Two DHH Subfamily 1 Proteins in Streptococcus pneumoniae Possess Cyclic Di-AMP Phosphodiesterase Activity and Affect Bacterial Growth and Virulence

Cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP) are signaling molecules that play important roles in bacterial biology and pathogenesis. However, these nucleotides have not been explored in Streptococcus pneumoniae, an important bacterial pathogen. In this study, we characterized the c-di-AMP-associated genes of S. pneumoniae. The results showed that SPD_1392 (DacA) is a diadenylate cyclase that converts ATP to c-di-AMP. Both SPD_2032 (Pde1) and SPD_1153 (Pde2), which belong to the DHH subfamily 1 proteins, displayed c-di-AMP phosphodiesterase activity. Pde1 cleaved c-di-AMP into phosphoadenylyl adenosine (pApA), whereas Pde2 directly hydrolyzed c-di-AMP into AMP. Additionally, Pde2, but not Pde1, degraded pApA into AMP. Our results also demonstrated that both Pde1 and Pde2 played roles in bacterial growth, resistance to UV treatment, and virulence in a mouse pneumonia model. These results indicate that c-di-AMP homeostasis is essential for pneumococcal biology and disease.     

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8–11 and SCR19–20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19–20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase.     

Protein Phosphorylation in Astrocytes Mediated by Protein Kinase C: Comparison with Phosphorylation by Cyclic AMP-Dependent Protein Kinase

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate-shaped, process-bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP-dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of 32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP-dependent mechanisms.     

Potassium Uptake Mediated by Trk1 Is Crucial for Candida glabrata Growth and Fitness

The maintenance of potassium homeostasis is crucial for all types of cells, including Candida glabrata. Three types of plasma-membrane systems mediating potassium influx with different transport mechanisms have been described in yeasts: the Trk1 uniporter, the Hak cation-proton symporter and the Acu ATPase. The C. glabrata genome contains only one gene encoding putative system for potassium uptake, the Trk1 uniporter. Therefore, its importance in maintaining adequate levels of intracellular potassium appears to be critical for C. glabrata cells. In this study, we first confirmed the potassium-uptake activity of the identified gene’s product by heterologous expression in a suitable S. cerevisiae mutant, further we generated a corresponding deletion mutant in C. glabrata and analysed its phenotype in detail. The obtained results show a pleiotropic effect on the cell physiology when CgTRK1 is deleted, affecting not only the ability of trk1Δ to grow at low potassium concentrations, but also the tolerance to toxic alkali-metal cations and cationic drugs, as well as the membrane potential and intracellular pH. Taken together, our results find the sole potassium uptake system in C. glabrata cells to be a promising target in the search for its specific inhibitors and in developing new antifungal drugs.     

Identification of a Protein That Inactivates the Competence-Stimulating Peptide of Streptococcus pneumoniae

Competence for genetic transformation of Streptococcus pneumoniae is a transient physiological property inducible by a competence-stimulating peptide (CSP). A 68-kDa CSP-inactivating protein was previously obtained following lithium chloride (LiCl) extraction. By the same protocol, a CSP-inactivating protein was purified and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry as an endopeptidase, PepO. Analysis of a pepO mutant provided no support for the hypothesis that PepO participates in competence regulation. To reconcile in vitro and in vivo data, we suggest that LiCl treatment results in the release of intracellular molecules, including PepO.     

Streptococcus pneumoniae Capsule Biosynthesis Protein CpsB Is a Novel Manganese-Dependent Phosphotyrosine-Protein Phosphatase

The first four genes of the capsule locus (cps) of Streptococcus pneumoniae (cpsA to cpsD) are common to most serotypes. We have previously determined that CpsD is an autophosphorylating protein-tyrosine kinase, demonstrated that CpsC is required for CpsD tyrosine-phosphorylation, and shown that CpsB is required for dephosphorylation of CpsD. In the present study we show that CpsB is a novel manganese-dependent phosphotyrosine-protein phosphatase that belongs to the PHP (polymerase and histidinol phosphatase) family of phosphoesterases. We also show that an S. pneumoniae strain with point mutations in cpsB, affecting one of the conserved motifs of CpsB, is unencapsulated and appears to be morphologically identical to a strain in which the cpsB gene had been deleted.     

