In Trans Complementation of Lethal Factor Reveal Roles in Colonization and Dissemination in a Murine Mouse Model

Lethal factor (LF) is a component of the B. anthracis exotoxin and critical for pathogenesis. The roles of LF in early anthrax pathogenesis, such as colonization and dissemination from the initial site of infection, are poorly understood. In mice models of infection, LF-deficient strains either have altered dissemination patterns or do not colonize, precluding analysis of the role of LF in colonization and dissemination from the portal of entry. Previous reports indicate rabbit and guinea pig models infected with LF-deficient strains have decreased virulence, yet the inability to use bioluminescent imaging techniques to track B. anthracis growth and dissemination in these hosts makes analysis of early pathogenesis challenging. In this study, the roles of LF early in infection were analyzed using bioluminescent signature tagged libraries of B. anthracis with varying ratios of LF-producing and LF-deficient clones. Populations where all clones produced LF and populations where only 40% of clones produce LF were equally virulent. The 40% LF-producing clones trans complimented the LF mutants and permitted them to colonize and disseminate. Decreases of the LF producing strains to 10% or 0.3% of the population led to increased host survival and decreased trans complementation of the LF mutants. A library with 10% LF producing clones could replicate and disseminate, but fewer clones disseminated and the mutant clones were less competitive than wild type. The inoculum with 0.3% LF producing clones could not colonize the host. This strongly suggests that between 10% and 0.3% of the population must produce LF in order to colonize. In total, these findings suggest that a threshold of LF must be produced in order for colonization and dissemination to occur in vivo. These observations suggest that LF has a major role in the early stages of colonization and dissemination.     

Identification of Functionally Important Residues of the Rat P2X4 Receptor by Alanine Scanning Mutagenesis of the Dorsal Fin and Left Flipper Domains

Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. Here, we focused on the role of these domains in receptor activation, responsiveness to orthosteric ATP analogue agonists, and desensitization. Alanine scanning mutagenesis of the R203-L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor showed that ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists 2-(methylthio)adenosine 5′-triphosphate, adenosine 5′-(γ-thio)triphosphate, 2′(3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate, and α,β-methyleneadenosine 5′-triphosphate. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. Among these mutants, D280A, T281A, R282A, H286A, G291A, and Y292A also exhibited increased time-constant of the desensitizing current response. These experiments, together with homology modeling, indicate that residues located in the upper part of the dorsal fin and left flipper domains, relative to distance from the channel pore, contribute to the organization of the ATP binding pocket and to the initiation of signal transmission towards residues in the lower part of both domains. The R203 and N204 residues, deeply buried in the protein, may integrate the output signal from these two domains towards the gate. In addition, the left flipper residues predominantly account for the control of transition of channels from an open to a desensitized state.     

Fluorescent Nanocrystals Reveal Regulated Portals of Entry into and Between the Cells of Hydra

Initially viewed as innovative carriers for biomedical applications, with unique photophysical properties and great versatility to be decorated at their surface with suitable molecules, nanoparticles can also play active roles in mediating biological effects, suggesting the need to deeply investigate the mechanisms underlying cell-nanoparticle interaction and to identify the molecular players. Here we show that the cell uptake of fluorescent CdSe/CdS quantum rods (QRs) by Hydra vulgaris, a simple model organism at the base of metazoan evolution, can be tuned by modifying nanoparticle surface charge. At acidic pH, amino-PEG coated QRs, showing positive surface charge, are actively internalized by tentacle and body ectodermal cells, while negatively charged nanoparticles are not uptaken. In order to identify the molecular factors underlying QR uptake at acidic pH, we provide functional evidence of annexins involvement and explain the QR uptake as the combined result of QR positive charge and annexin membrane insertion. Moreover, tracking QR labelled cells during development and regeneration allowed us to uncover novel intercellular trafficking and cell dynamics underlying the remarkable plasticity of this ancient organism.     

Biochemical Characterization of an Isolated and Functionally Reconstituted γ-Aminobutyric Acid/Benzodiazepine Receptor

We have solubilized, affinity-purified, and functionally reconstituted the gamma-aminobutyric acid/benzodiazepine (GABA/BDZ) receptor from rat brain into natural brain lipid liposomes. The detergent, 3-[(3-cholamidopropyl)-dimethylammonio] 1-propanesulphonate, was employed for the isolation of the receptor in the presence of a whole rat brain lipid extract supplemented with cholesteryl hemisuccinate. The soluble and reconstituted protein showed a homogeneous [3H]flunitrazepam binding population and the allosteric modulation of this binding site by GABA, by the pyrazolopyridine, cartazolate, and by the depressant barbiturate, pentobarbital. The purified GABA/BDZ receptor when incorporated into liposomes has been visualized by electron microscopy and reveals rosette structures, 8-9 nm in diameter, which appear to have a central pore. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the reconstituted GABA/BDZ receptor reveals three major protein bands of 41, 52-56, and 59-62 kDa, the latter two of which appears as doublets. Functional receptor reconstitution is demonstrated by the measurement of GABA-stimulated 36Cl- flux into the purified GABA/BDZ receptor incorporated liposomes and its modulation by the BDZs, barbiturates, and pyrazolopyridines.     

