Biomedical subjects

J J Ferretti

Publications and source records attributed to J J Ferretti.

At least 73 records · Page 4Linked to original sources

PubMed1991

Frequency of the erythrogenic toxin B and C genes (speB and speC) among clinical isolates of group A streptococci.

DNA probes corresponding to the internal region of the erythrogenic toxin B and C genes, speB and speC, were used in hybridization studies with clinical isolates of Streptococcus pyogenes to determine the frequency of occurrence of these genes in a large population of group A streptococci. More than 500 strains from different geographical locations throughout the world were used in this study. The results from colony-lift hybridization experiments indicated that the frequency of occurrence of each toxin gene among all of these strains was 100% for speB and 50% for speC. Division of these strains into subgroups of general group A strains and strains associated with scarlet fever or rheumatic fever resulted in a frequency of occurrence of speC of about 50% for all subgroups. The speC gene was found to be more frequently associated with serotype M2, M4, and M6 strains and less frequently associated with serotype M1, M3, and M49 strains. The results from a similar study with the speA gene have been previously reported (C.-E. Yu and J.J. Ferretti, Infect. Immun. 57:3715-3719, 1989).

Bacterial Proteins↗

PubMed1991

Chromosomal deletions in melibiose-negative isolates of Streptococcus mutans.

Isolates from a collection of phenotypically melibiose-negative (Mel-) Streptococcus mutans from widely-scattered geographical locations were examined and found to lack the activities of the enzymes alpha-galactosidase and alpha-glucosidase, in addition to being unable to transport melibiose. Cloned fragments of S. mutans DNA from the region of the chromosome carrying the genes for alpha-galactosidase (aga), sucrose phosphorylase (gtfA), and dextran glucosidase (dexB), as well as the genes encoding components of the binding-protein-dependent uptake system for raffinose and melibiose, were used in hybridization studies for investigation of the genetic basis of the Mel-phenotype. A region of at least 12 kilobases, containing all the above genes, was found to be deleted from the chromosome of the Mel- strains. It appears that this region of the chromosome is not essential for survival of S. mutants in the oral cavity. The reason for the frequent occurrence of deletions, as opposed to other forms of mutational events, is unknown.

Biological Transport↗

PubMed1990

Nucleotide sequence of the dextran glucosidase (dexB) gene of Streptococcus mutans.

Nucleotide sequencing of DNA in the region of the Streptococcus mutans chromosome adjacent to the previously characterized gtfA gene revealed the presence of two long open reading frames. One of these corresponds to the dexB gene and has been shown to encode an intracellular exodextranase (dextran glucosidase) which has short isomaltosaccharides as preferred substrates. Comparison with other published sequences showed that the dexB gene product shares regions of similarity with enzymes, from a variety of sources, which attack other glucose polymers.

Amino Acid Sequence↗

PubMed1990

Sequence analysis of the gene for the glucan-binding protein of Streptococcus mutans Ingbritt.

The nucleotide sequence of the gbp gene, which encodes the glucan-binding protein (GBP) of Streptococcus mutans, was determined. The reading frame for gbp was 1,689 bases. A ribosome-binding site and putative promoter preceded the start codon, and potential stem-loop structures were identified downstream from the termination codon. The deduced amino acid sequence of the GBP revealed the presence of a signal peptide of 35 amino acids. The molecular weight of the processed protein was calculated to be 59,039. Two series of repeats spanned three-quarters of the carboxy-terminal end of the protein. The repeats were 32 to 34 and 17 to 20 amino acids in length and shared partial identity within each series. The repeats were found to be homologous to sequences hypothesized to be involved in glucan binding in the GTF-I of S. downei and to sequences within the protein products encoded by gtfB and gtfC of S. mutans. The repeated sequences may represent peptide segments that are important to glucan binding and may be distributed among GBPs from other bacterial inhabitants of plaque or the oral cavity.

Amino Acid Sequence↗

PubMed1990

Analysis of the Streptococcus downei gtfS gene, which specifies a glucosyltransferase that synthesizes soluble glucans.

