The modification of the cell wall proteins in group A streptococci type M 29 under the influence of spermidine contained in the culture medium

The addition of spermidine into growth medium used for the cultivation of group A streptococci, type M 29, leads to changes in the amino acid composition of cell walls and surface proteins isolated by the method of E. H. Beachey et al. The separation of surface proteins into fibrinogen-binding proteins and fibrinogen receptors by affinity chromatography techniques on cellulose with covalently bound fibrinogen indicates that the proportion of these proteins in pepsin extracts obtained from different strains varies. Both spermidine and avirulent strains have similar content of fibrinogen-binding proteins, although these proteins are absent in virulent strains. Different amounts of fibrinogen receptors are extracted from all strains. As shown in the enzyme immunoassay, fibrinogen receptors contain no group-specific polysaccharide A, Fc-receptors and interact with total antiserum to group A streptococci, type M 29 [correction of 28]. Fibrinogen receptors isolated from the strains under study have been found to have similar amino acid composition. On the basis of these results we believe that neither receptor capacity to fibrinogen nor amino acid composition is indicative of the protective properties of protein M.     

The effect of blood serum components on the growth, biochemical and biological properties of streptococcus

The components of cattle blood serum, added to the medium for the cultivation of group A streptococci, considerably decrease the period of adaptation and increase the balanced growth rate of streptococci, which is manifested by changes in the surface structures of the cell wall: the absence or modification of protein M. Streptococci grown under these conditions lose their capacity for phagocytosis, and from the cell walls obtained from these streptococci no surface protein M can be isolated by pepsin treatment. Nevertheless, the ratio of the main cell-wall components (proteins, polysaccharide and peptidoglycan), the amino acid composition, as well as the resistance of the cell walls to the action of trypsin and endo-N-acetylmuramidase are the same in M+ and Mx variants, that makes it possible to infer that the modification of protein M or the inhibition of its synthesis occurs during the growth of streptococci in the presence of blood serum components.     

Immunochemical analysis in research on antibodies to Streptococcus group A ribosomes

The use of the competitive enzyme immunoassay (EIA) has made it possible to demonstrate that antiserum to the ribosomes of group A streptococcus, type 29 M, contains antibodies to homologous protein M and does not contain antibodies to group A polysaccharide, lipoteichoic acid and peptidoglycan. Ribosomal antiserum has been found to bind with the surface of heterologous type M streptococci in EIA, while forming no precipitation lines with heterologous proteins M in the double immunodiffusion test, which indicates that the binding of ribosomal antibodies with the surface of group A streptococcal cells is not type-specific. Antibodies to the ribosomes of group A streptococcus recognize some of the antigenic determinants of E. coli ribosomes.     

Type 1 M protein of Streptococcus pyogenes. N-terminal sequence and peptic fragments

Limited proteolysis of the surface of type 1 Streptococcus pyogenes by pepsin gives rise to fragment Pep M1 of Mr 20270 as the main product which covers the N-terminal part of the M protein. The amino acid sequence was determined of the N-terminal region of the M protein representing the most exposed part of the molecule on the surface fibrils of streptococcal cells, which seems to be very important for the differentiation of the individual serological types. The sequence differs from the homologous N-terminal sequences of types 5, 6 and 24, and shows a homology with sequences repeating in the chain of type 24. Fragment Pep M1 binds to fibrinogen; the absence of its 30 N-terminal amino acid residues, however, abolishes this interaction which is believed to play a role in the virulence of S. pyogenes.     

Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.     

Glycolipids from the cell walls of streptococci

A streptococcus, serologically defined as a Z₃III strain was compared with a mutant strain Z₃ lacking the type III polysaccharide antigen. The loss of type antigen represents a decrease in the carbohydrate content of the cell wall of the mutant and is accompanied by long-chain formation, increased sensitivity to streptomycin and agglutination in saline. Cell-wall preparations can be freed of membrane contamination by treatment with hot sodium dodecylsulfate (SDS). This resulted in a doubling of the ratio of muramic acid to lysine and in the disappearance of phospholipids. It could be shown that the membrane-free cell walls of these strains still contained appreciable amounts of glycolipids which could be identified as monoglucosyl glyceride and diglucosyl-diglyceride.     