Regulation of Epidermal Growth Factor Receptor Signaling by Endocytosis and Intracellular Trafficking

Ligand activation of the epidermal growth factor receptor (EGFR) leads to its rapid internalization and eventual delivery to lysosomes. This process is thought to be a mechanism to attenuate signaling, but signals could potentially be generated after endocytosis. To directly evaluate EGFR signaling during receptor trafficking, we developed a technique to rapidly and selectively isolate internalized EGFR and associated molecules with the use of reversibly biotinylated anti-EGFR antibodies. In addition, we developed antibodies specific to tyrosine-phosphorylated EGFR. With the use of a combination of fluorescence imaging and affinity precipitation approaches, we evaluated the state of EGFR activation and substrate association during trafficking in epithelial cells. We found that after internalization, EGFR remained active in the early endosomes. However, receptors were inactivated before degradation, apparently due to ligand removal from endosomes. Adapter molecules, such as Shc, were associated with EGFR both at the cell surface and within endosomes. Some molecules, such as Grb2, were primarily found associated with surface EGFR, whereas others, such as Eps8, were found only with intracellular receptors. During the inactivation phase, c-Cbl became EGFR associated, consistent with its postulated role in receptor attenuation. We conclude that the association of the EGFR with different proteins is compartment specific. In addition, ligand loss is the proximal cause of EGFR inactivation. Thus, regulated trafficking could potentially influence the pattern as well as the duration of signal transduction.     

Integrated Syntenic and Phylogenomic Analyses Reveal an Ancient Genome Duplication in Monocots

Unraveling widespread polyploidy events throughout plant evolution is a necessity for inferring the impacts of whole-genome duplication (WGD) on speciation, functional innovations, and to guide identification of true orthologs in divergent taxa. Here, we employed an integrated syntenic and phylogenomic analyses to reveal an ancient WGD that shaped the genomes of all commelinid monocots, including grasses, bromeliads, bananas (Musa acuminata), ginger, palms, and other plants of fundamental, agricultural, and/or horticultural interest. First, comprehensive phylogenomic analyses revealed 1421 putative gene families that retained ancient duplication shared by Musa (Zingiberales) and grass (Poales) genomes, indicating an ancient WGD in monocots. Intergenomic synteny blocks of Musa and Oryza were investigated, and 30 blocks were shown to be duplicated before Musa-Oryza divergence an estimated 120 to 150 million years ago. Synteny comparisons of four monocot (rice [Oryza sativa], sorghum [Sorghum bicolor], banana, and oil palm [Elaeis guineensis]) and two eudicot (grape [Vitis vinifera] and sacred lotus [Nelumbo nucifera]) genomes also support this additional WGD in monocots, herein called Tau (τ). Integrating synteny and phylogenomic comparisons achieves better resolution of ancient polyploidy events than either approach individually, a principle that is exemplified in the disambiguation of a WGD series of rho (ρ)-sigma (σ)-tau (τ) in the grass lineages that echoes the alpha (α)-beta (β)-gamma (γ) series previously revealed in the Arabidopsis thaliana lineage.     

人血管内皮生长因子受体Flt-1胞外配体结合域的筛选及其结构分析

 血管内皮生长因子受体 Flt- 1胞外区具有 7个免疫球蛋白样的袢 (Ig- like loop) ,氨基端 3个loop负责与其配体 VEGF的结合 .为了寻求能与配体结合的更小的 Flt- 1片段 ,在对 Flt- 1胞外前3个 loop氨基酸组成和晶体结构分析的基础上 ,应用酵母双杂交系统对 Flt- 1的配体结合域进行筛选 .利用 PCR技术 ,从人胎盘 c DNA文库扩增出 4个截短的 Flt- 1 c DNA,分别含胞外第 2 ,1 - 2 ,2 - 3和 1 - 3个 loop,构建酵母双杂交系统融合表达质粒 ,并将 p GBT9/h VEGF165与 p GAD42 4 /Flt- 1 s两两配对转化酵母菌 SFY52 6,采用滤纸法和液体培养定量检测法对阳性克隆进行β-半乳糖苷酶活性分析 .结果显示 ,Flt- 1胞外 loop 2 - 3与 loop 1 - 3的配体结合能力相差不大 ,loop 1 - 2的结合力较弱 ,单独第 2个 loop无配体结合能力 .     

lac Repressor Is an Antivirulence Factor of Salmonella enterica: Its Role in the Evolution of Virulence in Salmonella