The complete nucleotide sequence was determined for the Streptococcus downei (previously Streptococcus sobrinus) MFe28 gtfS gene which specifies a glucosyltransferase (GTF-S) producing water-soluble glucan. A single open reading frame which encodes a mature protein with a molecular weight of 147,408 (1,328 amino acids) and a putative signal peptide 36 or 37 amino acids in length was detected. GTF-S shares extensive sequence similarity with GTF-I (gtfI) from S. downei and GTF-I (gtfB) and GTF-SI (gtfC) from Streptococcus mutans. GTF-S contains a highly conserved enzymatic domain and C-terminal repeated sequences which appear to be involved in glucan binding. Comparison of the deduced GTF-S protein sequence with other sequenced GTF genes of mutans streptococci revealed that these C-terminal repeats occurred in all cases, although the patterns of repeated sequences varied with respect to each other and to the glucan-binding protein of S. mutans. GTF-S contains four C-terminal repeat sequences ranging from 49 to 51 amino acids in length and a partial repeat of 13 amino acids. Nuclear magnetic resonance analysis of the glucan produced by GTF-S revealed that the product consisted of more than 90% alpha-1,6-linked glucosyl residues.

Amino Acid Sequence↗

PubMed1989

Physical maps of the streptococcal bacteriophage A25 and C1 genomes.

A physical map of the group A streptococcal bacteriophage A25 genome was constructed using data obtained from restriction enzyme digestion analysis of purified DNA. Phage A25 was 34.6 kb in size and was digested with several enzymes which generated the following number of fragments: AvaI (3), EcoRI (2), HindIII (4), HaeII (10), HindII (22), HpaI (11), and PvuII (8). Restriction enzymes BamH1, PstI, and SaI did not cleave phage A25 DNA. A physical map of the group C streptococcal phage C1 genome was also constructed. Phage C1 was 16.9 kb in size and was cleaved by the following enzymes to give the indicated number of fragments: EcoRI (8), HaeII (10), HaeII (4), HaeII (10), HindIII (5), HpaI (8), PvuII (3), and SalI (2). Restriction enzymes AvaI, AvaII, BamH1, EcoRV, HpaII, and PstI did not cleave phage C1 DNA.

Bacteriophages↗

PubMed1989

Sequence analysis of the wall-associated protein precursor of Streptococcus mutans antigen A.

The nucleotide sequence has been determined for the Streptococcus mutans wall-associated protein A (wapA) gene from serotype c strains Ingbritt and GS5. The nucleotide sequence for each wapA gene was virtually identical, although the gene from strain GS5 contained a 24 base pair deletion. A 29 amino acid signal peptide was specified by each wapA gene with a mature protein of 424 amino acids (Mr, 45,276) for strain Ingbritt and 416 amino acids (Mr, 44,846) for strain GS5. In the C-terminal region of the wall-associated protein A, considerable sequence similarity was found with the membrane anchor region of proteins from other Gram-positive organisms such as the group A streptococcal M protein and the group G streptococcal IgG binding protein. Adjacent to the proposed membrane anchor is a highly hydrophilic region which may span the cell wall; both sequence data and experimental evidence indicate the existence of a region immediately outside the wall at which proteolytic cleavage occurs to release antigen A of Mr 29,000 into the culture supernatant. Thus, the wall-associated protein A is a precursor of the 29,000 Mr antigen A.

Amino Acid Sequence↗

PubMed1989

The streptokinase gene of group A streptococci: cloning, expression in Escherichia coli, and sequence analysis.

The gene specifying the group A streptokinase (ska) gene was cloned from an M type 49 strain of Streptococcus pyogenes and shown to express in Escherichia coli. The nucleotide sequence of the DNA fragment carrying ska was determined and compared to that of the group C streptokinase gene (skc). There is 90% sequence identity between the two genes, with highly conserved transcription and translation control regions. The deduced amino acid sequence of the group A streptokinase (SKA) contains the same number of amino acids as that of group C streptokinase, with 85% sequence identity between the two proteins. Among 440 amino acid residues specified by the coding sequence, there are 62 non-identical residues with 45 conserved and 17 non-conserved residues. The non-identical residues are located in two major regions, spanning residues 174 to 244 and 270 to 290, with 40 and 10 amino acid changes, respectively. The sequence differences provide an explanation at the molecular level for the previous findings of immunological and chemical heterogeneity among streptokinase produced by pathogenic streptococci.

Amino Acid Sequence↗

PubMed1989

Molecular cloning of the Streptococcus mutans gene specifying antigen A.

A gene encoding a Streptococcus mutans surface protein antigen has been isolated from a strain GS-5 gene bank constructed via the Streptococcus-Escherichia coli shuttle vector pSA3. This E. coli recombinant clone, designated 4B2, expressed S. mutans proteins, as shown by Western immunoblot analysis with a specific rabbit antibody to S. mutans surface antigens. Three bands were observed, including a 52-kilodalton (kDa) protein (pI 5.7), a 29-kDa protein (pI 4.2), and a 20-kDa protein usually present in lower amounts. The 52- and 29-kDa proteins both reacted with a monoclonal antibody to S. mutans antigen A, a 29-kDa protein which has been characterized and used as a vaccine for the prevention of induced caries in rodents and monkeys. The 52-kDa protein, but not the 29-kDa protein, showed a capacity to bind to a broad number of carbohydrate polymers. The results from this study suggest that the recombinant 4B2 clone specifies a 52-kDa protein which is a precursor to the 29-kDa antigen A.

