Distribution of S-layers on the surface of Bacillus cereus strains: phylogenetic origin and ecological pressure.

Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis have been described as members of the Bacillus cereus group but are, in fact, one species. B. anthracis is a mammal pathogen, B. thuringiensis an entomopathogen and B. cereus a ubiquitous soil bacterium and an occasional human pathogen. In two clinical isolates of B. cereus, in some B. thuringiensis strains and in B. anthracis, an S-layer has been described. We investigated how the S-layer is distributed in B. cereus, and whether phylogeny or ecology could explain its presence on the surface of some but not all strains. We first developed a simple biochemical assay to test for the presence of the S-layer. We then used the assay with 51 strains of known genetic relationship: 26 genetically diverse B. cereus and 25 non-B. anthracis of the B. anthracis cluster. When present, the genetic organization of the S-layer locus was analysed further. It was identical in B. cereus and B. anthracis. Nineteen strains harboured an S-layer, 16 of which belonged to the B. anthracis cluster. All 19 were B. cereus clinical isolates or B. thuringiensis, except for one soil and one dairy strain. These findings suggest a common phylogenetic origin for the S-layer at the surface of B. cereus strains and, presumably, ecological pressure on its maintenance.

Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments.

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.

Crucial residues in the carboxy-terminal end of C1 inhibitor revealed by pathogenic mutants impaired in secretion or function.

The last exon of the C1-1NH gene was screened for point mutations in 36 unrelated hereditary angioedema patients. Mutations were found in eight patients, predicting changes in the short COOH-terminal region which anchors the reactive site loop on its COOH-terminal side. The effects of each of these mutations were examined in transiently transfected Cos-7 cells. Complete intracellular retention or degradation was observed with substitutions in the COOH-terminal strands 4B or 5B: Leu459-->Pro, Leu459-->Arg, and Pro467-->Arg were all blocked at early stages of intracellular transport, but differences in the immunofluorescence patterns indicated that a significant fraction of the Leu459-->Pro and of the Pro467-->Arg proteins reached a compartment distinct from the endoplasmic reticulum. In line with previous findings with alpha 1-antitrypsin, chain termination within strand 5B resulted in rapid degradation. Mutant Val451-->Met, in strand 1C, and mutant Pro476-->Ser, replacing the invariant proline near the COOH terminus, yielded reduced secretion, but these extracellular proteins were unable to bind the target protease C1s. Presence of low levels of both dysfunctional proteins in patient plasmas defies the conventional classification of C1 inhibitor deficiencies as type I or type II. These data point to a key role of certain residues in the conserved COOH-terminal region of serpins in determining the protein foldings compatible with transport and proper exposure of the reactive site loop.

C1 inhibitor mutations which affect intracellular transport and secretion in type I hereditary angioedema.

A cluster of point mutations was found in the region of exon 8 of the C1 INH gene which codes for the 28 C-terminal amino acids. Seven of these mutations introduce amino acid changes and one results in a stop codon. Upon transient expression in monkey kidney Cos-7 cells all of these C1 inhibitor mutants showed an impaired intracellular transport and most of them failed to be secreted. Biochemical and immunofluorescence studies indicated that the defective proteins accumulate or are degraded mainly in the endoplasmic reticulum. The product of deletions of exons 4, which has an internal in frame deletion of 45 amino acids, also fails to be secreted.

Complement genes C1r and C1s feature an intronless serine protease domain closely related to haptoglobin.

The exon-intron structure of the human complement C1s gene displays a striking similarity with that of the gene encoding haptoglobin, a peculiar transport protein distantly related to the serine proteases. While the protease regions of the serine zymogens are typically encoded by multiple exons, the protease domains of C1s and of its genetically linked and functionally interacting homolog C1r are encoded as intronless domains, not unlike a region of haptoglobin, which in fact is devoid of proteolytic activity. The close similarity of the C1s gene with haptoglobin includes the precise conservation of exon-intron junctions and it extends to upstream exons encoding the short repeats typical of several complement components, but found also in other functionally unrelated proteins. Additional evidence of the common ancestry of C1r, C1s and haptoglobin is the presence, within the protease domain, of a set of sequence markers that distinguish these three proteins from all known serine proteases. The finding of vertebrate serine protease genes with an uninterrupted protease-encoding exon supports the definition of a novel evolutionary branch of this gene family and rules out the hypothesis that regards this unusual exon as an irrelevant byproduct of the extravagant functional divergence of haptoglobin.

Human complement C1r and C1s proteins and genes: studies with molecular probes.