The genus Salmonella includes many pathogens of great medical and veterinary importance. Bacteria belonging to this genus are very closely related to those belonging to the genus Escherichia. lacZYA operon and lacI are present in Escherichia coli, but not in Salmonella enterica. It has been proposed that Salmonella has lost lacZYA operon and lacI during evolution. In this study, we have investigated the physiological and evolutionary significance of the absence of lacI in Salmonella enterica. Using murine model of typhoid fever, we show that the expression of LacI causes a remarkable reduction in the virulence of Salmonella enterica. LacI also suppresses the ability of Salmonella enterica to proliferate inside murine macrophages. Microarray analysis revealed that LacI interferes with the expression of virulence genes of Salmonella pathogenicity island 2. This effect was confirmed by RT-PCR and Western blot analysis. Interestingly, we found that SBG0326 of Salmonella bongori is homologous to lacI of Escherichia coli. Salmonella bongori is the only other species of the genus Salmonella and it lacks the virulence genes of Salmonella pathogenicity island 2. Overall, our results demonstrate that LacI is an antivirulence factor of Salmonella enterica and suggest that absence of lacI has facilitated the acquisition of virulence genes of Salmonella pathogenicity island 2 in Salmonella enterica making it a successful systemic pathogen.     

Complete Cytolysis and Neonatal Lethality in Keratin 5 Knockout Mice Reveal Its Fundamental Role in Skin Integrity and in Epidermolysis Bullosa Simplex

In human patients, a wide range of mutations in keratin (K) 5 or K14 lead to the blistering skin disorder epidermolysis bullosa simplex. Given that K14 deficiency does not lead to the ablation of a basal cell cytoskeleton because of a compensatory role of K15, we have investigated the requirement for the keratin cytoskeleton in basal cells by inactivating the K5 gene in mice. We report that the K5(-/-) mice die shortly after birth, lack keratin filaments in the basal epidermis, and are more severely affected than K14(-/-) mice. In contrast to the K14(-/-) mice, we detected a strong induction of the wound-healing keratin K6 in the suprabasal epidermis of cytolyzed areas of postnatal K5(-/-) mice. In addition, K5 and K14 mice differed with respect to tongue lesions. Moreover, we show that in the absence of K5 and other type II keratins, residual K14 and K15 aggregated along hemidesmosomes, demonstrating that individual keratins without a partner are stable in vivo. Our data indicate that K5 may be the natural partner of K15 and K17. We suggest that K5 null mutations may be lethal in human epidermolysis bullosa simplex patients.     

EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance.     

Crystal Structures of YkuI and Its Complex with Second Messenger Cyclic Di-GMP Suggest Catalytic Mechanism of Phosphodiester Bond Cleavage by EAL Domains

Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that is involved in the regulation of cell surface-associated traits and the persistence of infections. Omnipresent GGDEF and EAL domains, which occur in various combinations with regulatory domains, catalyze c-di-GMP synthesis and degradation, respectively. The crystal structure of full-length YkuI from Bacillus subtilis, composed of an EAL domain and a C-terminal PAS-like domain, has been determined in its native form and in complex with c-di-GMP and Ca²⁺. The EAL domain exhibits a triose-phosphate isomerase-barrel fold with one antiparallel β-strand. The complex with c-di-GMP-Ca²⁺ defines the active site of the putative phosphodiesterase located at the C-terminal end of the β-barrel. The EAL motif is part of the active site with Glu-33 of the motif being involved in cation coordination. The structure of the complex allows the proposal of a phosphodiesterase mechanism, in which the divalent cation and the general base Glu-209 activate a catalytic water molecule for nucleophilic in-line attack on the phosphorus. The C-terminal domain closely resembles the PAS-fold. Its pocket-like structure could accommodate a yet unknown ligand. YkuI forms a tight dimer via EAL-EAL and trans EAL-PAS-like domain association. The possible regulatory significance of the EAL-EAL interface and a mechanism for signal transduction between sensory and catalytic domains of c-di-GMP-specific phosphodiesterases are discussed.The dinucleotide cyclic di-GMP (c-di-GMP) was discovered about 20 years ago when it was found to regulate the activity of cellulase synthase in Acetobacter xylinum (1). However, its prominent role as a global second messenger has been realized only upon the recent recognition of the omnipresence of genes coding for domains that catalyze c-di-GMP biosynthesis and degradation in eubacteria (2). GGDEF domains catalyze the condensation of two GTP molecules to the cyclic 2-fold symmetric dinucleotide (diguanylate cyclase activity (3-6)), whereas EAL domains are involved in its degradation to yield the linear dinucleotide pGpG (phosphodiesterase (PDE)⁴ A activity) (3, 7-9). Recently, also HD-GYP domains have been implicated in c-di-GMP-specific PDE activity (10). All the domains have been named according to their sequence signature motifs. They are typically found in combinations with various other, mostly sensory or regulatory, domains. It is thought that the balance between antagonistic diguanylate cyclase and PDE-A activities determines the cellular level of c-di-GMP and, thus, affects a variety of physiological processes in bacteria.It has been shown that, in general, c-di-GMP regulates cell surface-associated traits and community behavior such as biofilm formation (for reviews see Refs. 11-12), and its relevance to the virulence of pathogenic bacteria has been demonstrated (11, 13, 14). In particular, the dinucleotide has been proposed to orchestrate the switch between acute and persistent phase of infection.The best characterized diguanylate cyclase is PleD from Caulobacter crescentus with a Rec-Rec-GGDEF domain architecture (Rec indicates response regulator receiver domain). The structure of its GGDEF domain revealed a single GTP-binding site and suggested that dimerization is the prerequisite for enzymatic activity (4). This has been corroborated recently by crystallography showing directly that modification of the first Rec domain, mimicking phosphorylation by the cognate kinase, induces formation of a tightly packed dimer (15). Additionally, an upper limit of c-di-GMP levels in the cell seems to be ensured by potent allosteric product inhibition of the PleD cyclase (4, 15, 16). Recently, the crystal structure of another diguanylate cyclase, WspR from Pseudomonas aeruginosa with a Rec-GGDEF domain architecture, has been determined (17), which showed a tetrameric quaternary structure and active and feedback inhibition sites that are very similar to those in PleD.For EAL domains, it has been demonstrated that genetic knock-out results in phenotypes that are in line with the paradigm that an elevated cellular c-di-GMP concentration corresponds to a sessile and a low concentration to a motile bacterial life style (13, 18, 19). Only recently, EAL-mediated PDE-A activity has been measured in vitro (7-9, 20-22).The Bacillus subtilis YkuI protein was targeted for structure determination by the Midwest Center for Structural Genomics as a member of the large sequence family that contains EAL (Pfam number PF00563) domains. Here we report the crystal structure of YkuI showing the fold of the N-terminal EAL domain and the C-terminal PAS-like domain. Co-crystallization with c-di-GMP revealed the substrate binding mode and allows the proposal of a catalytic mechanism. The PAS-like domain most probably has regulatory function, which is discussed. Recently, another EAL structure has been deposited in the Protein Data Bank by the Midwest Center for Structural Genomics, the EAL domain of a GGDEF-EAL protein from Thiobacillus denitrificans (tdEAL; PDB code 2r6o). Comparison of the two structures suggests a possible regulatory mechanism.     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase : IV. Fusicoccin Induces the Association between the Plasma Membrane H+-ATPase and the Fusicoccin Receptor

Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H⁺-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H⁺-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H⁺-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H⁺-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H⁺-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H⁺-ATPase antiserum and with an anti-14–3-3 antiserum indicated an FC-induced association of FCBP with the PM H⁺-ATPase. Analysis of the activation state of PM H⁺-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.     

In Vitro Mutational and Bioinformatics Analysis of the M71 Odorant Receptor and Its Superfamily

We performed an extensive mutational analysis of the canonical mouse odorant receptor (OR) M71 to determine the properties of ORs that inhibit plasma membrane trafficking in heterologous expression systems. We employed the use of the M71::GFP fusion protein to directly assess plasma membrane localization and functionality of M71 in heterologous cells in vitro or in olfactory sensory neurons (OSNs) in vivo. OSN expression of M71::GFP show only small differences in activity compared to untagged M71. However, M71::GFP could not traffic to the plasma membrane even in the presence of proposed accessory proteins RTP1S or mβ2AR. To ask if ORs contain an internal “kill sequence”, we mutated ~15 of the most highly conserved OR specific amino acids not found amongst the trafficking non-OR GPCR superfamily; none of these mutants rescued trafficking. Addition of various amino terminal signal sequences or different glycosylation motifs all failed to produce trafficking. The addition of the amino and carboxy terminal domains of mβ2AR or the mutation Y289A in the highly conserved GPCR motif NPxxY does not rescue plasma membrane trafficking. The failure of targeted mutagenesis on rescuing plasma membrane localization in heterologous cells suggests that OR trafficking deficits may not be attributable to conserved collinear motifs, but rather the overall amino acid composition of the OR family. Thus, we performed an in silico analysis comparing the OR and other amine receptor superfamilies. We find that ORs contain fewer charged residues and more hydrophobic residues distributed throughout the protein and a conserved overall amino acid composition. From our analysis, we surmise that it may be difficult to traffic ORs at high levels to the cell surface in vitro, without making significant amino acid modifications. Finally, we observed specific increases in methionine and histidine residues as well as a marked decrease in tryptophan residues, suggesting that these changes provide ORs with special characteristics needed for them to function in olfactory neurons.