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V A Fischetti

Publications and source records attributed to V A Fischetti.

At least 55 records · Page 3Linked to original sources
PubMed1996

Gram-positive commensal bacteria for mucosal vaccine delivery.

To avoid the use of engineered pathogens for vaccine delivery, systems have been developed that allow the expression of heterologous antigens in commensal Gram-positive bacteria. In some cases, both a serum IgG and secretory IgA response are induced to the recombinant protein after vaccination, verifying the validity of the approach. These live recombinant bacteria may be used in the future to introduce a protective immune response to pathogenic microorganisms after mucosal colonization.

Adjuvants, Immunologic↗
PubMed1995

Mucosal and systemic immune responses to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii after oral colonization.

To circumvent the need to engineer pathogenic microorganisms as live vaccine-delivery vehicles, a system was developed which allowed for the stable expression of a wide range of protein antigens on the surface of Gram-positive commensal bacteria. The human oral commensal Streptococcus gordonii was engineered to surface express a 204-amino acid allergen from hornet venom (Ag5.2) as a fusion with the anchor region of the M6 protein of Streptococcus pyogenes. The immunogenicity of the M6-Ag5.2 fusion protein was assessed in mice inoculated orally and intranasally with a single dose of recombinant bacteria, resulting in the colonization of the oral/pharyngeal mucosa for 10-11 weeks. A significant increase of Ag5.2-specific IgA with relation to the total IgA was detected in saliva and lung lavages when compared with mice colonized with wild-type S. gordonii. A systemic IgG response to Ag5.2 was also induced after oral colonization. Thus, recombinant Gram-positive commensal bacteria may be a safe and effective way of inducing a local and systemic immune response.

Animals↗
PubMed1995

Serologic evidence for a class I group A streptococcal infection among rheumatic fever patients.

Group A streptococci (GAS) of serotypes most commonly associated with rheumatic fever (RF) outbreaks differ from many other serotypes by the presence of a unique, surface-exposed epitope on the M protein molecule. Based on the presence or absence of this epitope, GAS are categorized as class I or II, respectively. The objective of this study was to determine whether RF patients have an altered immune response to the class I-specific epitope. Immunoreactivity to class I- and class II-specific epitopes was determined for serum IgG derived from persons with a recent history of acute RF, uncomplicated GAS pharyngitis, and no known recent GAS infection. The results indicate that only RF patients display elevated levels of serum IgG directed towards the class I-specific epitope; they lack immunoreactivity to the class II epitope. The serologic findings strongly suggest that many of the RF patients were recently infected with a class I GAS isolate.

Antibodies, Bacterial↗
PubMed1995

Location of the complement factor H binding site on streptococcal M6 protein.

The surface M protein of group A streptococci binds factor H, a regulatory protein of the alternative complement pathway, which may contribute to the antiphagocytic activity of the M molecules. To locate the factor H binding domain in the alpha-helical coiled-coil structure of the M molecule, the M protein was cleaved with pepsin at pH 5.8, which separates the molecule approximately in half. Western blot (immunoblot), amino acid sequence, and mass spectrometric analyses revealed that factor H bound to a 14.6-kDa C-terminal fragment of the M molecule. Competitive inhibition of factor H binding to the 14.6-kDa fragment with M protein peptides localized the binding site to amino acids 256 to 292. This segment is located within the surface-exposed region of the M6 protein, identified as the C-repeat region, whose sequence is conserved among heterologous M and M-like molecules. These studies also identified a second pepsin-susceptible site with the sequence ELAK located within the cell wall-associated region of the M molecule.

Amino Acid Sequence↗
PubMed1995

DNA sequence of the serum opacity factor of group A streptococci: identification of a fibronectin-binding repeat domain.

The serum opacity factor (SOF) is a group A streptococcal protein that induces opacity of mammalian serum. The serum opacity factor 22 gene (sof22) from an M type 22 strain was cloned from an EMBL4 library by screening for plaques exhibiting serum opacity activity. DNA sequencing yielded an open reading frame of 3,075 bp. Its deduced amino acid sequence predicts a protein of 1,025 residues with a molecular weight of 112,735, a size that approximates that of the SOF22 protein isolated from both the original streptococcal strain and Escherichia coli harboring the cloned sof22 gene. The molecule is composed of three domains: an N-terminal domain responsible for the opacity reaction (opacity domain), a repeat domain with fibronectin-binding (Fn-binding) activity, and a C-terminal cell attachment domain. The C-terminal end of SOF22 is characterized by a hexameric LPXTGX motif, an adjacent hydrophobic region, and a charged C terminus, which are the hallmarks of cell-bound surface proteins found on nearly all gram-positive bacteria. Immediately upstream of this cell anchor region, SOF22 contains four tandem repeat sequence blocks, flanked by prolinerich segments. The repeats share up to 50% identity with a repeated motif found in other group A streptococcal Fn-binding proteins and exhibit Fn-binding activity, as shown by subcloning experiments. According to deletion analysis, the opacity domain is confined to the region N terminal to the repeat segment. Thus, SOF22 is unique among the known Fn-binding proteins from gram-positive bacteria in containing an independent module with a defined function in its N-terminal portion. Southern blot analysis with a probe from this N-terminal region indicates that the opacity domain of SOF varies extensively among different SOF-producing M types.

