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V A Fischetti

Publications and source records attributed to V A Fischetti.

At least 73 records · Page 4Linked to original sources
PubMed1992

Expression of M6 protein gene of Streptococcus pyogenes in Streptococcus gordonii after chromosomal integration and transcriptional fusion.

The M6 protein of Streptococcus pyogenes was expressed on the cell surface and secreted in Streptococcus gordonii Challis (formerly Streptococcus sanguis) after chromosomal integration of a promoterless M6 protein gene (emm-6.1). The ermC gene, conferring resistance to erythromycin, was cloned downstream of emm-6.1, within the same ClaI fragment. The initiation codon of emm-6.1 was 19 bp downstream of a ClaI site, so that ClaI cleavage would leave the gene promoterless. The ClaI fragment containing the promoterless emm-6.1 and ermC was ligated in vitro with a ClaI digest of S. gordonii chromosomal DNA. Random chromosomal integration of the heterologous DNA was obtained by using the ligation mixture to transform the naturally competent S. gordonii Challis. Twenty-eight percent of transformants selected for erythromycin resistance also expressed M6. Among the best M6 producers, 10 clones were selected for the stability of their phenotype. Nine of the 10 clones were shown to harbour one intact copy of the emm-6.1/ermC ClaI fragment integrated into the chromosome. These strains both expressed M6 protein on the surface and secreted different amounts of the molecule, since in each case the protein was produced after a transcriptional fusion of emm-6.1 with a different chromosomal promoter. A S. gordonii strain expressing large amounts of surface M6 protein, as judged by immunofluorescence and Western blot, was compared to the M- parental strain in a standard opsonophagocytosis assay. Of the isogenic pair, M6+ S. gordonii survived better in human blood and was phagocytosed at a slower rate.

Bacterial Outer Membrane Proteins↗
PubMed1992

Nucleotide sequences of two adjacent M or M-like protein genes of group A streptococci: different RNA transcript levels and identification of a unique immunoglobulin A-binding protein.

M protein is a key virulence factor present on the surface of group A streptococci. M protein is defined by its antiphagocytic function, whereas M-like proteins, while structurally related to M proteins, lack an established antiphagocytic function. Group A streptococci can be divided into two main groups (class I and II) on the basis of the presence or absence of certain antigenic epitopes within the M and M-like molecules, and importantly, the two classes correlate with the disease-causing potential of group A streptococci. In an effort to better understand this family of molecules, a 2.8-kb region containing the two M protein-like genes from a class II isolate (serotype 2) was cloned and sequenced. The two genes lie adjacent to one another on the chromosome, separated by 211 bp, and have many structural features in common. The emmL2.1-derived product (ML2.1 protein) is immunoreactive with type-specific antiserum, a property associated with M proteins. The cloned product of the downstream gene, emmL2.2 (ML2.2 protein), is an immunoglobulin A (IgA)-binding protein, binding human myeloma IgA. Interestingly, the RNA transcript levels of emmL2.1 exceed that of emmL2.2 by at least 32-fold. Northern (RNA) hybridization and primer extension studies suggest that the RNA transcripts of emmL2.1 and emmL2.2 are monocistronic. The ML2.1 and ML2.2 proteins exhibit 53% amino acid sequence identity and differ primarily in their amino termini and peptidoglycan-spanning domains and in a Glu-Gln-rich region present only in the ML2.1 protein. However, the previously described M-like, IgA-binding protein from a serotype 4 isolate (Arp4) displays a higher level of amino acid sequence homology with the ML2.1 molecule than with the IgA-binding ML2.2 protein. Amino acid sequence alignments between all M and M-like proteins characterized to date suggest the existence of two fundamental M or M-like gene subclasses within class II organisms, represented by emmL2.1 and emmL2.2. In addition, IgA-binding activity can be found within both types of molecules.

Amino Acid Sequence↗
PubMed1992

Role of fibrinogen in complement inhibition by streptococcal M protein.

