Detection and quantitative analysis of two independent binding modes of a small ligand responsible for DC-SIGN clustering.

DC-SIGN (dendritic cell-specific ICAM-3 grabbing non-integrin) is a C-type lectin receptor (CLR) present, mainly in dendritic cells (DCs), as one of the major pattern recognition receptors (PRRs). This receptor has a relevant role in viral infection processes. Recent approaches aiming to block DC-SIGN have been presented as attractive anti-HIV strategies. DC-SIGN binds mannose or fucose-containing carbohydrates from viral proteins such as the HIV envelope glycoprotein gp120. We have previously demonstrated that multivalent dendrons bearing multiple copies of glycomimetic ligands were able to inhibit DC-SIGN-dependent HIV infection in cervical explant models. Optimization of glycomimetic ligands requires detailed characterization and analysis of their binding modes because they notably influence binding affinities. In a previous study we characterized the binding mode of DC-SIGN with ligand 1, which shows a single binding mode as demonstrated by NMR and X-ray crystallography. In this work we report the binding studies of DC-SIGN with pseudotrisaccharide 2, which has a larger affinity. Their binding was analysed by TR-NOESY and STD NMR experiments, combined with the CORCEMA-ST protocol and molecular modelling. These studies demonstrate that in solution the complex cannot be explained by a single binding mode. We describe the ensemble of ligand bound modes that best fit the experimental data and explain the higher inhibition values found for ligand 2.

Periumbilical parasitic thumbprint purpura: strongyloides hyperinfection syndrome acquired from a cadaveric renal transplant.

Periumbilical parasitic thumbprint purpura may be a presenting sign of hyperinfection strongyloidiasis in the immunocompromised host. We report a case of fatal hyperinfection strongyloidiasis acquired from a cadaveric renal allograft, diagnosed by the pathognomonic periumbilical thumbprint purpuric eruption, confirmed by skin biopsy and laboratory testing.

Phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae contributes to persistence in the respiratory tract and sensitivity to serum killing mediated by C-reactive protein.

Haemophilus influenzae undergoes phase variation in expression of the phosphorylcholine (ChoP) epitope, a structure present on several invasive pathogens residing in the human respiratory tract. In this study, structural analysis comparing organisms with and without this epitope confirmed that variants differ in the presence of ChoP on the cell surface-exposed outer core of the lipopolysaccharide. During nasopharyngeal carriage in infant rats, there was a gradual selection for H. influenzae variants that express ChoP. In addition, genotypic analysis of the molecular switch that controls phase variation predicted that the ChoP+ phenotype was predominant in H. influenzae in human respiratory tract secretions. However, ChoP+ variants of nontypable H. influenzae were more sensitive to the bactericidal activity of human serum unrelated to the presence of naturally acquired antibody to ChoP. Serum bactericidal activity required the binding of C-reactive protein (CRP) with subsequent activation of complement through the classical pathway. Results of this study suggested that the ability of H. influenzae to vary expression of this unusual bacterial structure may correlate with its ability both to persist on the mucosal surface (ChoP+ phenotype) and to cause invasive infection by evading innate immunity mediated by CRP (ChoP- phenotype).

Mutations in the keratin 9 gene in Pakistani families with epidermolytic palmoplantar keratoderma.

BACKGROUND: Keratins are heteropolymeric proteins that form the intermediate filament cytoskeleton in epithelial cells. The common basic structure of all keratins is organized in a central α-helical rod domain flanked by nonhelical, variable head and tail regions. Most mutations in keratins are found in the central α-helical rod domain. Keratin 9 (K9) is expressed only in the suprabasal layers of palmoplantar epidermis. Mutations in the keratin 9 gene (KRT9) have been shown to cause epidermolytic palmoplantar keratoderma (EPPK; OMIM 144200), an autosomal dominant genodermatosis characterized clinically by diffuse hyperkeratosis limited to the palms and soles, and histologically by epidermolysis in suprabasal layers of the epidermis. AIM: To elucidate the genetic basis of EPPK in five Pakistani families. METHODS: Using microsatellite markers localized to the areas around the type I keratin gene cluster on chromosome 17q21, genotyping of these families was performed, followed by sequencing of the KRT9 gene. RESULTS: The analysis resulted in the identification of two novel (p.M157K and p.Y454H) and two recurrent (p.M157T and p.R163Q) mutations in the KRT9 of all five families. All mutations occurred within the highly conserved helix initiation or termination motif of K9. CONCLUSIONS: The affected members of all five families possess mutations in the KRT9 gene that severely affect heterodimer formation with the type II keratin partner. The results of our study further underscore the crucial role of K9 protein in the palmoplantar epidermis.

