophoretic mobility, the degrees of piliation and haemagglutination, and the pilE nucleotide sequence. These data demonstrate a central role for PilC in pilus-mediated adherence of N. gonorrhoeae to human epithelial cells and further indicate that neither PilC1 nor PilC2 is obligatory for the assembly of gonococcal pili.

Amino Acid Sequence↗
PubMed1992

Properties of IgA-binding receptors on murine T cells: relative importance of Fc alpha R, beta-galactosyltransferase and anti-secretory component reactive proteins (ASCP).

Murine T cells and T-cell lines express receptors for the Fc of IgA (Fc alpha R); however, their molecular properties remain to be elucidated. In the present study, we examined three candidate molecules for IgA-binding receptors including Fc alpha R, beta-galactosyltransferase (beta-GT) and anti-secretory component (SC) reactive proteins (ASCP) expressed on T cells which might participate in the binding of different molecular forms of IgA. T-cell lines derived from CD4+ T cells of mouse Peyer's patches (PP) (designated PPT 4-6 and PPT 4-16) and from cloned PP T helper (Th) cell lines (ThHA1 #9 and #10) bound both monomeric and dimeric IgA (mIgA and dIgA), while the fusion partners (BW 5147 and R1.1) did not. In contrast, both Fc alpha R+ and Fc alpha R- cell lines bound to high molecular weight polymeric or aggregated IgA (pIgA). All cell lines reacted with a monoclonal anti-beta-GT (MoAb) and beta-GT enzyme activity was associated with the cell lysates and membrane fractions of all cells tested. The anti-beta-GT MoAb stained a 47-kDa band on immunoblots which was identical to that seen with native enzyme. mRNA analysis with beta-GT cDNA showed that all cell lines constitutively produced enzyme-specific mRNA. Both Fc alpha R+ T cells and Fc alpha R- control cell lines showed cell surface specific beta-GT activity. This is the first study which shows that mouse T cells produce beta-GT. However, Fc alpha R and beta-GT appear to be separate receptors, because Fc alpha R+ T cells bound mIgA and dIgA, and this treatment did not affect staining with biotinylated anti-beta-GT MoAb. Further, preincubation of the Fc alpha R+ cells with anti-beta-GT MoAb did not block mIgA binding. However, the anti-beta-GT MoAb partially blocked binding of pIgA to both Fc alpha R+ and Fc alpha R- T cells, suggesting that beta-GT may be a receptor for pIgA. Others have shown that T cells may bind IgA through a receptor serologically related to SC. We found that antibodies both to human SC and to rat SC specifically bound to both Fc alpha R+ and Fc alpha R- T cells. Further, a 72-kDa band was detected when cell membrane fractions were analysed with these antisera (ASCP) by solid phase immunoisolation technique and immunoblot analysis. The ASCP is not an IgA-binding receptor, since anti-SC did not block either mIgA or pIgA binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Biochemical characterization of Porphyromonas (Bacteroides) gingivalis collagenase.

A protease was purified from Porphyromonas gingivalis 1101, a clinical isolate, by sequential sodium dodecyl sulfate-polyacrylamide gel electrophoresis, substrate diffusion gel electrophoresis, and electroelution. The enzyme cleaved radiolabeled human basement membrane type IV collagen and the synthetic collagen peptide substrate for eukaryotic collagenases. It was inactivated by the thiol protease inhibitor N-ethylmaleimide but not by EDTA or EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and activated by reducing agents such as beta-mercaptoethanol. The enzyme exists as an active precursor protein of molecular mass 94 kDa and undergoes proteolytic cleavage to 75-, 56-, and 19-kDa forms. Biotin-labeled collagen bound specifically to the 94-kDa form of the protein and to its cleavage products in ligand blots, suggesting a role for this enzyme not only in collagen degradation but also in adhesion to collagenous substrata.

Cell Fractionation↗