Crucial importance of PKC-beta(I) in LFA-1-mediated locomotion of activated T cells.

Crawling T cell locomotion in which activated lymphocyte function-associated antigen 1 (LFA-1) integrins participate is associated with translocation of the protein kinase C-beta (PKC-beta) isoenzyme to the microtubule cytoskeleton. In normal T cells and T lymphoma cell lines, this type of motility is accompanied by PKC-beta-sensitive cytoskeletal rearrangements and the formation of trailing cell extensions, which are supported by microtubules. Expression of PKC-beta(I) and enhanced green fluorescent protein (EGFP) in nonmotile PKC-beta-deficient T cells restored their locomotory behavior in response to a triggering stimulus delivered via LFA-1 and correlated directly with the degree of cell polarization. We have also shown that PKC-beta(I) is a component of the tubulin-enriched LFA-1-cytoskeletal complex assembled upon LFA-1 cross-linking. These observations may have physiological equivalents at advanced (post-integrin activation) stages of lymphocyte extravasation.

Contribution of clumping factor B to pathogenesis of experimental endocarditis due to Staphylococcus aureus.

Staphylococcus aureus Newman with an insertion mutation in clfB, the gene encoding clumping factor B, only marginally decreased infection rate (P>0.05) in rats with experimental endocarditis. In contrast, clfB complementation on a multicopy plasmid significantly increased infectivity (P<0.05) over the deleted mutants. Although clfB could affect endovascular infection, its importance in experimental endocarditis was limited.

The thioredoxin system of Helicobacter pylori.

This paper describes the purification of thioredoxin reductase (TR) and the characterization, purification, and cloning of thioredoxin (Trx) from Helicobacter pylori. Purification, amino acid sequence analysis, and molecular cloning of the gene encoding thioredoxin revealed that it is a 12-kDa protein which possesses the conserved redox active motif CGPC. The gene encoding Trx was amplified by polymerase chain reaction and inserted into a pET expression vector and used to transform Escherichia coli. Trx was overexpressed by induction with isopropyl-1-thio-beta-D-galactopyranoside as a decahistidine fusion protein and was recovered from the cytoplasm as a soluble and active protein. The redox activity of this protein was characterized using several mammalian proteins of different architecture but all containing disulfide bonds. H. pylori thioredoxin efficiently reduced insulin, human immunoglobulins (IgG/IgA/sIgA), and soluble mucin. Subcellular fractionation analysis of H. pylori revealed that thioredoxin was associated largely with the cytoplasm and inner membrane fractions of the cell in addition to being recovered in the phosphate-buffered saline-soluble fraction of freshly harvested cells. H. pylori TR was purified to homogeneity by chromatography on DEAE-52, Cibacron blue 3GA, and 2',5'-ADP-agarose. Gel filtration revealed that the native TR had a molecular mass of 70 kDa which represented a homodimer composed of two 35-kDa subunits, as determined by SDS-polyacrylamide gel electrophoresis. H. pylori TR (NADPH-dependent) efficiently catalyzed the reduction of 5,5'-dithiobis(nitrobenzoic acid) in the presence of either native or recombinant H. pylori Trx. H. pylori Trx behaved also as a stress response element as broth grown bacteria secreted Trx in response to chemical, biological, and environmental stresses. These observations suggest that Trx may conceivably assist H. pylori in the process of colonization by inducing focal disruption of the oligomeric structure of mucin while rendering host antibody inactive through catalytic reduction.

Clumping factor B (ClfB), a new surface-located fibrinogen-binding adhesin of Staphylococcus aureus.

The surface-located fibrinogen-binding protein (clumping factor; ClfA) of Staphylococcus aureus has an unusual dipeptide repeat linking the ligand binding domain to the wall-anchored region. Southern blotting experiments revealed several other loci in the S. aureus Newman genome that hybridized to a probe comprising DNA encoding the dipeptide repeat. One of these loci is analysed here. It also encodes a fibrinogen-binding protein, which we have called ClfB. The overall organization of ClfB is very similar to that of ClfA, and the proteins have considerable sequence identity in the signal sequence and wall attachment domains. However, the A regions are only 26% identical. Recombinant biotinylated ClfB protein bound to fibrinogen in Western ligand blots. ClfB reacted with the alpha- and beta-chains of fibrinogen in the ligand blots in contrast to ClfA, which binds exclusively to the gamma-chain. Analysis of proteins released from the cell wall of S. aureus Newman by Western immunoblotting using antibody raised against the recombinant A region of ClfB identified a 124 kDa protein as the clfB gene product. This protein was detectable only on cells that were grown to the early exponential phase. It was absent from cells from late exponential phase or stationary phase cultures. Using a clfB mutant isolated by allelic replacement alone and in combination with a clfA mutation, the ClfB protein was shown to promote (i) clumping of exponential-phase cells in a solution of fibrinogen, (ii) adherence of exponential-phase bacteria to immobilized fibrinogen in vitro, and (iii) bacterial adherence to ex vivo human haemodialysis tubing, suggesting that it could contribute to the pathogenicity of biomaterial-related infections. However, in wild-type exponential-phase S. aureus Newman cultures, ClfB activity was masked by the ClfA protein, and it did not contribute at all to interactions of cells from stationary-phase cultures with fibrinogen. ClfB-dependent bacterial adherence to immobilized fibrinogen was inhibited by millimolar concentrations of Ca2+ and Mn2+, which indicates that, like ClfA, ligand binding by ClfB is regulated by a low-affinity inhibitory cation binding site.