M protein, the major virulence factor of group A streptococci, has antiopsonic activity in that it inhibits activation of the alternative complement pathway on the streptococcal surface. Two properties of M protein have been claimed to account for the inhibitory activity, namely, (i) its binding affinity for complement factor H, which is an inhibitor of alternative pathway activation, and (ii) its high binding affinity for fibrinogen. We have recently shown that fibrinogen, like M protein, inhibits alternative pathway activation by possessing binding affinity for factor H. Here we report that fibrinogen effectively competes with factor H for binding to M protein but retains its own binding affinity for factor H. The presence of fibrinogen did not significantly affect alternative pathway inhibition on the streptococcal surface.

Antigens, Bacterial↗
PubMed1992

Delivery and expression of a heterologous antigen on the surface of streptococci.

We have developed a system in which a foreign antigen replaces nearly all of the surface-exposed region of the fibrillar M protein from Streptococcus pyogenes and is fused to the C-terminal attachment motif of the M molecule. The fusion protein is thus expressed on the surface of Streptococcus gordonii, a commensal organism of the oral cavity. The antigen chosen to be expressed within the context of the M6 molecule was the E7 protein (98 amino acids) of human papillomavirus type 16. Stable recombinant streptococci were obtained by integrating genetic constructs into the chromosome, exploiting in vivo homologous recombination. The M6-E7 fusion protein expressed on the S. gordonii surface was shown to be immunogenic in mice. This is the first step in the construction of recombinant live vaccines in which nonpathogenic streptococci as well as other gram-positive bacteria may be used as vectors to deliver heterologous antigens to the immune system.

Animals↗
PubMed1991

Assembly and analysis of a functional vaccinia virus "amplicon" containing the C-repeat region from the M protein of Streptococcus pyogenes.

Previous studies have shown that when inoculated intranasally into mice, vaccinia virus (VV) recombinants expressing the carboxyl half of the Streptococcus pyogenes M protein [which contains the C-repeat region (CRR)] could elicit a protective immune response against subsequent challenge by both homologous and heterologous serotypes of pathogenic group A streptococci. In the present study, an insertion plasmid was constructed that contained three tandem in-frame repeats of a 310-base-pair DNA sequence encoding the CRR from streptococcal M6 protein under control of a constitutive viral promoter. The plasmid was used to introduce the bacterial sequences into the VV genome by homologous recombination. Surprisingly, the recombinant VV:CRR3X virus that was isolated appeared to represent not an individual recombinant virus but a complex mixture of variants that contained from 1 to greater than 20 tandem copies of the CRR region at the insertion site. This genomic complexity was mirrored at the transcriptional level in that a nested set of coterminal transcripts was detected in VV:CRR3X-infected cells, which increased in size from 1400 to 6600 bases by increments of approximately 300 bases. All transcripts containing two or more CRR inserts appeared functional, as Western (immuno) blot analyses of VV:CRR3X-infected cell extracts revealed a family of CRR-related proteins with apparent molecular masses that increased from 30 kDa upward in increments of 10 kDa. All data are consistent with the hypothesis that variation in the VV:CRR3X recombinants is from random crossover events that occur within the CRR region during viral DNA replication. These results suggest that the genomic diversity generated by the "recombinogenic" properties of vaccinia recombinants containing tandem foreign inserts could be used to facilitate induction of a broadly protective immune response against antigenically diverse pathogenic agents.

Amino Acid Sequence↗
PubMed1991

A method to detect proteinase activity using unprocessed X-ray films.

Routine assays to detect proteinases in biological samples are generally tedious and time-consuming. To expedite the recognition of proteinases, we have developed an assay utilizing the gelatin on the surface of an unprocessed Kodak X-Omat AR film as the proteolytic substrate. A positive reaction is indicated by a clear zone on the film after it has been rinsed with running water. This proteinase assay has been found to be inexpensive, rapid, and simple. Besides its ease of use, this assay has been found to be quantitatively reproducible with a well-defined endpoint. More importantly, this assay method is applicable to a variety of proteolytic enzymes under diverse pH (5-8.5) and salt conditions (up to 5 M NaCl) and has a sensitivity similar to that of azocoll. Since the assay does not require sophisticated equipment, it is useful as a general laboratory procedure.

Azo Compounds↗
PubMed1991

The role of fibrinogen in mediating staphylococcal adherence to fibers.