Antibodies, Bacterial↗

PubMed1989

Molecular epidemiologic analysis of the type A streptococcal exotoxin (erythrogenic toxin) gene (speA) in clinical Streptococcus pyogenes strains.

A molecular epidemiology analysis was performed with over 440 clinical isolates of Streptococcus pyogenes obtained from 11 different countries in order to determine the frequency of occurrence of the type A streptococcal exotoxin (erythrogenic toxin) gene (speA) among group A strains. The colony hybridization technique employing a specific internal fragment of the speA gene was used for initial screening, and all positive results were further confirmed by the Southern hybridization technique. Among over 300 general strains obtained from patients with a variety of diseases, except scarlet fever (such as tonsillitis, impetigo, cellulitis, pyoderma, abscess, rheumatic fever, and glomerulonephritis), 15% were found to contain the speA gene. Among a group of 146 strains obtained from individuals described as having scarlet fever, 45% were shown to contain the speA gene. Further analysis of the data indicated that strains with certain M- or T-type surface antigens showed a higher (such as M and T types 1 and 3/13) or lower (such as M2, M12, T4, T5, and T28) tendency to contain the speA gene. No correlation was found between speA content of a strain and the ability to cause a specific disease, although strains possessing the speA gene were more likely to be associated with scarlet fever and rheumatic fever than with other types of disease.

Bacterial Proteins↗

PubMed1989

Heterogeneity of the streptokinase gene in group A streptococci.

A molecular epidemiological study was conducted to determine the distribution of the streptokinase gene in group A streptococcal strains of different M types and in other streptococcal species. Plasmid pNC1, containing only the internal coding sequence of the streptokinase gene from group C streptococcal strain H46A, was used as a DNA probe in colony and Southern hybridization experiments. Only the pathogenic group A, C, and G streptococci contained a streptokinase gene; 12 other Lancefield group strains did not. A total of 134 group A strains, including 61 M types and 6 T types, were tested. Although only 62% (83 of 134) of the strains tested showed positive streptokinase activity by the casein-plasminogen overlay assay, all strains contained the streptokinase gene as evidenced by strong hybridization with the pNC1 probe. Southern blot DNA hybridizations were carried out with 101 strains of group A streptococci. The restriction enzymes HindIII and HaeIII were used to digest the genomic DNA. Six hybridization patterns were observed after HindIII digestion. Double hybridization bands appeared in all of the patterns, which indicated the existence of a highly conserved HindIII site. More complex hybridization results were obtained after HaeIII digestion. Twelve hybridization patterns were observed; three were characterized by a single hybridization band, and nine were characterized by double bands. Variations in hybridization patterns were observed in strains of both the same and different serotypes. The overall results at the gene level indicate that there is considerable heterogeneity among the streptokinases of group A streptococci, consistent with previous findings of immunological and chemical differences among streptokinases of group A streptococci.

DNA Probes↗

PubMed1989

Sequence analysis and expression in Escherichia coli of the hyaluronidase gene of Streptococcus pyogenes bacteriophage H4489A.

The hyaluronidase gene (hylP) from Streptococcus pyogenes bacteriophage H4489A was previously cloned into Escherichia coli plasmid pUC8 as a 3.1-kilobase ThaI fragment. Southern hybridization experiments confirmed the origin of this fragment in bacteriophage H4489A before determination of the nucleotide sequence of the entire fragment. Two open reading frames (ORFs) were found, the first of which specified a 39,515-molecular-weight protein identified as the bacteriophage hyaluronidase. The second ORF encoded a 65,159-molecular-weight protein of unknown function. Putative transcription and translation control sequences for each ORF were identified by using a plasmid containing a promoterless chloramphenicol acetyltransferase gene. Controlled exclusive expression of the hylP gene via the T7 polymerase-promoter system in E. coli resulted in a 40,000-dalton protein, a result consistent with the coding capacity of the hylP gene.

Amino Acid Sequence↗

PubMed1988

Tripartite streptokinase gene fusion vectors for gram-positive and gram-negative procaryotes.