Adhesins, Bacterial↗
PubMed1995

Cloning, expression, and nucleotide sequence of a Staphylococcus aureus gene (fbpA) encoding a fibrinogen-binding protein.

Septicemia due to Staphylococcus aureus often begins as a focal infection (e.g., colonized wounds or catheters) from which the organism gains access to the bloodstream. On the basis of recent data from this laboratory, it is likely that S. aureus colonizes catheters and endothelium by using a fibrinogen-binding protein to mediate adhesion to fibrinogen-coated surfaces. To characterize the fibrinogen-reactive protein, we screened a lambda Zap library of S. aureus DB, a clinical isolate, for clones that were reactive with fibrinogen. Of 100,000 plaques screened, 3 were found to react with fibrinogen on immunoblots. Plasmid DNA prepared from clones 14, 30, and 36, upon digestion with EcoR1, which released the insert, revealed fragments of 4.6, 3.6, and 3.2 kb, respectively. To identify the cloned protein expressed in E. coli, cells were fractionated into periplasmic, membrane, and cytoplasmic fractions. Expression studies of clone 14, which comprised approximately two-thirds of the mature molecule, including the C terminus, revealed a 34-kDa fibrinogen-reactive protein in both the periplasmic and membrane fractions. This protein, designated FbpA, could be partially purified on a fibrinogen column. By using both clones 14 and 36 as templates, the complete DNA sequence of the fibrinogen-binding protein was obtained, yielding a molecule with a predicted size of 69,991 Da. Although sequence analysis revealed a high degree of homology with coagulase, there is a unique sequence of 11 amino acids that is not found in three known coagulases as well as two recently cloned fibrinogen-binding proteins. This unique sequence shares homology with a cell wall anchor motif found in other gram-positive surface proteins.

Amino Acid Sequence↗
PubMed1994

Characterization and biological properties of a new staphylococcal exotoxin.

Staphylococcus aureus strain D4508 is a toxic shock syndrome toxin 1-negative clinical isolate from a nonmenstrual case of toxic shock syndrome (TSS). In the present study, we have purified and characterized a new exotoxin from the extracellular products of this strain. This toxin was found to have a molecular mass of 25.14 kD by mass spectrometry and an isoelectric point of 5.65 by isoelectric focusing. We have also cloned and sequenced its corresponding genomic determinant. The DNA sequence encoding the mature protein was found to be 654 base pairs and is predicted to encode a polypeptide of 218 amino acids. The deduced protein contains an NH2-terminal sequence identical to that of the native protein. The calculated molecular weight (25.21 kD) of the recombinant mature protein is also consistent with that of the native molecules. When injected intravenously into rabbits, both the native and recombinant toxins induce an acute TSS-like illness characterized by high fever, hypotension, diarrhea, shock, and in some cases death, with classical histological findings of TSS. Furthermore, the activity of the toxin is specifically enhanced by low quantities of endotoxins. The toxicity can be blocked by rabbit immunoglobulin G antibody specific for the toxin. Western blotting and DNA sequencing data confirm that the protein is a unique staphylococcal exotoxin, yet shares significant sequence homology with known staphylococcal enterotoxins, especially the SEA, SED, and SEE toxins. We conclude therefore that this 25-kD protein belongs to the staphylococcal enterotoxin gene family that is capable of inducing a TSS-like illness in rabbits.

Amino Acid Sequence↗
PubMed1994

Human T-helper cell recognition of an immunodominant epitope of HIV-1 gp120 expressed on the surface of Streptococcus gordonii.

Our genetic system for expression of heterologous proteins on the surface of the Gram-positive bacterium Streptococcus gordonii was used to express a human T-helper epitope of HIV-1 envelope glycoprotein gp120. In previous work on the naive repertoire of human T-helper cells, it was shown that a 15-amino acid synthetic peptide of the HIV-1 gp120 sequence contained an immunodominant T-helper epitope. Synthetic DNA coding for this peptide was cloned in frame within the gene for the streptococcal surface protein M6, and the gene fusion was integrated by transformation into the chromosome of S. gordonii. The expected M6-gp120 fusion protein was found to be expressed on the surface of the recombinant streptococci. To test whether the T epitope could be recognized by T cells when expressed on the bacterial surface within the context of M6, recombinant bacteria were used as antigen in proliferation assays to stimulate the 15-amino acid-specific human T-helper clone, in the presence of autologous antigen-presenting cells. Bacteria expressing the T epitope were efficiently recognized by the T cells in culture. In proliferation assays, 10(6)-10(7) bacteria induced responses comparable to those obtained by standard amounts of synthetic peptide (0.02-0.2 micrograms). Recombinant S. gordonii, a candidate for a live vaccine vector, appeared suitable for delivering T epitopes to the immune system.