The use of tampons and surgical gauze pads and colonization with Staphylococcus aureus have been established as risk factors for the development of toxic shock syndrome. To elucidate the role of blood factors in the mediation of staphylococcal adherence to fibers used in tampons and surgical packing, an adherence assay with cotton fibers was developed. Results demonstrated that cotton disks precoated with fibrinogen in the presence of human serum albumin bound a significant percentage of the inoculum for both staphylococcal strains tested when compared to human serum albumin controls. Likewise, fibers pretreated with plasma or defibrinated blood containing a small amount of fibrin revealed comparable staphylococcal adherence to that of fibrinogen. In contrast, fibers pretreated with serum, fibronectin, or vitronectin did not exhibit significant augmentation in staphylococcal attachment in comparison to human serum albumin controls. The attachment of staphylococci to fibrinogen and/or fibrin appeared to be specific and is blocked by goat anti-human fibrinogen antibody, but not fibronectin, vitronectin, or nonimmune goat IgG. Thus, our data indicate that fibrinogen/fibrin is the dominant blood component in the mediation of staphylococcal adherence to fibers used in tampons and surgical gauze pads.

Animals↗
PubMed1991

Genetic diversity among the T-protein genes of group A streptococci.

T protein is a trypsin- and pepsin-resistant molecule on the surface of group A streptococci used as a serological tool to differentiate streptococci of this group. The purpose of this study was to determine the relatedness among the T protein genes of the 25 known T serotypes. DNA probes were constructed which represented various regions of the structural gene for the T6 protein, tee6. The probes were assayed for their ability to hybridize HindIII digests of chromosomal DNA from the 25 different T serotypes. Probe pTEE6.3, coding for the entire T6 protein, and pTEE6(1-299), coding for the amino-terminal half of T6, displayed the highest amount of homology, each binding to 10 of 25 T serotypes. Probes coding for sequences in the carboxy-terminal half of T6 showed considerably less homology among T serotypes with one probe hybridizing with only three out of 25. A synthetic oligonucleotide coding for the carboxy-terminal hydrophobic domain of T6, an area conserved to some degree among several bacterial surface proteins, showed homology with only seven out of 25 T serotypes. Hybridization with sequences outside the tee6 coding area provided additional information on the relatedness of certain sets of T serotypes according to restriction-fragment size heterogeneity. Clearly, there is considerable diversity among T-serotype genes. The data suggest that two or more families of structurally variant T proteins exist, which share only the property of proteolytic resistance and/or, perhaps, some biological function.

DNA Probes↗
PubMed1991

Recombinant human tumor necrosis factor alpha promotes adherence of Staphylococcus aureus to cultured human endothelial cells.

Tumor necrosis factor (TNF), a potent inflammatory mediator secreted by monocytes during inflammation, was shown to significantly increase the adherence of Staphylococcus aureus to cultured human umbilical vein endothelial cells in vitro. The stimulatory effect of TNF was dose dependent and was bimodal with respect to time; bacterial adhesion peaked after 4 and 16 h of stimulation with recombinant human TNF-alpha. The ability of TNF-alpha to augment staphylococcal adherence to endothelial cells was contingent upon the presence of plasma factors. Thus, the complex interaction among cytokines (such as TNF), plasma factor(s), and the endothelium serves to modulate bacterial adherence to endothelial cells.

Bacterial Adhesion↗
PubMed1991

Fibrinogen acts as a bridging molecule in the adherence of Staphylococcus aureus to cultured human endothelial cells.