Transduction of M+- and SOR+-markers in group A streptococci]

Phagolysates obtained in passage of the virulent phage CA1 on M+ SOR+-cultures of streptococcus, serotype 22, offered a possibility of transduction of the M22+-sign to M-SOR--strains of serotype T12. Transductants were selected in direct bactericidal test with subsequent determination of their type in the indirect bactericidal test with homo- and heterologous immune sera. The UV-lysates of the lysogenic M+ SOR+-cultures of serotypes 4, 22, and 49 offered a possibility of transducing both signs conjointly. In this case transductants were selected by the SOR-phenotype in the dishes with serum agar. All the transductants retained the T-serotype of the recipient's strain and segregated M-- and SOR--clones with high frequency. There were for the first time obtained in streptococcus of group A proofs of transduction of M+- and SOR+-signs pointing to their determination by different, but closely conjoint genes. Conditions of the marker transduction, selection and study of transductant were chosen, they opened new possibilities for genetic analysis of the virulence of streptococcus, group A.     

Psoralen-sensitive mutant pso9-1 of Saccharomyces cerevisiae contains a mutant allele of the DNA damage checkpoint gene MEC3

Complementation analysis of the pso9-1 yeast mutant strain sensitive to photoactivated mono- and bifunctional psoralens, UV-light 254 nm, and nitrosoguanidine, with pso1 to pso8 mutants, confirmed that it contains a novel pso mutation. Molecular cloning via the reverse genetics complementation approach using a yeast genomic library suggested pso9-1 to be a mutant allele of the DNA damage checkpoint control gene MEC3. Non-complementation of several sensitivity phenotypes in pso9-1/mec3Delta diploids confirmed allelism. The pso9-1 mutant allele contains a -1 frameshift mutation (deletion of one A) at nucleotide position 802 (802delA), resulting in nine different amino acid residues from that point and a premature termination. This mutation affected the binding properties of Pso9-1p, abolishing its interactions with both Rad17p and Ddc1p. Further interaction assays employing mec3 constructions lacking the last 25 and 75 amino acid carboxyl termini were also not able to maintain stable interactions. Moreover, the pso9-1 mutant strain could no longer sense DNA damage since it continued in the cell cycle after 8-MOP + UVA treatment. Taken together, these observations allowed us to propose a model for checkpoint activation generated by photo-induced adducts.     

The porin OmpC of Salmonella typhimurium mediates adherence to macrophages.

Macrophages recognize, adhere to, and phagocytose Salmonella typhimurium. The major outer membrane protein OmpC is a candidate ligand for macrophage recognition. To confirm this we used transposon mutagenesis to develop an ompC-deficient mutant in a known virulent strain of S. typhimurium; mutant and wild type were compared in macrophage adherence and association assays. Radiolabeled wild type S. typhimurium bound to macrophages at five-fold higher levels than did the ompC mutant. In association assays, macrophages in monolayers bound and internalized three-fold more wild type than mutant, while macrophages in suspension bound and internalized 40-fold more wild type than mutant. The ompC gene of our test strain of S. typhimurium contains several discrete differences compared with the ompC genes of Salmonella typhi and Escherichia coli. The deduced OmpC amino acid sequence of S. typhimurium shares 77 and 98% identity with OmpC amino acid sequence of E. coli and S. typhi, respectively. Evidence from this study supports a role for the OmpC protein in initial recognition by macrophages and distinguishes regions of this protein that potentially participate in host-cell recognition of bacteria by phagocytic cells.     

Isolation and partial characterization of exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A.