Amino Acid Sequence↗
PubMed1993

Glyceraldehyde-3-phosphate dehydrogenase on the surface of group A streptococci is also an ADP-ribosylating enzyme.

We recently identified an enzymatically active glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; GAPDH) as a major protein on the surface of group A streptococci (SDH), which exhibits multiple binding activity to various mammalian proteins. We now report that the SDH molecule also functions as an ADP-ribosylating enzyme, which, in the presence of NAD, is auto-ADP-ribosylated. In a crude cell wall extract of group A streptococci, SDH is the only protein that is ADP-ribosylated. SDH found in the streptococcal cytoplasmic fraction could not be ADP-ribosylated in the presence of NAD. Treatment of ADP-ribosylated SDH with the cytoplasmic fraction removed the ADP-ribose from SDH, suggesting the presence of an ADP-ribosyl hydrolase in the cytoplasmic compartment. The covalent linkage of ADP-ribose to SDH was stable to neutral hydroxylamine, sensitive to HgCl2, and inhibitable by free cysteine, indicating that the modification was at a cysteine residue of SDH. In addition to its auto-ADP-ribosylation activity, purified SDH or streptococcal cell wall extracts were able to transfer the ADP-ribose moiety of NAD specifically to free cysteine, resulting in a true thioglycosidic linkage. Treatment of purified SDH or the crude cell wall extract with sodium nitroprusside, which spontaneously generates nitric oxide, was found to stimulate the ADP-ribosylation of SDH in a time-dependent manner. ADP-ribosylation and nitric oxide treatment inhibited the GAPDH activity of SDH. Since ADP-ribosylation and nitric oxide are involved in signal transduction events, the ADP-ribosylating activity of SDH may enable communication between host and parasite during infection by group A streptococci.

Adenosine Diphosphate Ribose↗
PubMed1993

Surface-exposed conserved region of the streptococcal M protein induces antibodies cross-reactive with denatured forms of myosin.

Vaccines based on a highly conserved cell surface exposed C-repeat region of the group A streptococcal M protein molecule have been found to induce protection against mucosal challenge by homologous and heterologous streptococcal serotypes. Rabbit hyperimmune antisera were produced to four partially overlapping peptides of the C-repeat region of M6 protein. These were examined by both direct and competitive ELISA and by Western blotting for their reactivity against mammalian coiled coil proteins such as laminin, myosin, light meromyosin, heavy meromyosin, and cardiac tropomyosin, and to the denatured forms of some of these molecules. All sera reacted strongly with the recombinant M6 protein molecule. In addition, antibodies to three of the peptides displayed generally low levels of cross-reactivity with at least one of the mammalian proteins, whereas antibodies to one peptide did not cross-react with any of the proteins tested. The observed reactivity was found to be directed predominantly against denatured forms of the mammalian molecules. For instance, the cleaved forms of myosin bound better to the cross-reactive antibodies than the intact molecule. Furthermore, heat-denatured heavy meromyosin competed severalfold better in competitive ELISA than the non-heat-denatured "native" form. Our results demonstrate that M protein peptides corresponding to epitopes shared among rheumatic fever-associated strains of streptococci can lead to the production of low levels of antibodies reactive with mammalian coiled coil molecules. These antibodies are directed against the denatured forms of these molecules.

Amino Acid Sequence↗
PubMed1993

Expression of foreign proteins on gram-positive commensal bacteria for mucosal vaccine delivery.

Non-pathogenic Gram-positive oral commensal bacteria expressing recombinant fusion proteins on their cell surface have been successfully used to raise both a mucosal and a systemic immune response to foreign antigens while colonizing the oropharynx. In this system, fusion-protein vaccines are delivered and anchored to the surface of a commensal, which occupies the mucosal niche invaded by a particular pathogen. Surface expression of these foreign proteins is achieved by exploiting the common mechanism employed by Gram-positive bacteria for translocating and anchoring proteins to the cell surface. The process offers a safe alternative to the use of engineered pathogens as live vaccine delivery vehicles.

Administration, Oral↗
PubMed1992

A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate-dehydrogenase with multiple binding activity.

