scholarly journals Role of SraP, a Serine-Rich Surface Protein of Staphylococcus aureus, in Binding to Human Platelets

2005 ◽  
Vol 73 (4) ◽  
pp. 2273-2280 ◽  
Author(s):  
Ian R. Siboo ◽  
Henry F. Chambers ◽  
Paul M. Sullam
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Molecular Mass ◽  
Signal Sequence ◽  
Rabbit Model ◽  
Human Platelets ◽  
Ligand Interaction ◽  
Calculated Molecular Mass ◽  
Platelet Binding

ABSTRACT The binding of bacteria to platelets is a postulated central event in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus gordonii is mediated in large part by GspB, a high-molecular-mass cell wall glycoprotein. Although Staphylococcus aureus has a GspB homolog (SraP), little is known about its function. SraP has a calculated molecular mass of 227 kDa and, like GspB, is predicted to contain an atypical N-terminal signal sequence, two serine-rich repeat regions (srr1 and srr2) separated by a nonrepeat region, and a C-terminal cell wall anchoring motif (LPDTG). To assess whether SraP contributes to platelet binding, we compared the binding to human platelets of S. aureus strain ISP479C and of an isogenic variant (strain PS767) in which sraP had been disrupted by allelic replacement. Platelet binding in vitro by PS767 was 47% ± 17% (mean ± standard deviation) lower than that of ISP479C (P < 0.001). In addition, a recombinant fragment of SraP containing srr1 and the nonrepeat region was found to bind platelets directly. Binding was saturable, suggesting a receptor-ligand interaction. When tested in a rabbit model of endocarditis, in which each animal was simultaneously infected with ISP479C and PS767 at a ratio of approximately 1:1, the titers of the mutant strain within vegetations were significantly lower than those of the parent strain at 1 and 24 h postinfection. These results indicate that SraP can mediate the direct binding of S. aureus to platelets and that the platelet-binding domain of this glycoprotein is located within its N-terminal region. Moreover, the expression of SraP appears to be a virulence determinant in endovascular infection.

scholarly journals Successful Therapy of Experimental Endocarditis Caused by Vancomycin-Resistant Staphylococcus aureus with a Combination of Vancomycin and β-Lactam Antibiotics

2006 ◽  
Vol 50 (9) ◽  
pp. 2951-2956 ◽  
Author(s):  
Paige M. Fox ◽  
Russell J. Lampen ◽  
Katrina S. Stumpf ◽  
Gordon L. Archer ◽  
Michael W. Climo
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Rabbit Model ◽  
Bloodstream Infections ◽  
Aberrant Cell ◽  
Trial Treatment ◽  
Vancomycin Resistant ◽  
Vancomycin Treatment

ABSTRACT VRS1 is the first isolated strain of vancomycin-resistant Staphylococcus aureus (VRSA) found to carry the vanA gene complex previously described in Enterococcus. Under vancomycin pressure, VRS1 makes aberrant cell walls consisting of stem tetrapeptide and depsipeptide that lack the terminal d-Ala-d-Ala residues targeted by vancomycin. Previous data have suggested that this aberrant cell wall is not cross-linked by PBP2a, the enzyme responsible for cell wall transpeptidation in the presence of β-lactam antibiotics. We examined the efficacy of treating VRS1 with a combination of vancomycin and β-lactam antibiotics in vitro and in vivo. We found that the MIC of oxacillin for VRS1 decreased from >256 μg/ml to <1 μg/ml in the presence of vancomycin. Using the rabbit model of endocarditis, we treated VRS1-infected rabbits with nafcillin alone, vancomycin alone, or a combination of nafcillin and vancomycin. Treatment with nafcillin in combination with vancomycin cleared bloodstream infections within 24 h and sterilized 12/13 spleens (92%), as well as 8/13 kidneys (62%), following 3 days of treatment. Mean aortic valve vegetation counts were reduced 3.48 log10 CFU/g with the combination therapy (compared to untreated controls) and were significantly lower than with either vancomycin or nafcillin given alone. VRS1 was extremely virulent in this model, as no untreated rabbits survived the 3-day trial. Treatment of clinical infections due to VRSA with the combination of vancomycin and β-lactams may be an option, based on these results.

scholarly journals Comparison of Borate Bioactive Glass and Calcium Sulfate as Implants for the Local Delivery of Teicoplanin in the Treatment of Methicillin-Resistant Staphylococcus aureus-Induced Osteomyelitis in a Rabbit Model

2015 ◽  
Vol 59 (12) ◽  
pp. 7571-7580 ◽  
Author(s):  
Wei-Tao Jia ◽  
Qiang Fu ◽  
Wen-Hai Huang ◽  
Chang-Qing Zhang ◽  
Mohamed N. Rahaman
Keyword(s):  
Staphylococcus Aureus ◽  
Bioactive Glass ◽  
Calcium Sulfate ◽  
Rabbit Model ◽  
Local Delivery ◽  
Methicillin Resistant ◽  
Content Type ◽  
Borate Bioactive Glass ◽  
Phosphate Buffered Saline