Homogeneous fragments of exosporium were isolated and purified from mature spores of a highly sporogenic mutant derived from Clostridium botulinum type A strain 190L. The exosporium was composed of three lamellae and showed a hexagonal array when negatively stained. The hexagonal array of isolated exosporium was resistant to sodium dodecyl sulfate, urea, dithiothreitol, and proteolytic enzymes such as trypsin, pronase, and nagarse, except for pepsin. The hexagonal array was partially disintegrated with 5 M guanidine-HCl and almost completely disrupted with 8 M urea in combination with 1% mercaptoethanol under alkaline conditions. The purified exosporium fraction was composed mainly of protein (69.1%) and lipids (13.8%). A small amount of amino sugars (2.5%) was present, but neutral sugars could not be detected. The exosporium protein had a predominantly acidic amino acid composition accompanied by low levels of cystine, methionine, and histidine.     

Molecular characterization of a novel fibronectin-binding protein of Streptococcus pyogenes strains isolated from toxic shock-like syndrome patients

Group A Streptococcus pyogenes has surface-located fibronectin (Fn)-binding proteins known to be a major virulence factor, which adheres to and invades host cells. We present a novel Fn-binding protein of group A streptococcus serotype M3 and M18 strains isolated from patients with toxic shock-like syndrome (TSLS). By searching the whole genome sequence of an M3 strain from a TSLS patient, an open reading frame was found among the putative surface proteins. It possessed an LPXTG motif and Fn-binding repeat domains in the C-terminal region and was designated as FbaB (Fn-binding protein of group A streptococci type B). The fbaB gene was found in all M3 and M18 strains examined, although not in other M serotypes. Furthermore, FbaB protein was expressed on the cell surface of TSLS strains but not on non-TSLS ones. Enzyme-linked immunosorbent assay and ligand blotting revealed that recombinant FbaB exhibits a strong Fn-binding ability. An FbaB-deficient mutant strain showed 6-fold lower adhesion and invasion efficiencies to HEp-2 cells than the wild type. Moreover, mortality was decreased in mice infected with the mutant strain in comparison to the wild type. These data suggest that FbaB is etiologically involved in the development of invasive streptococcal diseases.     

A fibrinogen receptor from group B Streptococcus interacts with fibrinogen by repetitive units with novel ligand binding sites

Group B Streptococcus (GBS) is a frequent cause of bacterial sepsis and meningitis in neonates. During the course of infection, GBS colonizes and invades a number of host compartments, thereby interacting with different host proteins. In the present report, we describe the isolation of the fbsA gene, which encodes a fibrinogen receptor from GBS. The deduced FbsA protein is characterized by repetitive units, each 16 amino acids in length. Sequencing of the fbsA gene from five different GBS strains revealed significant variation in the number of repeat-encoding units. The deletion of the fbsA gene in the genome of GBS 6313 completely abolished fibrinogen binding, suggesting that FbsA is the major fibrinogen receptor in this strain. Growth of the fbsA deletion mutant in human blood was significantly impaired, indicating that FbsA protects GBS from opsonophagocytosis. In Western blot experiments with truncated FbsA -proteins, the repeat region of FbsA was identified as mediating fibrinogen binding. Using synthetic peptides, even a single repeat unit of FbsA was demonstrated to bind to fibrinogen. Spot membrane analysis and competitive binding experiments with peptides carrying single amino acid substitutions allowed the prediction of a fibrinogen-binding motif with the consensus sequence G-N/S/T-V-L-A/E/M/Q-R-R-X-K/R/W-A/D/E/N/Q-A/F/I/L/V/Y-X-X-K/R-X-X.     

