Anchoring of surface proteins to the cell wall of Staphylococcus aureus. III. Lipid II is an in vivo peptidoglycan substrate for sortase-catalyzed surface protein anchoring

Surface proteins of Staphylococcus aureus are anchored to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins are first synthesized in the bacterial cytoplasm and then transported across the cytoplasmic membrane. Cleavage of the N-terminal signal peptide of the cytoplasmic surface protein P1 precursor generates the extracellular P2 species, which is the substrate for the cell wall anchoring reaction. Sortase, a membrane-anchored transpeptidase, cleaves P2 between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine and the amino group of cell wall cross-bridges. We have used metabolic labeling of staphylococcal cultures with [(32)P]phosphoric acid to reveal a P3 intermediate. The (32)P-label of immunoprecipitated surface protein is removed by treatment with lysostaphin, a glycyl-glycine endopeptidase that separates the cell wall anchor structure. Furthermore, the appearance of P3 is prevented in the absence of sortase or by the inhibition of cell wall synthesis. (32)P-Labeled cell wall anchor species bind to nisin, an antibiotic that is known to form a complex with lipid II. Thus, it appears that the P3 intermediate represents surface protein linked to the lipid II peptidoglycan precursor. The data support a model whereby lipid II-linked polypeptides are incorporated into the growing peptidoglycan via the transpeptidation and transglycosylation reactions of cell wall synthesis, generating mature cell wall-linked surface protein.     

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Ion conduction across the cellular membrane requires the simultaneous opening of activation and inactivation gates of the K⁺ channel pore. The bacterial KcsA channel has served as a powerful system for dissecting the structural changes that are related to four major functional states associated with K⁺ gating. Yet, the direct observation of the full gating cycle of KcsA has remained structurally elusive, and crystal structures mimicking these gating events require mutations in or stabilization of functionally relevant channel segments. Here, we found that changes in lipid composition strongly increased the KcsA open probability. This enabled us to probe all four major gating states in native-like membranes by combining electrophysiological and solid-state NMR experiments. In contrast to previous crystallographic views, we found that the selectivity filter and turret region, coupled to the surrounding bilayer, were actively involved in channel gating. The increase in overall steady-state open probability was accompanied by a reduction in activation-gate opening, underscoring the important role of the surrounding lipid bilayer in the delicate conformational coupling of the inactivation and activation gates.     

Pharmacokinetics,Brain Delivery,and Efficacy in Brain Tumor-Bearing Mice of Glutathione Pegylated Liposomal Doxorubicin (2B3-101)

Brain cancer is a devastating disease affecting many people worldwide. Effective treatment with chemotherapeutics is limited due to the presence of the blood-brain barrier (BBB) that tightly regulates the diffusion of endogenous molecules but also xenobiotics. Glutathione pegylated liposomal doxorubicin (2B3-101) is being developed as a new treatment option for patients with brain cancer. It is based on already marketed pegylated liposomal doxorubicin (Doxil®/Caelyx®), with an additional glutathione coating that safely enhances drug delivery across the BBB.Uptake of 2B3-101 by human brain capillary endothelial cells in vitro was time-, concentration- and temperature-dependent, while pegylated liposomal doxorubicin mainly remained bound to the cells. In vivo, 2B3-101 and pegylated liposomal doxorubicin had a comparable plasma exposure in mice, yet brain retention 4 days after administration was higher for 2B3-101. 2B3-101 was overall well tolerated by athymic FVB mice with experimental human glioblastoma (luciferase transfected U87MG). In 2 independent experiments a strong inhibition of brain tumor growth was observed for 2B3-101 as measured by bioluminescence intensity. The effect of weekly administration of 5 mg/kg 2B3-101 was more pronounced compared to pegylated liposomal doxorubicin (p<0.05) and saline (p<0.01). Two out of 9 animals receiving 2B3-101 showed a complete tumor regression. Twice-weekly injections of 5 mg/kg 2B3-101 again had a significant effect in inhibiting brain tumor growth (p<0.001) compared to pegylated liposomal doxorubicin and saline, and a complete regression was observed in 1 animal treated with 2B3-101. In addition, twice-weekly dosing of 2B3-101 significantly increased the median survival time by 38.5% (p<0.001) and 16.1% (p<0.05) compared to saline and pegylated liposomal doxorubicin, respectively.Overall, these data demonstrate that glutathione pegylated liposomal doxorubicin enhances the effective delivery of doxorubicin to brain tumors and could become a promising new therapeutic option for the treatment of brain malignancies.     