The surface of streptococci presents an array of different proteins, each designed to perform a specific function. In an attempt to understand the early events in group A streptococci infection, we have identified and purified a major surface protein from group A type 6 streptococci that has both an enzymatic activity and a binding capacity for a variety of proteins. Mass spectrometric analysis of the purified molecule revealed a monomer of 35.8 kD. Molecular sieve chromatography and sodium dodecyl sulfate (SDS)-gel electrophoresis suggest that the native conformation of the protein is likely to be a tetramer of 156 kD. NH2-terminal amino acid sequence analysis revealed 83% homology in the first 18 residues and about 56% in the first 39 residues with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of eukaryotic or bacterial origin. This streptococcal surface GAPDH (SDH) exhibits a dose-dependent dehydrogenase activity on glyceraldehyde-3-phosphate in the presence of beta-nicotinamide adenine dinucleotide both in its pure form and on the streptococcal surface. Its sensitivity to trypsin on whole organism and its inability to be removed with 2 M NaCl or 2% SDS support its surface location and tight attachment to the streptococcal cell. Affinity-purified antibodies to SDH detected the presence of this protein on the surface of all M serotypes of group A streptococcal tested. Purified SDH was found to bind to fibronectin, lysozyme, as well as the cytoskeletal proteins myosin and actin. The binding activity to myosin was found to be localized to the globular heavy meromyosin domain. SDH did not bind to streptococcal M protein, tropomyosin, or the coiled-coil domain of myosin. The multiple binding capacity of the SDH in conjunction with its GAPDH activity may play a role in the colonization, internalization, and the subsequent proliferation of group A streptococci.

Amino Acid Sequence↗
PubMed1992

Sorting of protein A to the staphylococcal cell wall.

The cell wall of gram-positive bacteria can be thought of as representing a unique cell compartment, which contains anchored surface proteins that require specific sorting signals. Some biologically important products are anchored in this way, including protein A and fibronectin binding protein of Staphylococcus aureus and streptococcal M protein. Studies of staphylococcal protein A and Escherichia coli alkaline phosphatase show that the signal both necessary and sufficient for cell wall anchoring consists of an LPXTGX motif, a C-terminal hydrophobic domain, and a charged tail. These sequence elements are conserved in many surface proteins from different gram-positive bacteria. We propose the existence of a hitherto undescribed sorting mechanism that positions proteins on the surface of gram-positive bacteria.

Amino Acid Sequence↗
PubMed1992

Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr.

A single insertion of transposon Tn917LTV1 into the chromosome of a Staphylococcus aureus clinical isolate, strain DB, resulted in a pleiotropic effect on the expression of a number of extracellular and cell-wall-associated proteins. Detailed comparison of phenotypes associated with the mutant, 11D2, and the parent, DB, indicated that the chromosomal locus inactivated as a result of transposon mutagenesis differs from the S. aureus accessory gene regulator locus (agr). In particular, the expression of alpha-hemolysin, which is not detectable in Agr- mutants, was enhanced in mutant 11D2, while it remained at a low level in strain DB. Likewise, protease activity was significantly enhanced in 11D2 compared with DB. In addition, most of the cell-bound proteins were expressed at lower levels in the mutant than the parent strain. This pattern is contrary to that found in switching from Agr+ to Agr- phenotypes. Southern blot hybridization with an agr probe indicated that the inactivated chromosomal locus is distinct from agr. Transduction experiments demonstrated that the phenotypes associated with mutant 11D2 could be transferred to the parental strain DB as well as to RN450, an S. aureus strain with a genetic background similar to strain 8325-4. This locus on the S. aureus chromosome, possibly regulatory in nature, has been designated sar for staphylococcal accessory regulator.

Bacterial Proteins↗
PubMed1992

Cytotoxic and viral neutralizing antibodies crossreact with streptococcal M protein, enteroviruses, and human cardiac myosin.

The development of autoimmunity in certain instances is related to infectious agents. In this report, cytotoxic monoclonal antibodies (mAbs) that recognize epitopes on both enteroviruses and the bacterium Streptococcus pyogenes are described. Murine anti-streptococcal mAbs that were crossreactive with streptococcal M protein, human cardiac myosin, and other alpha-helical coiled-coil molecules were found to neutralize coxsackieviruses B3 and B4 or poliovirus type 1. The viral-neutralizing anti-streptococcal mAbs were also cytotoxic for heart and fibroblast cell lines and reacted with viral capsid proteins on a Western immunoblot. Alignment of amino acid sequences shared between streptococcal M protein, coxsackie-virus B3 capsid protein VP1, and myosin revealed 40% identity in a 14- to 15-amino acid overlap. Synthetic peptides containing these sequences blocked mAb reactivity with streptococcal M protein. The data show that antibodies against alpha-helical structures of bacterial and viral antigens can lead to cytotoxic reactions and may be one mechanism to explain the origin of autoimmune heart disease.

Amino Acid Sequence↗