trans-complementation of a Staphylococcus aureus agr mutant by Staphylococcus lugdunensis agr RNAIII

RNAIII from Staphylococcus lugdunensis (RNAIII-sl) in a Staphylococcus aureus agr mutant partially restored the Agr phenotype. A chimeric construct consisting of the 5' end of RNAIII-sl and the 3' end of RNAIII from S. aureus restored the Agr phenotype to a greater extent, suggesting the presence of independent regulatory domains.     

Elderberry (Sambucus nigra) bark contains two structurally different Neu5Ac(alpha2,6)Gal/GalNAc-binding type 2 ribosome-inactivating proteins

A second NeuAc(alpha2,6)Gal/GalNAc binding type 2 ribosome-inactivating protein (RIP), called SNAI' has been isolated from elderberry (Sambucus nigra) bark. SNAI' is a minor bark protein which closely resembles the previously described major Neu5Ac(alpha2,6)Gal/GalNAc binding type 2 RIP called SNAI with respect to its carbohydrate-binding specificity and ribosome-inactivating activity but has a different molecular structure. Molecular cloning revealed that the deduced amino acid sequence of SNAI' is highly similar to that of SNAI and that the difference in molecular structure between both proteins relies on a single cysteine residue present in the B chain of SNAI but absent from SNAI'. The isolation of SNAI' not only identifies a minor bark protein as a type 2 RIP but also further emphasizes the complexity of the type 2 RIP/lectin mixture present in the bark of elderberry.     

RAP46 is a negative regulator of glucocorticoid receptor action and hormone-induced apoptosis

RAP46 was first identified by its ability to bind the glucocorticoid receptor. It has since been reported to bind several cellular proteins, including the anti-apoptotic protein Bcl-2, but the biological significance of these interactions is unknown. Here we show that RAP46 binds the hinge region of the glucocorticoid receptor and inhibits DNA binding and transactivation by the receptor. We further show that overexpression of RAP46 in mouse thymoma S49.1 cells inhibits glucocorticoid-induced apoptosis. Conversely, glucocorticoid-induced apoptosis and transactivation were enhanced after treating S49.1 cells with the immunosuppressant rapamycin, which down-regulates cellular levels of BAG-1, the mouse homolog of RAP46. The effect of rapamycin can, however, be overcome by overexpression of RAP46. These results together identify RAP46 as a protein that controls glucocorticoid-induced apoptosis through its negative regulatory action on the transactivation property of the glucocorticoid receptor.     

A novel wound dressing with an antibiotic delivery system stimulated by microbial infection

The aim of this study was to develop a new wound dressing with controlled release of antibiotics only in the presence of infection. In the first experiment using an infected dorsal pouch of rats, exudate containing proteinases from pouches contaminated with Staphylococcus aureus or Pseudomonas aeruginosa showed significantly higher hydrolytic activity toward Boc-Val-Pro-Arg-MCA than that from noninfected wounds. The authors then developed a new type of wound dressing (AGX), a drug delivery system in which gentamicin is bound to polyvinylalcohol hydrogel through an enzymatically degradable peptide linker containing a -(D)-Phe-Pro-Arg-sequence. To investigate in vitro effectiveness, AGX was incubated with exudate from S. aureus infected or P. aeruginosa infected wounds. Gentamicin was selectively released from AGX in the presence of the exudate from S. aureus infected or P. aeruginosa infected wounds, but was not released in the presence of exudate from noninfected wounds. Next, AGX or the polyvinylalcohol hydrogel that served as control was incubated with S. aureus in the presence of human plasma. After 24 hours, S. aureus concentration was markedly lower in the case with AGX than in that with polyvinylalcohol hydrogel. These results indicate that proteinases from wounds infected with S. aureus or P. aeruginosa cleaved the linker and gentamicin was released.     