BslA,the S‐layer adhesin of B. anthracis,is a virulence factor for anthrax pathogenesis

Microbial pathogens use adhesive surface proteins to bind to and interact with host tissues, events that are universal for the pathogenesis of infectious diseases. A surface adhesin of Bacillus anthracis, the causative agent of anthrax, required to mediate these steps has not been discovered. Previous work identified BslA, an S‐layer protein, to be necessary and sufficient for adhesion of the anthrax vaccine strain, Bacillus anthracis Sterne, to host cells. Here we asked whether encapsulated bacilli require BslA for anthrax pathogenesis in guinea pigs. Compared with the highly virulent parent strain B. anthracis Ames, bslA mutants displayed a dramatic increase in the lethal dose and in mean time‐to‐death. Whereas all tissues of animals infected with B. anthracis Ames contained high numbers of bacilli, only few vegetative forms could be recovered from internal organs of animals infected with the bslA mutant. Surface display of BslA occurred at the poles of encapsulated bacilli and enabled the binding of vegetative forms to host cells. Together these results suggest that BslA functions as the surface adhesin of the anthrax pathogen B. anthracis strain Ames.     

Contitions for optimal growth of a PSTV-infected potato cell suspension and detection of viroid-complementary longer-than-unit-length RNA in these cells

A suspension culture from potato spindle tuber viroid (PSTV)-infected cells of the wild type potato (Solanum demissum) has been established, which is a suitable model system for studying PSTV replicationin vivo. The conditions for rapid growth of these cells and for permanent extensive viroid biosynthesis within them are described. Biosynthesis of PSTV in the potato cells was demonstrated by³²P-incorporation into nucleic acids and their subsequent electrophoretic analysis on polyacrylamide gels. Under optimum culture conditions the amount of³²P-orthophosphate incorporation into PSTV reached 10% of that incorporated into the 2 M LiCl-soluble cellular RNA. (+)PSTV and its complementary form, i.e. (?)PSTV were identified after their electrophoretic separation on polyacrylamide and agarose gels by molecular hybridization. This analysis revealed the presence of six high molecular weight(?)PSTV species, which are possibly multimers of the unit length(+)PSTV molecule consisting of 359 nucleotides.     

The multiple lives of NMD factors: balancing roles in gene and genome regulation

Nonsense-mediated mRNA decay (NMD) largely functions to ensure the quality of gene expression. However, NMD is also crucial to regulating appropriate expression levels for certain genes and for maintaining genome stability. Furthermore, just as NMD serves cells in multiple ways, so do its constituent proteins. Recent studies have clarified that UPF and SMG proteins, which were originally discovered to function in NMD, also have roles in other pathways, including specialized pathways of mRNA decay, DNA synthesis and cell-cycle progression, and the maintenance of telomeres. These findings suggest a delicate balance of metabolic events - some not obviously related to NMD - that can be influenced by the cellular abundance, location and activity of NMD factors and their binding partners.     

Carotenoid cleavage products modify respiratory burst and induce apoptosis of human neutrophils

Carotenoid supplementation in the treatment of diseases associated with oxidative stress has been recently questioned because of the cell damage and the increased risk of lung cancer in male smokers. Because of the complex role of neutrophils in lung diseases, we investigated whether carotenoid derivatives could affect respiratory burst and apoptosis of human neutrophils purified from peripheral blood. Stimulation of superoxide production was induced by nanomolar and micromolar concentrations of carotenoid cleavage products with aliphatic chains of different length, but not by carotenoids lacking the carbonyl moiety. The stimulatory effect of carotenoid cleavage products was observed in cells activated by phorbol myristate acetate (PMA), while a slight inhibition of superoxide production was noticed with cells activated by the chemotactic tripeptide N-formyl-Met-Leu-Phe (f-MLP). At higher concentrations, carotenoid cleavage products inhibited superoxide production in the presence of both PMA and f-MLP. In the presence of 20 microM carotenoid cleavage products, inhibition of superoxide production was accompanied by DNA fragmentation and increased level of intracellular caspase-3 activity.     

COP9 signalosome: a provider of DNA building blocks

In fission yeast, the COP9 signalosome is required to activate ribonucleotide reductase for DNA synthesis. This is mediated via the ubiquitin ligase Pcu4, activation of which leads to degradation of the scaffold protein Spd1, which anchors the small ribonucleotide reductase subunit in the nucleus away from the large subunit in the cytoplasm.