Staphylococcus aureus sortase mutants defective in the display of surface proteins and in the pathogenesis of animal infections.
(17/414)
Many gram-positive bacteria covalently tether their surface adhesins to the cell wall peptidoglycan. We find that surface proteins of Staphylococcus aureus are linked to the cell wall by sortase, an enzyme that cleaves polypeptides at a conserved LPXTG motif. S. aureus mutants lacking sortase fail to process and display surface proteins and are defective in the establishment of infections. Thus, the cell wall envelope of gram-positive bacteria represents a surface organelle responsible for interactions with the host environment during the pathogenesis of bacterial infections. (+info)
Mechanism of heavy metal ion activation of phytochelatin (PC) synthase: blocked thiols are sufficient for PC synthase-catalyzed transpeptidation of glutathione and related thiol peptides.
(18/414)
The dependence of phytochelatin synthase (gamma-glutamylcysteine dipeptidyltranspeptidase (PCS), EC ) on heavy metals for activity has invariably been interpreted in terms of direct metal binding to the enzyme. Here we show, through analyses of immunopurified, recombinant PCS1 from Arabidopsis thaliana (AtPCS1), that free metal ions are not essential for catalysis. Although AtPCS1 appears to be primarily activated posttranslationally in the intact plant and purified AtPCS1 is able to bind heavy metals directly, metal binding per se is not responsible for catalytic activation. As exemplified by Cd(2+)- and Zn(2+)-dependent AtPCS1-mediated catalysis, the kinetics of PC synthesis approximate a substituted enzyme mechanism in which micromolar heavy metal glutathione thiolate (e.g. Cd.GS(2) or Zn.GS(2)) and free glutathione act as gamma-Glu-Cys acceptor and donor. Further, as demonstrated by the facility of AtPCS1 for the net synthesis of S-alkyl-PCs from S-alkylglutathiones with biphasic kinetics, consistent with the sufficiency of S-alkylglutathiones as both gamma-Glu-Cys donors and acceptors in media devoid of metals, even heavy metal thiolates are dispensable. It is concluded that the dependence of AtPCS1 on the provision of heavy metal ions for activity in media containing glutathione and other thiol peptides is a reflection of this enzyme's requirement for glutathione-like peptides containing blocked thiol groups for activity. (+info)
Molecular epidemiology survey of penicillin-susceptible and -resistant Streptococcus pneumoniae recovered from patients with meningitis in France.
(19/414)
The genetic diversity of Streptococcus pneumoniae isolates (n=291) recovered from cerebrospinal fluid of patients with meningitis in France was investigated by restriction fragment length polymorphism analysis of the ribosomal RNA gene regions and of the pbp2b and 2x genes. Statistical analysis of the data by factorial analysis of correspondence established the following: penicillin-susceptible isolates had a high level of genetic diversity, especially those belonging to serogroups frequently associated with carriage; capsular serotype switches could occur among penicillin-susceptible and -resistant isolates; and the mechanisms of acquired penicillin resistance were clearly distinct in isolates with penicillin minimum inhibitory concentration (MIC) values <1 mg/L and isolates with penicillin MIC values >/=1 mg/L. Thus, an increase in the penicillin MIC for a given strain, from intermediate to high-level resistance would be a rare event. (+info)
Azuki bean cells are hypersensitive to cadmium and do not synthesize phytochelatins.
(20/414)
Suspension-cultured cells of azuki bean (Vigna angularis) as well as the original root tissues were hypersensitive to Cd (<10 microM). Repeated subculturings with a sublethal level of Cd (1-10 microM) did not affect the subsequent response of cells to inhibitory levels of Cd (10-100 microM). The azuki bean cells challenged to Cd did not contain phytochelatin (PC) peptides, unlike tomato (Lycopersicon esculentum) cells that have a substantial tolerance to Cd (>100 microM). Both of the cell suspensions contained a similar level of reduced glutathione (GSH) when grown in the absence of Cd. Externally applied GSH to azuki bean cells recovered neither Cd tolerance nor PC synthesis of the cells. Furthermore, enzyme assays in vitro revealed that the protein extracts of azuki bean cells had no activity converting GSH to PCs, unlike tomato. These results suggest that azuki bean cells are lacking in the PC synthase activity per se, hence being Cd hypersensitive. We concluded that the PC synthase has an important role in Cd tolerance of suspension-cultured cells. (+info)
Soluble GPI8 restores glycosylphosphatidylinositol anchoring in a trypanosome cell-free system depleted of lumenal endoplasmic reticulum proteins.