Staphylococci express a receptor for human transferrin: identification of a 42-kilodalton cell wall transferrin-binding protein

Staphylococcus aureus and the coagulase-negative staphylococci are commonly responsible for peritonitis in renal patients undergoing continuous ambulatory peritoneal dialysis. To simulate growth conditions in vivo, staphylococci isolated from peritoneal infections were cultured in used human peritoneal dialysate (HPD). Immunoblotting experiments using cell wall preparations from these staphylococci revealed the presence of the host iron-binding glycoprotein transferrin bound to S. aureus, S. epidermidis, S. capitis, S. haemolyticus, and S. hominis but not to S. warneri or S. saprophyticus. Similar results were obtained by incubating broth-grown staphylococci with human transferrin, although, in contrast to S. aureus, the coagulase-negative staphylococci bound more transferrin after growth in iron-restricted broth. To determine whether the staphylococci express a saturable specific receptor for human transferrin, the interaction of human 125I-transferrin with the staphylococci was examined. Both S. aureus and S. epidermidis bound the radiolabelled iron-saturated ligand in a time- and concentration-dependent manner. From competition binding assays, the affinity (Kd) and number of receptors were estimated for S. epidermidis (Kd, 0.27 microM; 4,200 receptors per cell) and S. aureus (Kd, 0.28 microM; 4,200 receptors per cell). S. epidermidis but not S. aureus receptor activity was partially iron regulated. Human apotransferrin and iron-saturated transferrin and rabbit and rat transferrins competed equally well for the staphylococcal receptor. Bovine and porcine transferrins and ovotransferrin as well as human and bovine lactoferrins were much less effective at competing with human transferrin. Treatment of whole staphylococci with protease abolished transferrin binding, indicating the involvement of cell surface protein. Western blots (immunoblots) of cell wall preparations probed with human transferrin revealed the presence of a 42-kDa transferrin-binding protein common to both S. aureus and S. epidermidis. On Western strip blots, the binding of human transferrin to this protein was blocked by labelled human transferrin but not by albumin, immunoglobulin G, or bovine transferrin or ovotransferrin. To assess the conservation of the 42-kDa transferrin-binding protein, cell wall proteins of S. epidermidis, S. haemolyticus, S. capitis, S. hominis, S. warneri, and S. saprophyticus were Western blotted and probed with human transferrin. Only S. warneri and S. saprophyticus lacked the 42-kDa wall protein, consistent with their inability to bind transferrin. These data show that the staphylococci express a specific receptor for human transferrin based at least in part on a common 42-kDa cell wall protein.     

A peptide derived from a B cell epitope of Plasmodium falciparum rhoptry associated protein 2 specifically raises antibodies to rhoptry associated protein 1

Mice immunised with a recombinant form of malarial antigen rhoptry-associated protein 2 (RAP2) produce antisera which recognise the native protein by indirect immunofluorescence and immunoblotting. Purified IgG components of the antisera partially inhibit erythrocyte invasion in vitro. This response was obtained only if the recombinant immunogen was presented to the mice in the presence of reducing and denaturing agents. An 8-mer epitope in RAP2 was recognised by antibodies in three of the antisera: E25TEFSKLY32. Immunisation with this octapeptide raised antibodies which strongly recognised reduced RAP2 in seven out of eight mice. However, this antisera either failed to recognise (five out of eight mice), or only weakly (three out of eight mice) recognised nonreduced RAP2. Examination of disulphide bonds in native RAP2 showed that the 4 cysteines of RAP2 form two disulphide bridges: Cys24-Cys88, and Cys277-Cys376. One of these (Cys24-Cys88) is adjacent to the octapeptide in the native protein. Surprisingly, seven out of eight mice immunised with the octapeptide also raised antibodies against native rhoptry-associated protein 1 (RAP1). The raising of antibodies which recognise RAP1 was induced specifically by the RAP2 octapeptide rather than the carrier protein used for immunisation. The epitope in RAP1 recognised by the antibodies was identified and shown not to be the result of any shared contiguous homologous sequence between the two proteins, but to shared homologous amino acids in critical positions within the epitope. Purified IgG components from the antisera of mice immunised with the octapeptide gave partial inhibition of erythrocyte invasion in vitro.     

Functional display of a heterologous protein on the surface of Lactococcus lactis by means of the cell wall anchor of Staphylococcus aureus protein A

In this study, we showed that the cell wall anchor of protein A from Staphylococcus aureus is functional in the food-grade organism Lactococcus lactis. A fusion protein composed of the lactococcal Usp45 secretion signal peptide, streptavidin monomer, and the S. aureus protein A anchor became covalently attached to the peptidoglycan when expressed in L. lactis. The streptavidin moiety of the fusion protein was functionally exposed at the cellular surface. L. lactis cells expressing the anchored fusion polypeptide could be specifically immobilized on a biotinylated alkaline phosphatase-coated polystyrene support.     