The lipopolysaccharide of Porphyromonas gingivalis is not antigenically cross-reactive with that of other species.

Large numbers of Porphyromonas, Prevotella, and Bacteroides strains were screened by 3 monoclonal antibodies (MAbs) and 8 rabbit antisera raised against Porphyromonas gingivalis, in order to detect any possible recognition of non-P. gingivalis surface antigens by these immunoreagents. All three MAbs, which were LPS-specific, extensively recognized LPS from 10 P. gingivalis strains in immunoblotting, whereas they recognized none of the 34 non-P. gingivalis strains. Rabbit antisera were similarly specific for P. gingivalis cells in immunofluorescence and with LPS in grid-blotting, but several of them recognized LPS from one Prevotella melaninogenica and 5 Prevotella intermedia strains in Western blotting. Since several pre-immune sera and an irrelevant serum raised to a Streptococcus species recognized up to 5 of these preparations, we exclude that the reactions were due to antigens shared by P. gingivalis and Prevotella. Rather, we consider that they were false-positive reactions due to natural antibodies, stimulated in a non-specific manner upon immunization with P. gingivalis, in animals whose immune systems were sensitized to Prevotella species before immunization.

The hemagglutinating adhesin HA-Ag2 of Bacteroides gingivalis is distinct from fimbrilin.

We carried out a series of immunoblots with antigenic preparations from the periodontal pathogen Bacteroides gingivalis using antisera of restricted specificity for the hemagglutinating adhesin HA-Ag2, and for the major structural subunit of the fimbriae (fimbrilin). We have been able to show that these 2 antigens are distinct. The fimbrilin subunit had an apparent molecular weight of 42 kDa in all of the bacterial preparations tested. HA-Ag2 occurred as a pair of bands at 43 and 49 kDa in outer membranes prepared as extracellular vesicles, and at 33 and 38 kDa in glass-bead-EDTA extracted antigens and in sheared-cell outer membranes prepared in the presence of EDTA. No HA-Ag2 was found in an enriched fimbrial preparation. The 2 antigens could thus be distinguished on the basis of their behaviour when subjected to different extraction techniques. The lower apparent molecular weight of HA-Ag2 (a pair of bands at 33 and 38 kDa) was invariably associated with the presence of EDTA in the buffers used to prepare the extracts, and the effect could be partially prevented by adding MgCl2 to the extraction buffer. The difference in apparent molecular weight of HA-Ag2 in the different extracts can thus be attributed either to an EDTA-sensitive tertiary conformation of its component polypeptides, or to an EDTA-sensitive linkage of each of these polypeptides to an unknown component of approximately 10 kDa.

SDS-PAGE analysis of protein and lipopolysaccharide of extracellular vesicules and Sarkosyl-insoluble membranes from Bacteroides gingivalis.

We have compared outer membranes (OM) of Bacteroides gingivalis ATCC 33277 isolated by the following 3 techniques: 1) high speed centrifugation after mechanical cell shearing; 2) sonication of the bacteria, followed by solubilization of the cytoplasmic membrane with N-Laurylsarconsinate (Sarkosyl), after which the Sarkosyl-insoluble membranes were recovered by centrifugation; 3) ammonium sulfate precipitation of extracellular vesicules from culture supernatant, followed by centrifugation and dialysis. Electron microscopy showed that the 3 preparations consisted of closed vesicules. Analysis by SDS-PAGE revealed that all 3 contained up to 28 polypeptides, most of which were common to each extract. The extracellular vesicules and Sarkosyl-insoluble preparation yielded similar protein patterns, although quantitative differences were observed. The sheared-cell preparation contained 8 additional proteins. The level of contamination of OM material by peptidoglycan and cytosol components was 1.8% in the sheared-cell preparation, and was null or lower than 0.8% in the other preparations. All 3 preparations showed the presence of LPS with a multiple banding pattern typical of smooth LPS. The sheared-cell preparation had a slightly lower LPS content than the other 2 preparations. Since extracellular vesicules are naturally released during bacterial growth, and are relatively simple to obtain, such native entities seem an appropriate source of OM components for use in studying the immunobiology of B. gingivalis surface antigens.

Improved performance of intensive semicontinuous cultures of Scenedesmus by biomass recirculation.

The microscopic green alga, Scenedesmus obliquus, was used in a semicontinuous culture system for the tertiary treatment of urban wastewater, with the simultaneous production of usable biomass. Partial biomass recycling was used to increase the productivity of the system by overcoming the limits imposed by the low maximal growth rate of the alga. The biomass to be recycled was collected by simple gravity settling of the removed culture.The culture system was operated at different dilution rates and its productivity measured at each rate. An evaluation of the crude nutrient composition of the algae produced at each dilution rate was also carried out.The system was found to operate stably at dilution rates of up to 0.8 day(-1) which represents a 20% net increase over the maximum dilution rate allowed under the same conditions in a system without recirculation. The composition of the biomass produced varied little over a range of dilution rates, which may be of relevance to its projected end-use.The study indicated that such a system can exploit available light to the full and should be of particular value for the treatment of low-strength wastes such as we employed.