Topology of Streptococcus pneumoniae CpsC,a Polysaccharide Copolymerase and Bacterial Protein Tyrosine Kinase Adaptor Protein

In Gram-positive bacteria, tyrosine kinases are split into two proteins, the cytoplasmic tyrosine kinase and a transmembrane adaptor protein. In Streptococcus pneumoniae, this transmembrane adaptor is CpsC, with the C terminus of CpsC critical for interaction and subsequent tyrosine kinase activity of CpsD. Topology predictions suggest that CpsC has two transmembrane domains, with the N and C termini present in the cytoplasm. In order to investigate CpsC topology, we used a chromosomal hemagglutinin (HA)-tagged Cps2C protein in S. pneumoniae strain D39. Incubation of both protoplasts and membranes with carboxypeptidase B (CP-B) resulted in complete degradation of HA-Cps2C in all cases, indicating that the C terminus of Cps2C was likely extracytoplasmic and hence that the protein''s topology was not as predicted. Similar results were seen with membranes from S. pneumoniae strain TIGR4, indicating that Cps4C also showed similar topology. A chromosomally encoded fusion of HA-Cps2C and Cps2D was not degraded by CP-B, suggesting that the fusion fixed the C terminus within the cytoplasm. However, capsule synthesis was unaltered by this fusion. Detection of the CpsC C terminus by flow cytometry indicated that it was extracytoplasmic in approximately 30% of cells. Interestingly, a mutant in the protein tyrosine phosphatase CpsB had a significantly greater proportion of positive cells, although this effect was independent of its phosphatase activity. Our data indicate that CpsC possesses a varied topology, with the C terminus flipping across the cytoplasmic membrane, where it interacts with CpsD in order to regulate tyrosine kinase activity.     

Binding of Complement Inhibitor C4b-binding Protein to a Highly Virulent Streptococcus pyogenes M1 Strain Is Mediated by Protein H and Enhances Adhesion to and Invasion of Endothelial Cells

Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonization with complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of ¹²⁵I-C4b. Furthermore, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18-amino acid-long peptide comprising residues 92–109, which specifically bound C4BP. Biacore was used to determine K_D = 6 × 10⁻⁷ m between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonization but also for invasion of endothelial cells by S. pyogenes.     

Structure of the Streptococcus pneumoniae Surface Protein and Adhesin PfbA

PfbA (plasmin- and fibronectin-binding protein A) is an extracellular Streptococcus pneumoniae cell-wall attached surface protein that binds to fibronectin, plasmin, and plasminogen. Here we present a structural analysis of the surface exposed domains of PfbA using a combined approach of X-ray crystallography and small-angle X-ray scattering (SAXS). The crystal structure of the PfbA core domain, here called PfbAβ, determined to 2.28 Å resolution revealed an elongated 12-stranded parallel β-helix fold, which structure-based comparisons reveal is most similar to proteins with carbohydrate modifying activity. A notable feature of the PfbAβ is an extensive cleft on one face of the protein with electrochemical and spatial features that are analogous to structurally similar carbohydrate-active enzymes utilizing this feature for substrate accommodation. Though this cleft displays a combination of basic amino acid residues and solvent exposed aromatic amino acids that are distinct features for recognition of carbohydrates, no obvious arrangement of amino acid side chains that would constitute catalytic machinery is evident. The pseudo-atomic SAXS model of a larger fragment of PfbA suggests that it has a relatively well-ordered structure with the N-terminal and core domains of PfbA adopting an extend organization and reveals a novel structural class of surface exposed pneumococcal matrix molecule adhesins.     