Pneumococcal trafficking across the blood-brain barrier. Molecular analysis of a novel bidirectional pathway.

Although Streptococcus pneumoniae is a major cause of meningitis in humans, the mechanisms underlying its traversal from the circulation across the blood-brain barrier (BBB) into the subarachnoid space are poorly understood. One mechanism might involve transcytosis through microvascular endothelial cells. In this study we investigated the ability of pneumococci to invade and transmigrate through monolayers of rat and human brain microvascular endothelial cells (BMEC). Significant variability was found in the invasive capacity of clinical isolates. Phase variation to the transparent phenotype increased invasion as much as 6-fold and loss of capsule approximately 200-fold. Invasion of transparent pneumococci required choline in the pneumococcal cell wall, and invasion was partially inhibited by antagonists of the platelet-activating factor (PAF) receptor on the BMEC. Pneumococci that gained access to an intracellular vesicle from the apical side of the monolayer subsequently were subject to three fates. Most opaque variants were killed. In contrast, the transparent phase variants were able to transcytose to the basal surface of rat and human BMEC in a manner dependent on the PAF receptor and the presence of pneumococcal choline-binding protein A. The remaining transparent bacteria entering the cell underwent a previously unrecognized recycling to the apical surface. Transcytosis eventually becomes a dominating process accounting for up to 80% of intracellular bacteria. Our data suggest that interaction of pneumococci with the PAF receptor results in sorting so as to transcytose bacteria across the cell while non-PAF receptor entry shunts bacteria for exit and reentry on the apical surface in a novel recycling pathway.

Synthesis and secretion of corticosteroid-binding globulin by rat liver. A source of heterogeneity of hepatic corticosteroid-binders.

Classical glucocorticoid receptors (type II) have a high affinity for synthetic and natural glucocorticoids. We have previously demonstrated an additional binding site in kidney cytosol (type III) which has a high affinity for corticosterone but a low affinity for dexamethasone. In many ways, this binder resembles plasma corticosteroid-binding globulin (CBG). The first goal of this study was to determine the organ distribution of the type III binding sites. Cytosol was prepared from isolated cells to avoid plasma contamination. Of the tissues examined, type III sites were found only in liver and kidney; sites were absent from thymocytes, IM-9 lymphocytes, adipocytes, and bone cells. The second goal of this study was to ascertain whether CBG is synthesized in liver and kidney. Liver and kidney slices were incubated in vitro and the concentration of type III sites was seen to rise in hepatic cytosol and incubating medium but not kidney. To verify the impression that liver was synthesizing and secreting CBG, the following experiments were performed: (a) To demonstrate that type III sites were CBG, steroid-binding profiles and migration on polyacrylamide gel electrophoresis were shown to be identical for hepatic type III sites and serum. (b) To indicate that the rise in type III sites was dependent on protein synthesis, it was shown that cycloheximide blocked the appearance of new type III sites. (c) To establish that the type III sites were being secreted, in situ liver perfusion experiments showed time-dependent release of new sites into the perfusate. In conclusion, liver synthesizes and secretes type III sites, a finding previously suspected but never proved. The presence of type III sites in kidney remains to be explained.

Protein synthesis elongation factor Tu present in spores of Streptomyces coelicolor can be phosphorylated in vitro by the spore protein kinase.

In vitro phosphorylation of EF-Tu was shown in cell-free extract from dormant spores of Streptomyces coelicolor by a protein kinase present in spores. EF-Tu phosphorylation was observed on both intrinsic S. coelicolor factor and externally added purified EF-Tu from S. aureofaciens, on two isoforms. Putative serine and threonine residues as potential phosphorylation targets were determined in primary sequence and demonstrated on 3D structure model of EF-Tu.

Cross-protective mucosal immunity mediated by memory Th17 cells against Streptococcus pneumoniae lung infection.

Pneumonia caused by Streptococcus pneumoniae (Sp) remains a leading cause of serious illness and death worldwide. Immunization with conjugated pneumococcal vaccine has lowered the colonization rate and consequently invasive diseases by inducing serotype-specific antibodies. However, many of the current pneumonia cases result from infection by serotype strains not included in the vaccine. In this study, we asked if cross-protection against lung infection by heterologous strains can be induced, and investigated the underlying immune mechanism. We found that immune mice recovered from a prior infection were protected against heterologous Sp strains in the pneumonia challenge model, as evident by accelerated bacterial clearance, reduced pathology, and apoptosis of lung epithelial cells. Sp infection in the lung induced strong T-helper type 17 (Th17) responses at the lung mucosal site. Transfer of CD4+ T cells from immune mice provided heterologous protection against pneumonia, and this protection was abrogated by interleukin-17A (IL-17A) blockade. Transfer of memory CD4+ T cells from IL-17A-knockout mice failed to provide protection. These results indicate that memory Th17 cells had a key role in providing protection against pneumonia in a serotype-independent manner and suggest the feasibility of developing a broadly protective vaccine against bacterial pneumonia by targeting mucosal Th17 T cells.

