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T F Meyer

Publications and source records attributed to T F Meyer.

At least 19 recordsLinked to original sources
PubMed1995

Role of pili and the phase-variable PilC protein in natural competence for transformation of Neisseria gonorrhoeae.

The Gram-negative bacterial pathogen Neisseria gonorrhoeae is naturally competent for transformation with species-related DNA. We show here that two phase-variable pilus-associated proteins, the major pilus subunit (pilin, or PilE) and PilC, a factor known to function in the assembly and adherence of gonococcal pili, are essential for transformation competence. The PilE and PilC proteins are necessary for the conversion of linearized plasmid DNA carrying the Neisseria-specific DNA uptake signal into a DNase-resistant form. The biogenesis of typical pilus fibers is neither essential nor sufficient for this process. DNA uptake deficiency of defined piliated pilC1,2 double mutants can be complemented by expression of a cloned pilC2 gene in trans. The PilC defect can also be restored by the addition of purified PilC protein, or better, pili containing PilC protein, to the mutant gonococci. Our data suggest that the two phase-variable Pil proteins act on the bacterial cell surface and cooperate in DNA recognition and/or outer membrane translocation.

Bacterial Proteins↗
PubMed1995

C-terminal glycine-histidine tagging of the outer membrane protein Iga beta of Neisseria gonorrhoeae.

A glycine-histidine tag (Gly3His6) was added to the C-terminus of a fusion protein consisting of the cholera toxin B-subunit (CtxB) and the IgA protease beta-domain (Iga beta). The aim was to facilitate single-step purification and to create a suitable tool for kinetic and structural studies on Iga beta-driven protein translocation across the outer membrane of Gram-negative bacteria. We demonstrate that the glycine-histidine tag does not interfere with the assembly of Iga beta in the outer membrane and that the translocator function of the modified Iga beta is maintained. The applicability of the new construct for the dissection of the Iga beta mediated translocation process and general aspects of C-terminal histidine tagging of outer membrane proteins are discussed.

Amino Acid Sequence↗
PubMed1995

Neisseria PilC protein identified as type-4 pilus tip-located adhesin.

Type-4 pilus-mediated adherence of Neisseria gonorrhoeae and Neisseria meningitidis is considered to be a crucial early event in neisserial infections. In addition to the principal pilus subunit (pilin or PilE), both pathogens produce low quantities of a phase-variable PilC protein which is implicated in pilus biogenesis and pilus-mediated epithelial cell adherence. The identity, however, of the pilus adhesin has remained obscure. Here we describe the isolation of a PilC protein from a gonococcal overproducing strain and demonstrate its specific interaction with human epithelial cells. Our results are consistent with the cell and species tropisms of neisserial infections. Binding of PilC effectively competes with pilus-mediated, but not Opa-mediated, attachment of N. gonorrhoeae and of N. meningitidis, indicating that both pathogens interact with identical or very similar epithelial cell receptors. Immunogold electron microscopy using antisera raised against purified PilC and synthetic peptides locates PilC at the tip of gonococcal pili. PilC thus represents an essential pilus-associated adhesin, providing a rationale for selective protection against neisserial infections.

Adhesins, Bacterial↗
PubMed1994

Construction and evaluation of an expression vector allowing the stable expression of foreign antigens in a Salmonella typhimurium vaccine strain.

Salmonella strains have great potential as live carriers of heterologous antigens to induce immunity against a variety of infectious diseases. However, the amount of heterologous antigen required to induce an adequate immune response may be toxic for the bacterium and result in cell death, overattenuation or loss of expression of the heterologous antigen. To solve this problem an expression vector was developed with a strong promoter located on a DNA fragment which is inverted at random. Antigen is only expressed in one particular orientation of the promoter. Thus a bacterial population harbouring the plasmid will consist of a subpopulation which does not produce heterologous antigen, and is therefore not affected in growth, persistence and dissemination within the host. Further, this non-producing population will continuously segregate antigen-producing bacteria. To evaluate the system, CtxB was used as a model antigen. Analysis of the plasmid DNA isolated from Salmonella revealed a selection against the promoter orientation that directs transcription of the ctxB gene. In spite of this, the vector was stably maintained in vivo and induced CtxB-specific IgA and IgG in mice. These results indicate that this kind of expression vector may offer a solution to the problem of unstable expression of foreign antigens in live bacterial vaccine strains.