Relation of arterial geometry to luminal narrowing and histologic markers for plaque vulnerability: the remodeling paradox

The agr P2 operon in Staphylococcus aureus codes for the elements of a density-sensing cassette made up of a typical two-component signalling system and its corresponding inducer. It is postulated that the autoinducer, a post-translationally modified octapeptide generated from the AgrD peptide, interacts with a receptor protein, coded by agrC, to transmit a signal via AgrA regulating expression of staphylococcal virulence genes through expression of agr RNA III. We show by analysis of PhoA fusions that AgrC is a transmembrane protein, and confirm using Western blotting that a 46 kDa protein corresponding to AgrC is present in the bacterial membrane. This protein is autophosphorylated on a histidine residue only in response to supernatants from an agr+ strain, and can also respond to the purified native octapeptide. A recombinant fusion protein where most of the N-terminal region of AgrC is replaced by the Escherichia coli maltose-binding protein is also autophosphorylated in response to stimulation by agr+ supernatants or purified octapeptide. We conclude that AgrC is the sensor molecule of a typical two-component signal system in S. aureus, and that the ligand-binding site of AgrC is probably located in the third extracellular loop of the protein.     

A high molecular weight protein from Staphylococcus intermedius cross-reacts with Staphylococcus aureus enterotoxin antibodies

Enterotoxin production by Staphylococcus species other than Staphylococcus aureus has been reported. Staphylococcus strains (104 in toto) representing twelve species and subspecies were examined for enterotoxins using a commercial staphylococcal enterotoxin ELISA immunoassay (TECRA, International Bioproducts). Staphylococcus intermedius (24 strains) and S. aureus (7 strains) were positive with this test. Western blots of S. aureus exoproteins demonstrated proteins of approximately 30 kD, consistent with known staphylococcal enterotoxins. The major antigen in all S. intermedius strains, a 75 kD protein, was not analogous to previously described staphylococcal enterotoxins. This protein was unique to S. intermedius. Gel filtration data indicate that the protein is a subunit of a larger protein in vivo. The 75 kD protein cross-reacts with several enterotoxin antibodies. It is unclear whether the protein is a toxin, but its homology with S. aureus enterotoxins may indicate a shared toxic region, or this protein may create false positive results in screening for enterotoxin.     

New ribosome-inactivating proteins with polynucleotide:adenosine glycosidase and antiviral activities from Basella rubra L. and bougainvillea spectabilis Willd

New single-chain (type 1) ribosome-inactivating proteins (RIPs) were isolated from the seeds of Basella rubra L. (two proteins) and from the leaves of Bougainvillea spectabilis Willd. (one protein). These RIPs inhibit protein synthesis both in a cell-free system, with an IC50 (concentration causing 50% inhibition) in the 10(-10) M range, and by various cell lines, with IC50S in the 10(-8)-10(-6) M range. All three RIPs released adenine not only from rat liver ribosomes but also from Escherichia coli rRNA, polyadenylic acid, herring sperm DNA, and artichoke mottled crinkle virus (AMCV) genomic RNA, thus being polynucleotide:adenosine glycosidases. The proteins from Basella rubra had toxicity to mice similar to that of most type 1 RIPs (Barbieri et al., 1993, Biochim Biophys Acta 1154: 237-282) with an LD50 (concentration that is 50% lethal) < or = 8 mg.kg-1 body weight, whilst the RIP from Bougainvillea spectabilis had an LD50 > 32 mg.kg-1. The N-terminal sequence of the two RIPs from Basella rubra had 80-93% identity, whereas it differed from the sequence of the RIP from Bougainvillea spectabilis. When tested with antibodies against various RIPs, the RIPs from Basella gave some cross-reactivity with sera against dianthin 32, and weak cross-reactivity with momordin I and momorcochin-S, whilst the RIP from Bougainvillea did not cross-react with any antiserum tested. An RIP from Basella rubra and one from Bougainvillea spectabilis were tested for antiviral activity, and both inhibited infection of Nicotiana benthamiana by AMCV.     