The low-molecular protein of the cell wall in streptococci group A devoid of serological type specificity]

The scheme for the isolation and purification of low-molecular cell-wall protein without type specificity, including the extraction of the cell walls of group A streptococci, type M 29, with 1% solution of Triton X-100, the separation of the extract by ion-exchange chromatography in DEAE-trisacryl M with the subsequent two-stage gel filtration in superfine Sephadex G-50, is described. The isolated protein had a molecular weight of 4,000 daltons and contained no admixtures of group-specific polysaccharide A, phosphorus, nucleic acids and Fc receptors and interacted with antisera to group A streptococcal cells of heterologous type M in the enzyme immunoassay (EIA). Purified protein was characterized by a high content of glycine. The antigenic determinants of immobilized protein, recognized by antibodies in EIA, were sensitive to the action of trypsin and resistant to the action of pepsin, papain, pronase E and sodium periodate.     

A single-base change within the DNA polymerase locus of herpes simplex virus type 2 can confer resistance to aphidicolin.

An aphidicolin-resistant (Aphr) mutant of herpes simplex virus (HSV) type 2 strain 186 previously has been shown to induce an altered viral DNA polymerase that is more resistant to aphidicolin and more sensitive to phosphonoacetic acid (PAA) than is wild-type DNA polymerase. In this study the mutation responsible for the aphidicolin-resistant phenotype was physically mapped by marker transfer experiments. The physical map limits for the Aphr mutation were contained in a 1.1-kilobase pair region within the HSV DNA polymerase locus. The 1.1-kilobase-pair fragment of the Aphr mutant also conferred hypersensitivity to PAA, and DNA sequence analysis revealed an AT to GC transition within this fragment of the Aphr mutant. Analysis of the three potential open reading frames within the 1,147-base-pair fragment and comparison with the amino acid sequence of DNA polymerase of HSV type 1 indicated that the Aphr mutant polymerase had an amino acid substitution from a tyrosine to a histidine in the well-conserved region of the DNA polymerase. These results indicate that this single amino acid change can confer altered sensitivity to aphidicolin and PAA and suggest that this region may form a domain that contains the binding sites for substrates, PPi, and aphidicolin.     

Bioinformatic analysis of structural proteins of paramyxovirus Tianjin strain

The amino acid sequences of the NP,P, M, F,HN and L proteins of the paramyxovirus Tianjin strain were analyzed by using the bioinformatics methods. Phylogenetic analysis based on 6 structural proteins among the Tianjin strain and 25 paramyxoviruses showed that the Tianjin strain belonged to the genus Respirovirus, in the subfamily Paramyxovirinae, and was most closely related to Sendal virus (SeV). Phylogenetic analysis with 14 known SeVs showed that Tianjin strain represented a new evolutionary lineage. Similarities comparisons indicated that Tianjin strain P protein was poorly conserved, sharing only 78.7%-91.9% amino acid identity with the known SeVs, while the L protein was the most conserved, having 96.0%-98.0% amino acid identity with the known SeVs. Alignments of amino acid sequences of 6 structural proteins clearly showed that Tianjin strain possessed many unique amino acid substitutions in their protein sequences, 15 in NP, 29 in P, 6 in M, 13 in F, 18 in HN, and 29 in L. These results revealed that Tianjin strain was most likely a new genotype of SeV. The presence of unique amino acid substitutions suggests that Tianjin strain maybe has a significant difference in biological, pathological, immunological, or epidemiological characteristics from the known SeVs.     

Peptide inhibitors of coagulative transformation of fibrinogen

The human blood plasma has peptide inhibitors of fibrinogen coagulative transformation that differ in molecular weight. Amino acid composition and N-terminal amino acid sequence of the largest peptide show that it precedes the lighter ones. The blood plasma of white rats and a bull contains inhibitors being alike in the action upon hemostasis and in amino acidic composition. As shown in investigations of white rats, up to 97% of pulled peptides, inhibitors of fibrinogen coagulative transformation is deposited in the viscera tissues. The mobile mechanism of the inhibitor redistribution in blood and tissue proves their great role in blood stabilization. The inhibitors of the peptide nature are believed to supply basic antipolymerisation plasma activity preventing fibrinogen formation in constant blood coagulation with slow intensity.     