A specific 1,596 bp HincII fragment ('skc) from the chromosome of Streptococcus equisimilis contains an active streptokinase (SK) gene (skc) lacking, in addition to the expression signals, codons 1 through 39 of wild-type skc but retaining the remainder of the skc coding sequence together with the transcription terminator. Using this fragment as an indicator gene, we constructed two types of vectors which in appropriate hosts resulted in the synthesis of SK fusion proteins after insertional activation of 'skc. The first type are open reading frame (ORF) vectors in which 'skc was inserted into pUC18 out of frame with respect to lacZ', thus conferring an SK-negative phenotype. Any DNA fragments representing ORFs inserted between the lacZ' expression signals and 'skc such that the skc reading frame was restored resulted in the production of tripartite proteins which exhibited SK activity. The second type of vector, which functioned in both gram-positive and gram-negative bacteria, used the streptococcal speA expression and secretion signals in front of the ORF to activate 'skc insertionally. Using a large fragment from the chymosin gene as the target sequence, the usefulness of these vectors for studying foreign gene expression in streptococci as well as Escherichia coli was demonstrated.

Amino Acid Sequence↗

PubMed1988

Streptococcus mutans gtfA gene specifies sucrose phosphorylase.

The ethanol-insoluble product formed from sucrose by purified enzyme encoded by the gtfA gene from Streptococcus mutans, expressed in recombinant Escherichia coli, was analyzed by 13C and 31P nuclear magnetic resonance. The product was identified as alpha-D-glucose-1-phosphate, and it was concluded that the GTF-A enzyme is sucrose phosphorylase (sucrose:orthophosphate alpha-D-glucosyltransferase [EC 2.4.1.7]).

DNA, Bacterial↗

PubMed1988

Sequence analysis of the glucosyltransferase A gene (gtfA) from Streptococcus mutans Ingbritt.

The complete nucleotide sequence of a 2.4-kilobase fragment containing the glucosyltransferase A gene (gtfA) of Streptococcus mutans Ingbritt (serotype c) was determined. The gtfA gene contains 481 codons and specifies a protein of molecular weight 55,665. There is no evidence of a signal peptide in the protein or that the glucosyltransferase A enzyme is secreted. No sequence homologies were observed between the gtfA gene or protein and the gtfI or gtfB gene and its protein.

Amino Acid Sequence↗

PubMed1988

Homology of glucosyltransferase gene and protein sequences from Streptococcus sobrinus and Streptococcus mutans.

The sequences of glucosyltransferase genes from Streptococcus sobrinus (gtfI) and Streptococcus mutans (gftB) were compared and show a high degree of homology. There is a 57.7% homology of nucleotides in the genes and a 56.7% homology of amino acids in the deduced protein sequences. The G + C content for the protein-coding region is 43.6% for S. sobrinus and 41.2% for S. mutans. Internal repeating sequences present in both proteins exhibit some difference in sequence pattern.

Amino Acid Sequence↗

PubMed1987

Purification and characterization of Streptococcus pyogenes erythrogenic toxin type A produced by a cloned gene in Streptococcus sanguis.

The gene of Streptococcus pyogenes erythrogenic toxin type A (speA) has been previously cloned in Streptococcus sanguis (Challis) and produces extracellular erythrogenic toxin type A (ET A). The ET A produced and secreted by this heterologous host was purified to homogeneity and shown to have properties identical to ET A produced by S. pyogenes strain NY-5; i.e., serological identity in immunodiffusion, migration in SDS-polyacrylamide gel electrophoresis, mitogenic activity, inhibition of mitogenic activity by specific antibody, and precipitation by an international scarlatina antitoxin preparation. The cloned speA gene specified an ET A which had a molecular weight identical to that of ET A from S. pyogenes previously reported from this laboratory. NH2-terminal sequence determination of the purified protein showed the first nine residues to be gln gln asp pro asp pro ser gln leu; this is consistent with predictions made from the nucleotide sequence of the speA gene according to Weeks and Ferretti and different from the sequence published by Johnson et al.

Amino Acids↗

PubMed1987

Immunologic cross-reactivity of type A streptococcal exotoxin (erythrogenic toxin) and staphylococcal enterotoxins B and C1.

Immunologic cross-reactivity between Streptococcus pyogenes type A exotoxin (erythrogenic toxin) and Staphylococcus aureus enterotoxins B and C1 was demonstrated by Ouchterlony double diffusion, Western immunoblot, and immunodot analyses. Specific antiserum to type A streptococcal exotoxin reacted more strongly with staphylococcal enterotoxin B than with enterotoxin C1. The reactivity of type A streptococcal exotoxin with antiserum to staphylococcal enterotoxin B was greater than that of antiserum to enterotoxin C1. These results suggest that a conserved domain is present in the three exotoxins, which most likely originated from a common evolutionary ancestor.

Antibodies, Bacterial↗