Animals↗
PubMed1994

A new method for the 3-D in vitro growth of human RT112 bladder carcinoma cells using the alginate culture technique.

We studied the response to different in vitro culture conditions and the ability for polarization in three-dimensional (3-D) and two-dimensional (2-D) cell culture systems of the commonly used human bladder carcinoma cell line RT112. In the case of 2-D culture the cells were grown on glass or plastic coverslips, filter membranes, or bovine lens capsules. The alginate culture technique (ACT) was used to test the effects of 3-D cell culture on the polarization of the RT112 epithelial cells. Our studies show clear differences in the arrangement of the cells depending on the cultivation procedure. The RT112 cells cultured on glass and plastic supports were irregular and flattened in shape whereas the cells grown on filters or on lens capsules developed into 2-3 layers consisting of markedly polarized cells. However, ACT was superior to cell culture on either artificial supports or even on lens capsule. During the 3-D cultivation the transformed epithelial cells regenerated multicellular spheroids which maintained a tissue-like, geometrically well-ordered and highly prismatic organization. This ACT-induced morphogenesis is novel and distinct from that reported with conventional culture conditions. Microscopic investigations showed that RT112 cells grown in alginate were both much more tightly packed and more regularly organized compared to 2-D cultures. In addition, the spheroidal organized cells exhibited well developed cell-cell contacts, a distinct endoplasmatic reticulum, and a marked Golgi apparatus. In summary, ACT can be used for 3-D in vitro growth of the transformed human epithelial cell line RT112 that offers substantial advantages over conventional cell culture methods.

Alginates↗
PubMed1994

Immunogenicity and evolutionary variability of epitopes within IgA1 protease from serogroup A Neisseria meningitidis.

Five murine epitopes were defined and mapped within IgA1 protease produced by Neisseria meningitidis. Epitopes 1 and 2 were present in IgA1 protease from all strains, and from Neisseria gonorrhoeae. Epitopes 3 through to 5 varied between subgroups of serogroup A meningococci, but have remained constant over decades within the subgroups, except for epitope 4, which changed between 1983 and 1987 during the spread of subgroup III meningococci from Asia to Africa. Binding of monoclonal antibodies to epitopes 1, 4 and 5 neutralized enzymatic function. Human sera containing antibodies to IgA1 protease as a result of natural infection inhibited binding of monoclonal antibodies to epitope 4 but not to the other epitopes.

Animals↗
PubMed1994

Generalized transposon shuttle mutagenesis in Neisseria gonorrhoeae: a method for isolating epithelial cell invasion-defective mutants.

One requirement for the invasion of, and tight adherence to, human epithelial cells by Neisseria gonorrhoeae is the synthesis of distinct opacity (Opa) outer membrane proteins, encoded by a family of phase-variable chromosomal genes. However, cloning and surface expression of invasion-promoting Opas in Escherichia coli is not sufficient for the efficient invasion of epithelial cells: additional factors besides Opa may be involved in this process. Using the phoA mini-transposon TnMax4, a library of gonococcal mutants affected in the expression of genes encoding exported proteins was generated through shuttle mutagenesis. Of a total of 608 PhoA+ plasmid clones identified in E. coli E145 approximately 40% were used successfully in transforming N. gonorrhoeae and in activating the corresponding chromosomal genes. Gonococci producing the invasion-promoting Opa50 served as the genetic background to identify 51 mutants unable to enter Chang human epithelial cells. We expect some of these mutations affect the interaction of N. gonorrhoeae with epithelial cells directly, while other mutants may carry defects in general house-keeping, secretory and/or regulatory determinants. In some mutants the loss of invasiveness appears to be due to a negative dominant effect of the PhoA+ fusions produced in these mutants. Some of the identified genes display a phase-variation phenomenon in E. coli and several genes are found in multiple copies in N. gonorrhoeae and/or present only in pathogenic Neisseria species.

Biological Transport↗
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

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

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↗
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↗