Cloning and characterization of the glutamate dehydrogenase gene in Bacillus licheniformis

The gdhA genes of IRC-3 GDH-strain and IRC-8 GDH+ strain were cloned,and they both successfully complemented the nutritional lesion of an E.coli glutamate auxotroph,Q100 GDH-.However,the gdhA gene from the mutant IRC-8 GDH+ strain failed to complement the glutamate deficiency of the wild type strain IRC-3.The gdhA genes of the wild type and mutant origin were sequenced separately.No nucleotide difference was detected between them.Further investigations indicated that the gdhA genes were actively expressed in both the wild type and the mutant.Additionally,no GDH inhibitor was found in the wild type strain IRC-3.It is thus proposed that the inactivity of GDH in wild type is the result of the deficiency at the post-translational level of the gdhA expression.Examination of the deduced amino acid sequence of Bacillus licheniformis GDH revealed the presence of the motifs characteristic of the familyⅠ-type hexameric protein,while the GDH of Bacillus subtilis belongs to family II.     

Repeating covalent structure and protective immunogenicity of native and synthetic polypeptide fragments of type 24 streptococcal M protein. Mapping of protective and nonprotective epitopes with monoclonal antibodies

The complete amino acid sequences of three cyanogen bromide peptide fragments (CB3, CB4, and CB50 of type 24 M protein extracted from Streptococcus pyogenes by limited pepsin digestion were determined by automated Edman degradation of the uncleaved peptides and their tryptic peptides. CB3 and CB4 each contain 35 amino acid residues, whereas CB5 contains 37. The sequence of CB3 was found to be: (formula: see text) (where Hse represents homoserine). The sequence of CB4 was identical except for amino acid substitutions of arginine and glutamine at positions 23 and 24, respectively. The sequence of CB5 also was identical with that of CB3 except for substitutions of aspartic acids at positions 28 and 29; leucine, glutamic acid, and glycine at positions 33, 34, and 35, respectively; and an additional two amino acids, alanine and homoserine, at positions 36 and 37, respectively. A comparison of the structures of these three peptide fragments with those previously reported for CB6 and CB7 revealed as few as one to six amino acid substitutions among the five repeating peptides; CB4 and CB6 differed only by a single Asp/Glu substitution at position 26. When covalently linked to polylysine and injected as an emulsion in complete Freund's adjuvant, CB3, CB4, and CB5 each evoked high titers of type-specific opsonic and bactericidal antibodies in rabbits. A chemically synthesized peptide identical with native CB3 except that it contained methionine instead of homoserine at its COOH terminus was similarly immunogenic. None of the conjugated native or synthetic peptides raised antibodies at reacted in immunofluorescence tests with sarcolemmal membranes of human heart tissue. Mapping studies with monoclonal antibodies revealed a number of distinct protective and nonprotective epitopes. The single Asp/Glu substitution between CB4 and CB4 rendered the 35-residue peptide unrecognizable by protective monoclonal antibodies but recognizable by a nonprotective one. Our studies demonstrate that the repeating covalent structures of native and chemically synthesized polypeptide fragments of streptococcal M protein possess several unique as well as repeating epitopes that evoke opsonic and presumably protective, but not heart cross-reactive, antibodies against a rheumatogenic strain of S. pyogenes.     

The amino acid sequence of the enterotoxin from Clostridium perfringens type A

The amino acid sequence of the enterotoxin from Clostridium perfringens type A was determined by analysis of peptides derived from the protein by digestion with trypsin chymotrypsin, thermolysin, pepsin, a lysine-specific protease. S. aureus V8 protease and a proline-specific protease, and fragments generated by cleavage with cyanogen bromide or by dilute acetic acid in 7 M guanidine HCl. The sequence which is complete except for the definite order of 3 small peptides between residues 88 and 103 consists of 309 amino acids and contains a correction to our preliminary announcement [(1984) FEMS Symp. 24, 329-330].