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Vincent A Fischetti

Publications and source records attributed to Vincent A Fischetti.

At least 19 recordsLinked to original sources
PubMed2006

Mutagenesis of a bacteriophage lytic enzyme PlyGBS significantly increases its antibacterial activity against group B streptococci.

Group B streptococci (GBS) are the leading cause of neonatal meningitis and sepsis worldwide. Intrapartum antibiotic prophylaxis (IAP) is the current prevention strategy given to pregnant women with confirmed vaginal GBS colonization. Due to antibiotic resistance identified in GBS, we previously developed another strategy using a bacteriophage lytic enzyme, PlyGBS, to reduce vaginal GBS colonization. In this study, various DNA mutagenesis methods were explored to produce PlyGBS mutants with increased lytic activity against GBS. Several hyperactive mutants were identified that contain only the endopeptidase domain found in the N-terminal region of PlyGBS and represent only about one-third of the wild-type PlyGBS in length. Significantly, these mutants not only have 18-28-fold increases in specific activities compared to PlyGBS, but they also have a similar activity spectrum against several streptococcal species. One of the hyperactive mutants, PlyGBS90-1, reduced the GBS colonization from >5 logs of growth per mouse to <50 colony-forming units (cfu) 4 h post treatment ( approximately 4-log reduction) using a single dose in a mouse vaginal model. A reduction in GBS colonization before delivery should significantly reduce neonatal GBS infection providing a safe alternative to IAP.

Animals↗
PubMed2006

First complete genome sequence of two Staphylococcus epidermidis bacteriophages.

Staphylococcus epidermidis is an important opportunistic pathogen causing nosocomial infections and is often associated with infections in patients with implanted prosthetic devices. A number of virulence determinants have been identified in S. epidermidis, which are typically acquired through horizontal gene transfer. Due to the high recombination potential, bacteriophages play an important role in these transfer events. Knowledge of phage genome sequences provides insights into phage-host biology and evolution. We present the complete genome sequence and a molecular characterization of two S. epidermidis phages, phiPH15 (PH15) and phiCNPH82 (CNPH82). Both phages belonged to the Siphoviridae family and produced stable lysogens. The PH15 and CNPH82 genomes displayed high sequence homology; however, our analyses also revealed important functional differences. The PH15 genome contained two introns, and in vivo splicing of phage mRNAs was demonstrated for both introns. Secondary structures for both introns were also predicted and showed high similarity to those of Streptococcus thermophilus phage 2972 introns. An additional finding was differential superinfection inhibition between the two phages that corresponded with differences in nucleotide sequence and overall gene content within the lysogeny module. We conducted phylogenetic analyses on all known Siphoviridae, which showed PH15 and CNPH82 clustering with Staphylococcus aureus, creating a novel clade within the S. aureus group and providing a higher overall resolution of the siphophage branch of the phage proteomic tree than previous studies. Until now, no S. epidermidis phage genome sequences have been reported in the literature, and thus this study represents the first complete genomic and molecular description of two S. epidermidis phages.

Base Sequence↗
PubMed2006

The 1.6 A crystal structure of the catalytic domain of PlyB, a bacteriophage lysin active against Bacillus anthracis.

Lysins are peptidoglycan hydrolases that are produced by bacteriophage and act to lyse the bacterial host cell wall during progeny phage release. Here, we describe the structure and function of a novel bacteriophage-derived lysin, PlyB, which displays potent lytic activity against the Bacillus anthracis-like strain ATCC 4342. This molecule comprises an N-terminal catalytic domain (PlyB(cat)) and a C-terminal bacterial SH3-like domain, SH3b. It is shown that both domains are required for effective catalytic activity against ATCC 4342. Further, PlyB has specific activity comparable to the phage lysin PlyG, an amidase being developed as a therapeutic against anthrax. In contrast to PlyG, however, the 1.6 A X-ray crystal structure of PlyB(cat) reveals that the catalytic domain adopts the glycosyl hydrolase (GH)-25, rather than phage T7 lysozyme-like fold. PlyB therefore represents a new class of anthrax lysin and a new defensive tool in the armament against anthrax-mediated bioterrorism.