The propensity of Staphylococcus aureus to cause acute endovascular infections during transient bacteremia is poorly understood. To examine the events leading to the attachment of staphylococci to endothelium, adherence assays were developed to study the role of blood factors in the mediation of staphylococcal adherence to cultured human umbilical vein endothelium in vitro. Results indicate that the preferential attachment of S. aureus to endothelial cells is mediated by fibrinogen adsorbed from plasma onto the endothelial surface. The binding is apparently specific because it could be blocked by goat anti-human fibrinogen antibody in a dose-dependent fashion while nonimmune goat IgG, mouse MAb against AG-1 (a platelet antigen found on the endothelial cell surface), nonspecific mouse MAb and rabbit antibodies to human vitronectin and fibronectin were not inhibitory. Our data also indicate that fibrinogen is a necessary but not the only blood constituent in the mediation of binding of S. aureus to endothelium. This was supported by the finding that fibrinogen alone, at a concentration equivalent to that in plasma, did not potentiate staphylococcal adherence as much as plasma while afibrinogenemic plasma reconstituted with fibrinogen did. Because fibrinogen is known to bind to endothelial cells, our data is consistent with the hypothesis that fibrinogen and additional plasma factor(s), possibly activated during inflammation, promote staphylococcal adherence to endothelium. In addition, the role of the fibrinogen binding receptor of S. aureus as an adherence factor to native endothelium is also suggested.

Antibodies↗
PubMed1990

Differentiation between two biologically distinct classes of group A streptococci by limited substitutions of amino acids within the shared region of M protein-like molecules.

Group A streptococci can be categorized into two classes (I and II) based on immunodeterminants contained within a surface-exposed, conserved region (C repeat domain) of the major virulence factor, M protein. Previous studies have shown that several biological properties correlate strongly with streptococcal class, and thus, there is a strong impetus to precisely define the antigenic epitopes unique to class I and II M proteins. Using synthetic peptides, the binding sites of two class I-specific mAbs were mapped to distinct epitopes within the C repeat region of type 6 M protein (class I). A class II M protein-like gene (type 2) was cloned and sequenced, and the predicted amino acid sequence was compared for homology to class I and II molecules, whose sequences were previously reported. For a given C repeat block 35 amino acid residues in length, 20 residue positions were conserved among all sequences analyzed. Of the 15 variable amino acid positions, only four were class specific, and three of the four positions were localized in the area to which the class I-specific mAbs bound. The predicted secondary structures of class I and II C repeat blocks reveals that they are alpha-helical, except for a single area of disruption. In the class I molecules, the area of disruption corresponds to the class I-specific mAb binding sites. Importantly, the predicted conformational characteristics of this disruption differs for class I and II molecules. The data suggest that only limited changes in amino acid residues differentiate between class I and II molecules in the C repeat region. Therefore, selective (biological) pressures may have contributed to the evolution of these two classes of molecules.

Amino Acid Sequence↗
PubMed1990

Synthetic peptide vaccine against mucosal colonization by group A streptococci. I. Protection against a heterologous M serotype with shared C repeat region epitopes.

M protein is an antigenically variable virulence determinant present on the surface of group A streptococci, and it provides the basis for the serologic typing scheme. Type-specific serum antibodies afford strong protection against infection by the homologous serotype. Non-type-specific antigenic epitopes also exist within the surface-exposed portion of M protein. Previous studies demonstrated that intranasal immunization with Ag corresponding to sequences within the non-type-specific pepsin-susceptible site and adjacent C repeat regions of M6 protein, evoke protective immunity against pharyngeal colonization by type 6 streptococci in a mouse model. Although the protective immunogens are not type-specific, the pepsin site region of M6 is shared among less than 20% of serotypes examined. Therefore it was necessary to determine whether more highly conserved M protein epitopes elicit mucosal protection against group A streptococci, and if protective immunity extends to heterologous serotypes. In this report, peptides were synthesized that correspond to sequences completely contained within the highly conserved C repeat region of M6 protein. Peptide Ag were covalently coupled to the mucosal adjuvant, cholera toxin B subunit (CTB), and mice immunized intranasally and orally with peptide-CTB conjugates were compared to control groups that received CTB only. Immunization with the peptide-CTB conjugates led to significant protection against pharyngeal colonization by group A streptococci. Furthermore, protection was observed against the heterologous M serotype, type 14. These findings suggest that protection against multiple serotypes of group A streptococci can be achieved with a vaccine consisting of the widely shared C repeat region of M6 protein.

Amino Acid Sequence↗
PubMed1990

A human IgG receptor of group A streptococci is associated with tissue site of infection and streptococcal class.