ABSTRACTThere is growing interest in biomaterials that can cure bone infection and also regenerate bone. In this study, two groups of implants composed of 10% (wt/wt) teicoplanin (TEC)-loaded borate bioactive glass (designated TBG) or calcium sulfate (TCS) were created and evaluated for their ability to release TECin vitroand to cure methicillin-resistantStaphylococcus aureus(MRSA)-induced osteomyelitis in a rabbit model. When immersed in phosphate-buffered saline (PBS), both groups of implants provided a sustained release of TEC at a therapeutic level for up to 3 to 4 weeks while they were gradually degraded and converted to hydroxyapatite. The TBG implants showed a longer duration of TEC release and better retention of strength as a function of immersion time in PBS. Infected rabbit tibiae were treated by debridement, followed by implantation of TBG or TCS pellets or intravenous injection with TEC, or were left untreated. Evaluation at 6 weeks postimplantation showed that the animals implanted with TBG or TCS pellets had significantly lower radiological and histological scores, lower rates of MRSA-positive cultures, and lower bacterial loads than those preoperatively and those of animals treated intravenously. The level of bone regeneration was also higher in the defects treated with the TBG pellets. The results showed that local TEC delivery was more effective than intravenous administration for the treatment of MRSA-induced osteomyelitis. Borate glass has the advantages of better mechanical strength, more desirable kinetics of release of TEC, and a higher osteogenic capacity and thus could be an effective alternative to calcium sulfate for local delivery of TEC.

scholarly journals A genomic and proteomic analysis of surface proteins in bovine-adapted lineages of Staphylococcus aureus

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Shauna D. Drumm ◽  
Rebecca Owens ◽  
Jennifer Mitchell ◽  
Orla M. Keane
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Surface Proteins ◽  
Host Cells ◽  
Mass Spectrometry Analysis ◽  
In Vitro Assays ◽  
Immune Recognition ◽  
Independent Manner ◽  
Genes Encoding

In Ireland, Staphylococcus aureus is the most common cause of intramammary infection (IMI) in cattle with the bovine-adapted lineages CC151 and CC97 most commonly found. Surface proteins play a major role in establishing and maintaining the infection. A previous study revealed that a strain from the CC151 lineage showed significant decay in genes encoding predicted surface proteins. Twenty-three S. aureus strains, twelve belonging to CC151 and eleven belonging to CC97, isolated from clinical IMI, were sequenced and genes encoding cell wall anchored (CWA) proteins predicted. Analysis showed that a minority of genes encoding putative CWA proteins were intact in the CC151 strains compared to CC97. Of the 26 known CWA proteins in S. aureus, the CC151 strains only encoded 10 intact genes while CC97 encoded on average 18 genes. Also within the CC97 lineage, the repertoire of genes varied depending on individual strains, with strains encoding between 17-20 intact genes. Although CC151 is reported to internalize within bovine host cells, it does so in a fibronectin-binding protein (FnBPA and FnBPB) independent manner. In-vitro assays were performed and results showed that strains from CC151, and surprisingly also CC97, weakly bound bovine fibronectin and that the FnBPs were poorly expressed in both these lineages. Mass spectrometry analysis of cell wall extracts revealed that SdrE and AdsA were the most highly expressed CWA proteins in both lineages. These results demonstrate significant differences between CC151 and CC97 in their repertoire of genes encoding CWA proteins, which may impact immune recognition of these strains and their interactions with host cells.

The yeast acid phosphatase can enter the secretory pathway without its N-terminal signal sequence

1987 ◽  
Vol 7 (9) ◽  
pp. 3306-3314
Author(s):  
S Silve ◽  
M Monod ◽  
A Hinnen ◽  
R Haguenauer-Tsapis
Keyword(s):  
Cell Wall ◽  
Acid Phosphatase ◽  
Gene Product ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
In Vitro Mutagenesis ◽  
Heterologous System ◽  
Pho5 Gene

The repressible Saccharomyces cerevisiae acid phosphatase (APase) coded by the PHO5 gene is a cell wall glycoprotein that follows the yeast secretory pathway. We used in vitro mutagenesis to construct a deletion (delta SP) including the entire signal sequence and four amino acids of the mature sequence of APase. An APase-deficient yeast strain was transformed with a high-copy-number plasmid carrying the PHO5/delta SP gene. When expressed in vivo, the PHO5/delta SP gene product accumulated predominantly as an inactive, unglycosylated form located inside the cell. A large part of this unglycosylated precursor underwent proteolytic degradation, but up to 30% of it was translocated, core glycosylated, and matured by the addition of mannose residues, before reaching the cell wall. It appears, therefore, that the signal sequence is important for efficient translocation and core glycosylation of yeast APase but that it is not absolutely necessary for entry of the protein into the yeast secretory pathway. mRNA obtained by in vitro transcription of PHO5 and PHO5/delta SP genes were translated in vitro in the presence of either reticulocyte lysate and dog pancreatic microsomes or yeast lysate and yeast microsomes. The PHO5 gene product was translocated and core glycosylated in the heterologous system and less efficiently in the homologous system. We were not able to detect any translocation or glycosylation of PHO5/delta SP gene product in the heterologous system, but a very small amount of core suppression of glycosylated material could be evidenced in the homologous system.