The isolation of complementary DNA clones for both enzymic subcomponents of C1 has made it possible to derive their complete amino acid sequences and to verify and extend previous protein data. We review here recent advances in studies of the C1r and C1s proteins and of the corresponding genes, using molecular probes. The mosaic structure of these proteins has been compared to the exon-intron organization of the C1s gene. Surprisingly, the C1r and the C1s genes feature an intronless serine protease domain, at variance with all vertebrate serine proteases. Moreover, C1r and C1s are related in evolution to haptoglobin, a serine protease analog lacking enzymic activity. The C1r and C1s genes are closely linked in an unusual tail to tail orientation. These findings are discussed with regard to the apparently coordinate expression of these complement components and to the combined nature of most C1r and C1s deficiencies. We also discuss the implications of the successful production of C1r protein using recombinant DNA technology.

The structure of tissue on cell culture-extracted thyroglobulin is independent of its iodine content.

The major protein synthesized in vitro by the ovine thyroid cell line OVNIS 6H is the prothyroid hormone thyroglobulin. Purified from serum-free cell culture media using sucrose gradient centrifugation, the thyroglobulin dimer was analysed for iodine content and observed by electron microscopy. In their usual medium, the OVNIS 6H cells produce a very poorly iodinated thyroglobulin containing 0.05 I atom per molecule. When cultured with methimazole or propylthiouracil, two inhibitors of iodide organification, less than 0.007 I atom/molecules was found. These molecules purified from cell cultures were compared to those purified from ovine thyroid tissue containing 26 I atoms/mol. Despite large differences in iodine content, the three preparations all consist of 19 S thyroglobulin dimers with the classical ovoidal shape. The variability in size measurements remains in a 2% range for all thyroglobulin types. Consequently, no real significant variation can be found between the highly iodinated thyroglobulin isolated from tissue, and the poorly or non-iodinated thyroglobulins isolated from cells cultured with or without methimazole or propylthiouracil.

Ultrastructural and anti-proteinase activity modifications of human alpha 2-macroglobulin induced by zinc and other divalent cations.

Native tetrameric alpha 2-macroglobulin molecules (alpha 2M) can be converted into a population of dimers by incubation with various divalent cations such as Zn, Cd, Mg, Cu, Ni, Co. This dissociation is completed within 30 min at 37 degrees C. These dimers have a characteristic shape and a size of about 16 X 8 nm, and appear to be the half of the native alpha 2M molecule which has a clear tetrameric structure as seen in the electron microscope. At room temperature or below, dimers obtained with 5 to 100 mM Zn++ can reassociate in long linear polymers which display a regular chain-like arrangement and a helical periodicity. The structural characteristics of this polymer are described. The trypsin inhibitory capacity of Zn++-treated alpha 2M has been studied in an attempt to correlate its Zn++-induced conformational changes with its functional modifications.

Electron microscopy of the conformational changes of alpha 2-macroglobulin from human plasma.

High resolution electron microscopy reveals that fully active alpha 2-macroglobulin (alpha2M) from fresh human plasma presents a very characteristic tetrameric structure. This native conformation of the alpha2M molecule is described here for the first time, along with its various orientations in negatively stained preparations. Although the native form is sensitive to inactivation, glutaraldehyde fixation is not necessary for its observation except when ammonium salts are used. The tetrameric structure of alpha2M undergoes a drastic conformational change when the protein is treated either with trypsin, thrombin or methylamine, as evidenced by the appearance of the typical)+(structure already described in the literature. The various aspects of this second conformation correspond to different orientations of the molecules in the stain film, and depend upon the nature of the support.

Immunolabelling of bacteriophage lambda receptor protein (LamB) on thin sections of E. coli embedded in Lowicryl.

LamB is one of the major cellular proteins when E. coli is grown in the presence of maltose and is localized in the outer membrane. Previous immunolabellings obtained with monoclonal antibodies showed that this protein is a transmembrane protein and led to the detection of 4 epitopes exposed on the cell surface and 2 located on the inner surface of the outer membrane (Scheckman et al., 1983). In the present study, we have used this biological model in order to see whether these two classes of epitopes could be distinguished by immunocytochemical labelling performed on thin sections of E. coli embedded in Lowicryl K4M (Carleman et al., 1982). The optimal conditions of fixation and embedding were first established for labelling with poly- or monoclonal antibodies detected by Protein A-gold complexes. The analysis of gold particle distribution on each side of the outer membrane after labelling with a polyclonal serum or after its adsorption on intact bacteria allowed us to conclude that the resolution of immunolabelling on thin sections was about 20 nm. The use monoclonal antibodies met with difficulties due mostly to the nonspecific labelling of the cytoplasm. Although this nonospecific labelling was decreased by fixing bacteria with paraformaldehyde alone, only one antibody gave a correct specific labelling after high dilution (1/3000). The gold particle distribution obtained with this antibody confirmed the location on the cell surface of this epitope.