The distribution of receptors for immunoglobulins of several different isotypes was examined for group A streptococcal isolates derived from skin and nasopharyngeal sites. Although human IgG-Fc receptor activity was a variable property of group A streptococci, found among 61% of all isolates tested, it was largely restricted to well-defined subpopulations. Human IgG-binding activity was observed among nearly all impetigo isolates examined. In addition, the expression of the class II M protein molecule (one of two broad antigenic classes of the major virulence factor) and opacity factor (a lipoproteinase) was almost invariably accompanied by human IgG binding, regardless of tissue site of infection. In contrast to class I impetigo isolates, class I nasopharyngeal isolates were relatively devoid of human IgG-binding activity. The data suggest that the presence or absence of human IgG-binding activity correlates with certain diseases caused by group A streptococci.

Animals↗
PubMed1990

The role of fibrinogen in staphylococcal adherence to catheters in vitro.

A study was conducted to examine the factors involved in adherence of Staphylococcus aureus to various catheter materials. Staphylococcal adherence to catheters coated with fibrinogen was specific and significantly increased compared with that to control catheters (preincubated in phosphate-buffered saline). The adherence of S. aureus to fibrinogen-coated catheters was blocked by goat anti-human fibrinogen antibody, but not fibronectin, vitronectin, or nonimmune goat IgG. Adherence to catheter tubings coated with fibrin did not differ markedly from that to those coated with fibrinogen alone, suggesting the conservation of the staphylococcal binding domain during the conversion of fibrinogen to fibrin. Plasma proteins such as fibronectin or factor XIII that interacted with fibrin did not modify staphylococcal adherence to fibrin. Adherence studies with staphylococcal cells treated with proteinases revealed that staphylococcal surface protein(s) were responsible for fibrinogen binding. These data indicate that fibrinogen or fibrin is the dominant plasma mediator of staphylococcal adherence to intravascular catheters.

Bacterial Adhesion↗
PubMed1990

Conservation of a hexapeptide sequence in the anchor region of surface proteins from gram-positive cocci.

A highly conserved hexapeptide sequence (both at the protein- and DNA level) has been identified within the C-terminal end of all 11 known surface proteins from Gram-positive cocci. The hexapeptide, with the consensus sequence LPXTGE, is located about 9 amino acids N-terminal from the C-terminal hydrophobic domain which is found in all these surface molecules. The conservation of the hexapeptide, despite sequence variation within the regions flanking it, suggests that it is important for the attachment of these proteins within the cell.

Amino Acid Sequence↗
PubMed1990

Sequence and structural characteristics of the trypsin-resistant T6 surface protein of group A streptococci.

The gene for the trypsin-resistant surface T6 protein of Streptococcus pyogenes D471 (M type 6) was cloned and expressed in Escherichia coli. The complete nucleotide sequence of the gene (tee6) and its flanking regions was determined and found to include only one major open reading frame coding for a protein of 537 amino acids (Mr, 57,675). The N terminus of the deduced protein sequence exhibits features of a typical signal sequence, and the C-terminal segment was found to have a high degree of homology with the membrane anchor region of other gram-positive surface proteins, such as streptococcal M protein, wapA protein from Streptococcus mutans and staphylococcal protein A. A hexapeptide having the consensus sequence LPSTGE and located immediately upstream of the C-terminal hydrophobic segment showed the highest degree of conservation at both the protein and DNA levels, with nearly all reported surface proteins from gram-positive cocci. The amino acid composition of the T6 protein revealed 21% serine and threonine residues distributed nearly regularly throughout the molecule, and analysis of the secondary structure predicted a conformation composed of greater than 70% beta-sheet potential interrupted by beta-turns or random coils. Localization experiments in E. coli show very little T6 protein in the periplasmic space. When found here, however, this T6 protein had a molecular mass of 55 kilodaltons, similar to that extracted from the streptococci by nonionic detergent. Most of the T6 protein was found localized in the membrane fraction, where it was composed of a triple band of 60, 58, and 57 kilodaltons. The coexistence of streptococcal surface proteins which are either resistant (T protein) or sensitive (M protein) to proteolytic enzymes may offer a new dimension to the modulation of these antigens under specific biological conditions.

Amino Acid Sequence↗