Amino Acid Sequence↗
PubMed2006

M.SpyI, a DNA methyltransferase encoded on a mefA chimeric element, modifies the genome of Streptococcus pyogenes.

While screening the clonality of Streptococcus pyogenes isolates from an outbreak of erythromycin-resistant pharyngitis in Pittsburgh, PA, we found a correlation between the presence of the chimeric element Phi10394.4 (carrying the macrolide efflux gene, mefA) and genomic DNA being resistant to cleavage by SmaI restriction endonuclease. A search of the open reading frames in Phi10394.4 identified a putative type II restriction-modification (R-M) cassette containing a cytosine methyltransferase gene (spyIM). Heterologous expression of the cloned spyIM gene, as well as allelic-replacement experiments, showed that the action of this methyltransferase (M.SpyI) was responsible for the inhibition of SmaI digestion of genomic DNA in the Phi10394.4-containing isolates. Analysis of the methylation patterns of streptococcal genomic DNA from spyIM-positive strains, a spyIM deletion mutant, and a spyIM-negative strain determined that M.SpyI specifically recognized and methylated the DNA sequence to generate 5'-C(m)CNGG. To our knowledge, this is the first methyltransferase gene from S. pyogenes to be cloned and to have its activity characterized. These results reveal why pulsed field gel electrophoresis analysis of SmaI-digested genomic DNA cannot be used to analyze the clonality of some streptococci containing Phi10394.4 and may explain the inability of previous epidemiological studies to use SmaI to analyze DNAs from macrolide-resistant streptococci. The presence of the SpyI R-M cassette in Phi10394.4 could impart a selective advantage to host strain survival and may provide another explanation for the observed increase in macrolide-resistant streptococci.

Alleles↗
PubMed2006

PlyC: a multimeric bacteriophage lysin.

Lysins are murein hydrolases produced by bacteriophage that act on the bacterial host cell wall to release progeny phage. When added extrinsically in their purified form, these enzymes produce total lysis of susceptible Gram-positive bacteria within seconds, suggesting a unique antimicrobial strategy. All known Gram-positive lysins are produced as a single polypeptide containing a catalytic activity domain, which cleaves one of the four major peptidoglycan bonds, and a cell-wall-binding domain, which may bind a species-specific carbohydrate epitope in the cell wall. Here, we have cloned and expressed a unique lysin from the streptococcal bacteriophage C(1), termed PlyC. Molecular characterization of the plyC operon reveals that PlyC is, surprisingly, composed of two separate gene products, PlyCA and PlyCB. Based on biochemical and biophysical studies, the catalytically active PlyC holoenzyme is composed of eight PlyCB subunits for each PlyCA. Inhibitor studies predicted the presence of an active-site cysteine, and bioinformatic analysis revealed a cysteine, histidine-dependent amidohydrolase/peptidase domain within PlyCA. Point mutagenesis confirmed that PlyCA is responsible for the observed catalytic activity, and Cys-333 and His-420 are the active-site residues. PlyCB was found to self-assemble into an octamer, and this complex alone was able to direct streptococcal cell-wall-specific binding. Similar to no other proteins in sequence databases, PlyC defines a previously uncharacterized structural family of cell-wall hydrolases.

Cell Wall↗
PubMed2006

Using phage lytic enzymes to control pathogenic bacteria.

Our laboratory has developed phage lytic enzymes to prevent infection by specifically destroying disease bacteria on mucous membranes and in blood. Enzymes specific for S. pneumoniae and S. pyogenes have been developed to be used nasally and orally to control these organisms in environments such as hospitals and nursing homes to prevent or markedly reduce serious infections by these pathogens. In addition, a B. anthracis-specific enzyme was developed to kill the vegetative forms of these bacteria in the blood of infected individuals. In animal studies, >80% of mice colonized mucosally or infected intravenously with pathogenic bacteria were decolonized or survived after a single enzyme treatment delivered to the same site of colonization or infection.

Journal Article↗
PubMed2006

Lysogeny of Streptococcus pneumoniae with MM1 phage: improved adherence and other phenotypic changes.