THE MODE OF ACTION OF THE ANTIBIOTIC PAROMOMYCIN ON STAPHYLOCOCCUS AUREUS

1966 ◽  
Vol 12 (6) ◽  
pp. 1157-1165 ◽  
Author(s):  
A. von Seefried ◽  
D. C. Jordan
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Binding Sites ◽  
Bactericidal Action ◽  
Plate Method ◽  
Vitro Binding ◽  
Resistant Mutants ◽  
Intracellular Binding

Paromomycin (Humatin, Parke Davis & Co.), a broad-spectrum aminoglycosidic antibiotic, inhibits the incorporation of amino acids into the trypsinsoluble protein fraction of Staphylococcus aureus 257. Protein synthesis is inhibited immediately, but the synthesis of cell-wall mucopeptide and alcohol-soluble proteins and lipids is not affected for approximately 35 min after antibiotic addition to actively growing cells. Paromomycin, at the ribosomal level, prevents the attachment of amino acyl-s-RNA and causes accumulation of m-RNA.Divalent cations (Ca++ and Mg++) antagonize the bactericidal action of paromomycin and interfere with the in vivo binding of the antibiotic on both the cell surface and the intracellular binding sites. In vitro binding to free ribosomes can be prevented and reversed by both monovalent and divalent cations.Using a "cylinder-plate" method, involving the displacement of antibiotic from cellular fractions by 0.2 M MgCl2, the antibiotic can be recovered from the ribosomes, cytoplasm, and the cell wall of paromomycin-sensitive S. aureus cells, but is not found in any of these fractions isolated from paromomycin-resistant cells developed from the sensitive parent strain. The resistant mutants apparently have lost the ability to adsorb and transport the antibiotic into the cell.

scholarly journals Molecular Analysis of the Gene Encoding a Novel Chitin-Binding Protease from Alteromonas sp. Strain O-7 and Its Role in the Chitinolytic System

2002 ◽  
Vol 184 (7) ◽  
pp. 1865-1872 ◽  
Author(s):  
Katsushiro Miyamoto ◽  
Eiji Nukui ◽  
Hiroyuki Itoh ◽  
Takaji Sato ◽  
Takeshi Kobayashi ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Signal Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chitinase Activity ◽  
Maximum Activity ◽  
Sequencing Analysis ◽  
Gene Encoding ◽  
Calculated Molecular Mass ◽  
Chitin Binding Domain

ABSTRACT Alteromonas sp. strain O-7 secretes several proteins in response to chitin induction. We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity. The gene encoding AprIV (aprIV) was cloned in Escherichia coli. DNA sequencing analysis revealed that the open reading frame of aprIV encoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da. AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3). Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed. The PkdD-ChtBD but not PkdD exhibited strong binding to α-chitin and β-chitin. Western and Northern analyses demonstrated that aprIV was induced in the presence of N-acetylglucosamine, N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity from Alteromonas sp. strain O-7 and characterized. The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of AprIV were pH 11.5 and 35°C, respectively, and even at 10°C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity.

Increase in the in vitro susceptibility of Staphylococcus aureus to antimicrobial agents in the presence of Candida albicans

1979 ◽  
Vol 25 (4) ◽  
pp. 429-435 ◽  
Author(s):  
J. deRepentigny ◽  
R. Lévesque ◽  
L. G. Mathieu
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Candida Albicans ◽  
Inhibitory Activity ◽  
Antimicrobial Agents ◽  
Mixed Cultures ◽  
Alpha Toxin ◽  
In Vitro Susceptibility ◽  
Pure Cultures