Kinetics of Ligand-Receptor Interaction Reveals an Induced-Fit Mode of Binding in a Cyclic Nucleotide-Activated Protein

Many receptors and ion channels are activated by ligands. One key question concerns the binding mechanism. Does the ligand induce conformational changes in the protein via the induced-fit mechanism? Or does the protein preexist as an ensemble of conformers and the ligand selects the most complementary one, via the conformational selection mechanism? Here, we study ligand binding of a tetrameric cyclic nucleotide-gated channel from Mesorhizobium loti and of its monomeric binding domain (CNBD) using rapid mixing, mutagenesis, and structure-based computational biology. Association rate constants of ∼10⁷ M⁻¹ s⁻¹ are compatible with diffusion-limited binding. Ligand binding to the full-length CNG channel and the isolated CNBD differ, revealing allosteric control of the CNBD by the effector domain. Finally, mutagenesis of allosteric residues affects only the dissociation rate constant, suggesting that binding follows the induced-fit mechanism. This study illustrates the strength of combining mutational, kinetic, and computational approaches to unravel important mechanistic features of ligand binding.     

Ammonium and Potassium Uptake by Citrus Roots

Multiphasic kinetics were indicated for uptake of ammonium and potassium by intact cirtrus seedlings as measured by a continuous flow technique. Uptake at low concentration, below 10^?3M, was biphasis. The patterns for ammonium uptake by 60-day-old and 180-day-old seedlings were similar, but the rates of uptake per g dry weight of root were greater for the younger plants.     

Studies on the Cellular Uptake of Retinol Binding Protein and Retinol

The uptake and release of (125)I-RBP and of holoRBP labeled with [(3)H]retinol ((3)H-ROH) were studied in two cell lines which synthesize and secrete RBP, the HepG2 hepatocarcinoma cell line and the Caki-1 kidney adenocarcinoma cell line, and in HeLa cells that do not express the endogenous RBP gene. In all three cell lines a part of endocytosed (125)I-RBP is recycled to the extracellular medium and part is degraded. Nonspecific endocytosis of (125)I-RBP was estimated to be approximately 10% of total endocytosed (125)I-RBP. In HepG2 cells the (3)H-ROH from the [(3)H]retinol-RBP complex ((3)H-ROH-RBP) is recycled bound to RBP into serum-free chase medium. This (3)H-ROH recycling is blocked in HepG2 cells by cyclohexymide and by brefeldin A, an inhibitor of protein export from the main secretory route, and is absent in HeLa cells, which do not synthesize RBP. These data suggest that at least part of retinol taken up from exogenous holoRBP is delivered to newly synthesized RBP. (3)H-ROH recycled by HeLa cells is bound to serum albumin, as is a portion of that recycled by HepG2 cells. Transfer of (3)H-ROH from RBP to serum albumin does not occur in the absence of cells. We conclude that RBP is endocytosed through a specific pathway and that the RBP-associated retinol is transferred to newly synthesized RBP or to serum albumin.     

Blood group antigen recognition by a Streptococcus pneumoniae virulence factor

The Streptococcus pneumoniae fucose utilization operon includes a gene encoding a virulence factor that belongs to family 98 in the glycoside hydrolase classification. This protein contains a C-terminal triplet of fucose binding modules that have significant amino acid sequence identity with the Anguilla anguilla fucolectin. Functional studies of these fucose binding modules reveal binding to fucosylated oligosaccharides and suggest the importance of multivalent binding. The high resolution crystal structures of ligand bound forms of one fucose binding module uncovers the molecular basis of fucose, ABH blood group antigen, and Lewisy antigen binding. These studies are extended by fluorescence microscopy to show specific binding to mouse lung tissue. These modules define a new family of carbohydrate binding modules now classified as family 47.     

PrPc介导的细胞信号转导

朊病毒病的发生是由于细胞正常朊蛋白PrPc转变成了异常构象的PrPc形式。PrPc的生理学功能目前尚不完全明确,可能与铜离子代谢、脂质摄取以及细胞信号传递有关。PrPc可以与小窝蛋白相互作用而活化Fyn非受体酪氨酸激酶从而引起下游信号通路的转导;可以作为受体与PrPc键合多肽结合后激活cAMP／PKA信号通路;以及引起细胞内钙离子浓度变化而活化信号通路。