Pneumococcal prophages are extremely frequent, but no role in pathogenesis has so far been attributed to them. We isolated a variant of phage MM1, named MM1-1998, from a serotype 24 strain of Streptococcus pneumoniae. We created three isogenic strain pairs (serotypes 3, 4, and 24) that differed only by the lysogenic presence of the MM1-1998 phage and did a phenotypic comparison. Lysogeny led to improved adherence to inert surfaces and pharyngeal cells compared to that with the cured variants of the strains. We found that lysogeny with MM1-1998 coincided with a more transparent phenotype and phage curing with more opaque colonies in all strain pairs, and we discovered that transparency was associated with more successful and stable lysogeny. Since transparency alone was possibly responsible for the adherence difference, we further compared the TIGR4 lysogen with an equally transparent variant of TIGR4 in order to reassess the role of phage or transparency separately. The results revealed that improved adherence was independently associated with lysogeny with the MM1-1998 phage. Other phenotypic differences such as faster growth, increased autolysis, and decreased intracellular hemolytic activity were more likely due to transparency. By improving the adherence of pneumococci, this prophage may contribute to their fitness and possibly to their persistence in humans.

Animals↗
PubMed2006

Purification and characterization of LPXTGase from Staphylococcus aureus: the amino acid composition mirrors that found in the peptidoglycan.

Bacterial surface proteins are important molecules in the infectivity and survival of pathogens. Surface proteins on gram-positive bacteria have been shown to attach via a transpeptidase, termed sortase, that cleaves an LPXTG sequence found close to the C termini of nearly all surface proteins on these bacteria. We previously identified a unique enzyme (LPXTGase) from Streptococcus pyogenes that also cleaves the LPXTG motif with a catalytic activity higher than that of sortase, suggesting that it plays an important role in the attachment process. We have now purified and characterized an LPXTGase from Staphylococcus aureus and found that it has both similar and unique features compared to the S. pyogenes enzyme. The S. aureus enzyme is glycosylated and contains unusual amino acids, like its streptococcal counterpart. Like the streptococcal enzyme, staphylococcal LPXTGase has an overrepresentation of amino acids found in the peptidoglycan, i.e., glutamine/glutamic acid, glycine, alanine, and lysine, and furthermore, we find that these amino acids are present in the enzyme at precisely the same ratio at which they are found in the peptidoglycan for the respective organism. This suggests that enzymes responsible for wall assembly may also play a role in the construction of LPXTGase.

Amino Acids↗
PubMed2006

Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: implications for evolution of environmental fitness and antibiotic resistance.

Phage-mediated lysis has been an essential laboratory tool for rapidly identifying Bacillus anthracis for more than 40 years, relying on the gamma phage derivative of a Bacillus cereus prophage called W. The complete genomic sequences of the temperate W phage, referred to as Wbeta, and its lytic variant gamma were determined and found to encode 53 open reading frames each, spanning 40,864 bp and 37,373 bp, respectively. Direct comparison of the genomes showed that gamma evolved through mutations at key loci controlling host recognition, lysogenic growth, and possibly host phenotypic modification. Included are a cluster of point mutations at the gp14 tail fiber locus of gamma, encoding a protein that, when fused to green fluorescent protein, binds specifically to B. anthracis. A large 2,003-bp deletion was also identified at the gamma lysogeny module, explaining its shift from a temperate to a lytic lifestyle. Finally, evidence of recombination was observed at a dicistronic Wbeta locus, encoding putative bacterial cell surface-modifying proteins, replaced in gamma with a locus, likely obtained from a B. anthracis prophage, encoding demonstrable fosfomycin resistance. Reverse transcriptase PCR analysis confirmed strong induction at the dicistronic Wbeta locus and at four other phage loci in B. anthracis and/or B. cereus lysogens. In all, this study represents the first genomic and functional description of two historically important phages and is part of a broader investigation into contributions of phage to the B. anthracis life cycle. Initial findings suggest that lysogeny of B. anthracis promotes ecological adaptation, rather than virulence, as with other gram-positive pathogens.