(21/414)
We previously established an in vitro assay for glycosylphosphatidylinositol (GPI) anchoring of proteins using trypanosome membranes. We now show that GPI anchoring is lost when the membranes are washed at high pH and restored to physiological pH prior to assay. We show that soluble component(s) of the endoplasmic reticulum that are lost in the high-pH wash are required for GPI anchoring. We reconstituted the high-pH extract with high-pH-treated membranes and demonstrated restoration of activity. Size fractionation of the high-pH extract indicated that the active component(s) was 30-50 kDa in size and was inactivated by iodoacetamide. Activity could also be restored by reconstituting the inactivated membranes with Escherichia coli-expressed, polyhistidine-tagged Leishmania mexicana GPI8 (GPI8-His; L. mexicana GPI8 is a soluble homologue of yeast and mammalian Gpi8p). No activity was seen when iodoacetamide-treated GPI8-His was used; however, GPI8-His could restore activity to iodoacetamide-treated membranes. Antibodies raised against L. mexicana GPI8 detected a protein of approx. 38 kDa in an immunoblot of the high-pH extract of trypanosome membranes. Our data indicate (1) that trypanosome GPI8 is a soluble lumenal protein, (2) that the interaction between GPI8 and other putative components of the transamidase may be dynamic, and (3) that GPI anchoring can be biochemically reconstituted using an isolated transamidase component. (+info)
Molecular epidemiology of penicillin-nonsusceptible Streptococcus pneumoniae among children in Greece.
(22/414)
A total of 145 penicillin-nonsusceptible Streptococcus pneumoniae strains were isolated from young carriers in Greece and analyzed by antibiotic susceptibility testing, serotyping, restriction fragment end labeling (RFEL), and penicillin-binding protein (PBP) genotyping. The serotypes 23A and 23F (54%), 19A and 19F (25%), 9V (5%), 15A, 15B, and 15C (4%), 6A and 6B (4%), and 21 (4%) were most prevalent in this collection. Fifty-three distinct RFEL types were identified. Sixteen different RFEL clusters, harboring 2 to 32 strains each, accounted for 82% of all strains. Eight of these genetic clusters representing 60% of the strains were previously identified in other countries. A predominant lineage of 66 strains (46%) harboring five RFEL types and the serotypes 19F and 23F was closely related to the pandemic clone Spain(23F)-1 (genetic relatedness of > or =85%). Another lineage, representing 11 strains, showed close genetic relatedness to the pandemic clone France(9V)-3. Another lineage of 8 serotype 21 strains was Greece specific since the RFEL types were not observed in an international collection of 193 genotypes from 16 different countries. Characterization of the PBP genes pbp1a, pbp2b, and pbp2x revealed 20 distinct PBP genotypes of which PBP type 1-1-1, initially observed in the pandemic clones 23F and 9V, was predominantly present in 11 RFEL types in this Greek collection of penicillin-nonsusceptible strains (55%). Sixteen PBP types covering 52 strains (36%) were Greece specific. This study underlines the strong contribution of penicillin-resistant international clones to the prevalence and spread of penicillin-nonsusceptible pneumococci among young children in Greece. (+info)
Inactivation of the srtA gene in Streptococcus gordonii inhibits cell wall anchoring of surface proteins and decreases in vitro and in vivo adhesion.
(23/414)
The srtA gene product, SrtA, has been shown to be required for cell wall anchoring of protein A as well as virulence in the pathogenic bacterium Staphylococcus aureus. There are five major mechanisms for displaying proteins at the surface of gram-positive bacteria (P. Cossart and R. Jonquieres, Proc. Natl. Acad. Sci. USA 97:5013-5015, 2000). However, since many of the known surface proteins of gram-positive bacteria are believed to be exported and anchored via the sortase pathway, it was of interest to determine if srtA plays a similar role in other gram-positive bacteria. To that end, the srtA gene in the human oral commensal organism Streptococcus gordonii was insertionally inactivated. The srtA mutant S. gordonii exhibited a marked reduction in quantity of a specific anchored surface protein. Furthermore, the srtA mutant had reduced binding to immobilized human fibronectin and had a decreased ability to colonize the oral mucosa of mice. Taken together, these results suggest that the activity of SrtA plays an important role in the biology of nonpathogenic as well as pathogenic gram-positive cocci. (+info)
A new pathway for heavy metal detoxification in animals. Phytochelatin synthase is required for cadmium tolerance in Caenorhabditis elegans.
(24/414)
Increasing emissions of heavy metals such as cadmium, mercury, and arsenic into the environment pose an acute problem for all organisms. Considerations of the biochemical basis of heavy metal detoxification in animals have focused exclusively on two classes of peptides, the thiol tripeptide, glutathione (GSH, gamma-Glu-Cys-Gly), and a diverse family of cysteine-rich low molecular weight proteins, the metallothioneins. Plants and some fungi, however, not only deploy GSH and metallothioneins for metal detoxification but also synthesize another class of heavy metal binding peptides termed phytochelatins (PCs) from GSH. Here we show that PC-mediated heavy metal detoxification is not restricted to plants and some fungi but extends to animals by demonstrating that the ce-pcs-1 gene of the nematode worm Caenorhabditis elegans encodes a functional PC synthase whose activity is critical for heavy metal tolerance in the intact organism. (+info)