In experiments with mixed cultures of Staphylococcus aureus and Candida albicans both in the absence and in the presence of 5-fluorocytosine (5-FC), we have observed that (1) there is an inhibition of S. aureus growth in mixed cultures with C. albicans in media supplemented with 1 μg/mL of 5-FC and that 5-FC has no effect on staphylococci in pure cultures; (2) this inhibition occurred with clinically isolated and laboratory strains and could be reversed by specific metabolites; (3) Staphylococcus aureus was inhibited by filtrates of C. albicans cultures treated with 5-FC and this seemed to be favored by some C. albicans filterable product which can affect the cell wall and the permeability of the staphylococcal cells since they become sensitive to 5-FC; (4) nine other commonly used antimicrobials showed an increased inhibitory activity against S. aureus in mixed cultures with C. albicans; and (5) there is a decrease in the number of precipitating antigens of S. aureus and of the activity of alpha toxin when this species was grown with both C. albicans and 5-FC. Our results indicate that the susceptibility of some species to antimicrobials could be significantly modified in the presence of other species. One cannot exclude that a similar phenomenon could happen in hosts under treatment with antibiotics against infection.

scholarly journals Beneficial Influence of Platelets on Antibiotic Efficacy in an In Vitro Model of Staphylococcus aureus-Induced Endocarditis

2004 ◽  
Vol 48 (7) ◽  
pp. 2551-2557 ◽  
Author(s):  
Renee-Claude Mercier ◽  
Robert M. Dietz ◽  
Jory L. Mazzola ◽  
Arnold S. Bayer ◽  
Michael R. Yeaman
Keyword(s):  
Staphylococcus Aureus ◽  
In Vitro Model ◽  
Antimicrobial Effect ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Activated Platelets ◽  
Vitro Model ◽  
Antibiotic Efficacy ◽  
Chamber Fluid

ABSTRACT Platelets contribute to antimicrobial host defense against infective endocarditis (IE) by releasing platelet microbicidal proteins (PMPs). We investigated the influence of thrombin-stimulated human platelets on the evolution of simulated IE in the presence and absence of vancomycin or nafcillin. Staphylococcus aureus strains differing in intrinsic susceptibility to PMPs or antibiotics were studied: ISP479C (thrombin-induced PMP-1 [tPMP-1] susceptible; nafcillin and vancomycin susceptible), ISP479R (tPMP-1 resistant; nafcillin and vancomycin susceptible), and GISA-NJ (tPMP-1 intermediate-susceptible; vancomycin intermediate-susceptible). Platelets were introduced and thrombin activated within the in vitro IE model 30 min prior to inoculation with S. aureus. At 0 to 24 h postinoculation, bacterial densities in chamber fluid and simulated endocardial vegetations (SEVs) were quantified and compared among groups. Activated platelets alone, or in combination with antibiotics, inhibited the proliferation of ISP479C in chamber fluid or SEVs over the initial 4-h period (P < 0.05 versus controls). Moreover, nafcillin-containing regimens exerted inhibitory effects beyond 4 h against ISP479C in both model phases. By comparison, activated platelets inhibited GISA-NJ proliferation in SEVs but not in chamber fluid. The combination of platelets plus nafcillin or vancomycin significantly inhibited proliferation of the GISA-NJ strain in SEVs compared to the effect of platelets or antibiotics alone (P < 0.05). In contrast, platelets did not significantly alter the antistaphylococcal efficacies of nafcillin or vancomycin against ISP479R. These data support our hypothesis that a beneficial antimicrobial effect may result from the interaction among platelets, PMPs, and anti-infective agents against antibiotic-susceptible or -resistant staphylococci that exhibit a tPMP-1-susceptible or -intermediate-susceptible phenotype.

scholarly journals Molecular Characterization of Saccharomyces cerevisiae TFIID

2002 ◽  
Vol 22 (16) ◽  
pp. 6000-6013 ◽  
Author(s):  
Steven L. Sanders ◽  
Krassimira A. Garbett ◽  
P. Anthony Weil
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Molecular Mass ◽  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Calculated Molecular Mass ◽  
General Transcription Factor

ABSTRACT We previously defined Saccharomyces cerevisiae TFIID as a 15-subunit complex comprised of the TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). In this report we give a detailed biochemical characterization of this general transcription factor. We have shown that yeast TFIID efficiently mediates both basal and activator-dependent transcription in vitro and displays TATA box binding activity that is functionally distinct from that of TBP. Analyses of the stoichiometry of TFIID subunits indicated that several TAFs are present at more than 1 copy per TFIID complex. This conclusion was further supported by coimmunoprecipitation experiments with a systematic family of (pseudo)diploid yeast strains that expressed epitope-tagged and untagged alleles of the genes encoding TFIID subunits. Based on these data, we calculated a native molecular mass for monomeric TFIID. Purified TFIID behaved in a fashion consistent with this calculated molecular mass in both gel filtration and rate-zonal sedimentation experiments. Quite surprisingly, although the TAF subunits of TFIID cofractionated as a single complex, TBP did not comigrate with the TAFs during either gel filtration chromatography or rate-zonal sedimentation, suggesting that TBP has the ability to dynamically associate with the TFIID TAFs. The results of direct biochemical exchange experiments confirmed this hypothesis. Together, our results represent a concise molecular characterization of the general transcription factor TFIID from S. cerevisiae.

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