Amino Acid Sequence↗
PubMed2005

T cell mimicry and epitope specificity of cross-reactive T cell clones from rheumatic heart disease.

Mimicry between streptococcal M protein and cardiac myosin is important in the pathogenesis of rheumatic heart disease. M protein-specific human T cell clones derived from rheumatic carditis were cross-reactive with human cardiac myosin, and laminin, a valve protein. Among the 11 CD4(+) and CD8(+) cross-reactive T cell clones, at least 6 different reactivity patterns were distinguished, suggesting different degrees of cross-reactivity and a very diverse T cell repertoire. The latter was confirmed by a heterogeneous Vbeta gene and CDR3 usage. HLA restriction and Th1 cytokine production in response to rM6 protein were preserved when the T cell clones were stimulated by human cardiac myosin or other alpha-helical proteins, such as tropomyosin and laminin. The cross-reactive human T cell clones proliferated to B2 and B3A, dominant peptide epitopes in the B repeat region of streptococcal M protein. In human cardiac myosin, epitopes were demonstrated in the S2 and light meromyosin regions. In our study, T cell mimicry was defined as recognition of structurally related Ags involved in disease and recognized by the same T cell. Mimicry in our study was related to alpha-helical coiled coil proteins which have a repetitive seven-aa residue periodicity that maintains alpha-helical structure and thus creates a high number of degenerate possibilities for recognition by T cells. The study of human T cell clones from rheumatic heart disease revealed potential sites of T cell mimicry between streptococcal M protein and human cardiac myosin and represents some of the most well-defined T cell mimicry in human autoimmune disease.

Amino Acid Sequence↗
PubMed2005

The career of Maclyn McCarty.

On January 2, 2005, the scientific community lost a valued colleague and friend. Maclyn McCarty, or "Mac," as he was better known, was perhaps most recognized for his part in the discovery of DNA as the carrier of genetic information. But McCarty's scientific career was long and fruitful, and his contributions to science were vast. This retrospective offers a look at some of Mac's other notable scientific achievements.

Biochemistry↗
PubMed2005

Bacteriophage lytic enzymes: novel anti-infectives.

Bacteriophage lytic enzymes, or lysins, are highly evolved molecules produced by bacterial viruses (bacteriophage) to digest the bacterial cell wall for bacteriophage progeny release. Small quantities of purified recombinant lysin added to gram-positive bacteria causes immediate lysis resulting in log-fold death of the target bacterium. Lysins have now been used successfully in animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and in blood. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance to lysins and their ability to kill colonizing pathogens on mucosal surfaces, capabilities that were previously unavailable. Thus, lysins could be an effective anti-infective in an age of mounting antibiotic resistance.

Amidohydrolases↗
PubMed2005

Removal of group B streptococci colonizing the vagina and oropharynx of mice with a bacteriophage lytic enzyme.

Group B streptococci (GBS) are the leading cause of neonatal meningitis and sepsis worldwide. The current treatment strategy is limited to intrapartum antibiotic prophylaxis in pregnant women to prevent early-onset neonatal diseases, but considering the potential for antibiotic resistance, the risk of losing control over the disease is high. To approach this problem, we have developed a bacteriophage (phage) lytic enzyme to remove colonizing GBS. Bacteriophage muralytic enzymes, termed lysins, are highly evolved molecules designed to degrade the cell wall of host bacteria to release phage particles from the bacterial cytoplasm. Several different lysins have been developed to specifically kill bacterial pathogens both on mucosal surfaces and in blood and represent a novel approach to control infection. A lysin cloned from a phage infecting GBS was found to contain two putative catalytic domains and one putative binding domain, which is similar to the domain organization of some staphylococcal phage lysins. The lysin (named PlyGBS) was recombinantly expressed in Escherichia coli, and purified PlyGBS efficiently killed all tested GBS serotypes in vitro. In a mouse model, a single dose of PlyGBS significantly reduced bacterial colonization in both the vagina and oropharynx. As an alternative strategy for intrapartum antibiotic prophylaxis, this approach may be used to reduce vaginal GBS colonization in pregnant women before delivery or to decontaminate newborns, thus reducing the incidence of GBS-associated neonatal meningitis and sepsis.