Synthetic peptides of human lysosomal cathepsin G with potent antipseudomonal activity

Enzymatically active and inactive (diisopropylfluorophosphate-treated) cathepsin G exerted antibacterial action in vitro against Staphylococcus aureus, whereas only enzymatically active cathepsin G displayed bactericidal action against Pseudomonas aeruginosa. In order to further test the requirement for protease activity for the antipseudomonal action of cathepsin G, synthetic peptides spanning the full-length mature protein were prepared and examined for antibacterial action. Surprisingly, three structurally distinct peptides that correspond to residues 61 to 80, 117 to 136, and 198 to 223 within the full-length protein were found to exert potent antipseudomonal action (> 4.5 logs of killing at 500 micrograms/ml) against P. aeruginosa ATCC 27853 and four mucoid clinical isolates. Only the peptide (CG117-136) corresponding to residues 117 to 136 (117-RPGTLCTVAGWGRVSMRRGT-136) within cathepsin G exerted antibacterial action against the gram-positive pathogen S. aureus. The antipseudomonal action of CG117-136 was rapid and could be inhibited either by increasing concentrations of NaCl or by 0.5 mM MgCl2 plus 0.5 mM CaCl2, and these conditions appeared to reduce binding of the peptide to whole bacteria. Variants of peptide CG117-136 lacking either a hydrophobic N-terminal domain or a positively charged C-terminal domain were found to have significantly less antipseudomonal action than CG117-136. The antibacterial capacity of the all-D-enantiomeric form of peptide CG117-136 was found to be identical to that of the all-L-peptide, suggesting that the mechanism of killing does not require the recognition of a target site possessing a chiral center.     

The Working Group report of Science-Based Categories for abstracts submitted to the annual Scientific Sessions. The Committee on Scientific Sessions Program (CSSP), American Heart Association

The presence of staphylococcal superantigenic toxins in the supernatants of liquid cultures was detected by an easy and rapid method assessing the activation of T lymphocytes by cytofluorimetric measurement of CD69 expression. Staphylococcus aureus cells were grown in Eagle's minimum essential medium supplemented with 5% heat-inactivated fetal calf serum. Supernatant fluids from all S. aureus strains producing superantigen-related toxins, including enterotoxins A to E, toxic shock syndrome toxin, and exfoliative toxins A and B, induced CD69 expression in a significantly higher number of T cells than a cutoff of 2%. This CD69 assay might be used for initial detection of superantigens from S. aureus strains isolated in the context of staphylococcal toxemia or related chronic human diseases such as atopic dermatitis or Kawasaki syndrome.     

A methylated Neurospora 5S rRNA pseudogene contains a transposable element inactivated by repeat-induced point mutation

In an analysis of 22 of the roughly 100 dispersed 5S rRNA genes in Neurospora crassa, a methylated 5S rRNA pseudogene, Psi63, was identified. We characterized the Psi63 region to better understand the control and function of DNA methylation. The 120-bp 5S rRNA-like region of Psi63 is interrupted by a 1.9-kb insertion that has characteristics of sequences that have been modified by repeat-induced point mutation (RIP). We found sequences related to this insertion in wild-type strains of N. crassa and other Neurospora species. Most showed evidence of RIP; but one, isolated from the N. crassa host of Psi63, showed no evidence of RIP. A deletion from near the center of this sequence apparently rendered it incapable of participating in RIP with the related full-length copies. The Psi63 insertion and the related sequences have features of transposons and are related to the Fot1 class of fungal transposable elements. Apparently Psi63 was generated by insertion of a previously unrecognized Neurospora transposable element into a 5S rRNA gene, followed by RIP. We name the resulting inactivated Neurospora transposon PuntRIP1 and the related sequence showing no evidence of RIP, but harboring a deletion that presumably rendered it defective for transposition, dPunt.     

Spondylitis and medullary transverse syndrome caused by Staphylococcus aureus

Three cases of haematogenous osteomyelitis in the vertebral column caused by Staphylococcus aureus are reported. The cases, which were associated with severe neurological symptoms and/or death, were initially characterized by a long period with no or discrete local signs of infection and by values of temperature and leucocyte counts within or close to normal values. In this period measurements of the sedimentation reaction and C-reactive protein were elevated, and were markers of persistent infection. At the Department of Clinical Microbiology in the county of Copenhagen blood cultures from a total of 49 patients were found to be positive for S. aureus during the period January to March 1996. Six patients were found to have osteomyelitis (12%, including four cases of spondylitis) and nine patients were suspected of having osteomyelitis. This frequency of patients with S. aureus bacteraemia having osteomyelitis was significantly higher than reported in another Danish study (10), which together with the severe outcome of the infection emphasizes the need for attentiveness to these serious complications of S. aureus bacteraemia.