Animals↗
PubMed2005

Synergistic killing of Streptococcus pneumoniae with the bacteriophage lytic enzyme Cpl-1 and penicillin or gentamicin depends on the level of penicillin resistance.

A combination of Cpl-1, a bacteriophage lytic enzyme, and penicillin, gentamicin, levofloxacin, or azithromycin was tested against Streptococcus pneumoniae strains with various susceptibilities to penicillin. Activities of Cpl-1 and gentamicin were increasingly synergistic with a decreasing penicillin MIC, while Cpl-1 and penicillin showed synergy against an extremely penicillin-resistant strain.

Drug Synergism↗
PubMed2005

Clinical and microbiological responses of volunteers to combined intranasal and oral inoculation with a Streptococcus gordonii carrier strain intended for future use as a group A streptococcus vaccine.

Streptococcus gordonii shows promise as a live mucosal vaccine vector for immunization against respiratory pathogens. In preparation for clinical trials to evaluate S. gordonii engineered to express group A streptococcal M protein antigens, we characterized the responses of 150 healthy volunteers to combined nasal and oral inoculation with approximately 1.5 x 10(9) CFU of SP204(1-1), an S. gordonii strain not bearing vaccine antigens. SP204(1-1) was selected for resistance to streptomycin and 5-fluoro-2-deoxyuridine to distinguish it from indigenous flora. In two antibiotic treatment studies, we performed serial culturing of nose, mouth, and saliva samples from 120 subjects treated with azithromycin beginning 5 days after inoculation to determine whether SP204(1-1) could be rapidly eliminated should safety concerns arise. A natural history study was performed to assess the time until spontaneous eradication in the remaining 30 subjects, who did not receive the antibiotic and who were monitored with repeated culturing for 14 weeks after inoculation. SP204(1-1) was generally well tolerated. Symptoms reported most often within 5 days of inoculation were nasal congestion (36%), headache (30%), and sore throat (19%). The strain was detected by culturing in 98% of subjects. A single dose of azithromycin eliminated colonization in 95% of subjects; all subjects receiving a 5-day course of an antibiotic showed clearance by day 11. Without the antibiotic, 82% of subjects showed spontaneous eradication of the implanted strain within 7 days, and all showed clearance by 35 days. The results of these clinical trials provide encouragement that the use of S. gordonii as a live mucosal vaccine vector is a feasible strategy.

Administration, Intranasal↗
PubMed2004

A novel secreted endoglycosidase from Enterococcus faecalis with activity on human immunoglobulin G and ribonuclease B.

The human pathogen Enterococcus faecalis can degrade the N-linked glycans of human RNase B to acquire nutrients, but no gene or protein has been associated with this activity. We identified an 88-kDa secreted protein, endoglycosidase (Endo) E, which is most likely responsible for this activity. EndoE, encoded by ndoE, consists of an alpha-domain with a family 18 glycosyl hydrolase motif and a beta-domain similar to family 20 glycosyl hydrolases. Phylogenetic analysis of EndoE indicates that the alpha-domain is related to human chitobiases, and the beta-domain is related to bacterial and human hexosaminidases. Recombinant expression of full-length EndoE or EndoEalpha, site-directed mutagenesis of the catalytic residues, mass spectroscopy, and homology modeling shows that EndoEalpha hydrolyzes the glycan on human RNase B, whereas EndoEbeta hydrolyzes the conserved glycan on IgG. Denaturation experiments indicate that the chitinase activity on RNase B is not dependent on the tertiary structure, although it is on IgG. The ndoE gene and secreted EndoE are present in most E. faecalis but not in Enterococcus faecium isolates. Correspondingly, E. faecalis, but not E. faecium, degrades the glycan on RNase B during growth. Thus, we have identified a secreted enzyme from E. faecalis, EndoE, which by two distinct activities hydrolyzes the glycans on RNase B and IgG. Both activities could be important for the molecular pathogenesis and persistence of E. faecalis during human infections.

Amino Acid Sequence↗