Agglutination by anti-capsular polysaccharide antibody is associated with protection against experimental human pneumococcal carriage.

The ability of pneumococcal conjugate vaccine (PCV) to decrease transmission by blocking the acquisition of colonization has been attributed to herd immunity. We describe the role of mucosal immunoglobulin G (IgG) to capsular polysaccharide (CPS) in mediating protection from carriage, translating our findings from a murine model to humans. We used a flow cytometric assay to quantify antibody-mediated agglutination demonstrating that hyperimmune sera generated against an unencapsulated mutant was poorly agglutinating. Passive immunization with this antiserum was ineffective to block acquisition of colonization compared to agglutinating antisera raised against the encapsulated parent strain. In the human challenge model, samples were collected from PCV and control-vaccinated adults. In PCV-vaccinated subjects, IgG levels to CPS were increased in serum and nasal wash (NW). IgG to the inoculated strain CPS dropped in NW samples after inoculation suggesting its sequestration by colonizing pneumococci. In post-vaccination NW samples pneumococci were heavily agglutinated compared with pre-vaccination samples in subjects protected against carriage. Our results indicate that pneumococcal agglutination mediated by CPS-specific antibodies is a key mechanism of protection against acquisition of carriage. Capsule may be the only vaccine target that can elicit strong agglutinating antibody responses, leading to protection against carriage acquisition and generation of herd immunity.

Construction and testing of a bacterial luciferase reporter gene system for in vivo measurement of nonsense suppression in Streptomyces.

A reporter gene system, based on luciferase genes from Vibrio harvei, was constructed for measurement of translation nonsense suppression in Streptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of the luxB gene was replaced by all of the three stop codons individually. By cloning of luxA and luxB genes under the control of strong constitutive Streptomyces promoter ermE* in plasmid pUWL201 we created Wluxl with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively. Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal in Streptomyces, the effect was negligible.

Ribosomal proteins of Streptomyces aureofaciens producing tetracycline.

Three different two-dimensional polyacrylamide gel electrophoretic systems were employed for identification of individual ribosomal proteins of Streptomyces aureofaciens. Proteins of small subunits were resolved into 21 spots. Larger ribosomal subunits contained 35 proteins. The separated ribosomal proteins from 50 S subunits were transferred on nitrocellulose membranes for immunochemical estimations. Antibodies developed against 50 S proteins of S. aureofaciens and Escherichia coli were used for identification of structural homologies between 50 S proteins of the two species. Results of the experiments indicate that about one half of the 50 S proteins of S. aureofaciens share common immunochemical determinants with corresponding proteins of 50 S subunits of E. coli. Evidence is presented that acidic ribosomal protein SL5 of large ribosomal subunits of S. aureofaciens can be assembled to E. coli P0 cores lacking proteins L7/L12. Reconstitution of the P0 cores with proteins SL5 or L7/L12 led to restoration of 78% activity in polyphenylalanine synthesis.

How many EF-Tu molecules participate in aminoacyl-tRNA binding and peptide bond formation in Escherichia coli translation?

We have observed that two EF-Tu.GTP cycles are required to make one peptide bond during steady-state translation in an accurate and fast poly(U) translation system prepared from Escherichia coli. We have also found that there are two complexes of EF-Tu.GTP bound to one molecule of aminoacyl-tRNA under our experimental conditions. We suggest, on the basis of these data, that aminoacyl-tRNA enters the ribosomal A-site in a pentameric complex together with two EF-Tu and two GTP molecules. When the tRNA is delivered to the ribosome two GTP molecules are hydrolyzed. It is possible that the functional role of such an EF-Tu dimer is related to the function of the two L7/L12 dimers in the large ribosomal subunit.

Post-translational modification(s) and cell distribution of Streptomyces aureofaciens translation elongation factor Tu overproduced in Escherichia coli.

We cloned EF-Tu from Streptomyces aureofaciens on a pET plasmid and overproduced it using the T7 RNA polymerase system in Escherichia coli. Streptomyces EF-Tu represented more than 40% of the total cell protein and was stored mostly in inclusion bodies formed apically at both ends of E. coli cells. Analysis of the inclusion bodies by transmission and scanning electron microscopy did not reveal any internal or surface ultrastructures. We developed the method for purification of S. aureofaciens EF-Tu from isolated inclusion bodies based on the ability of the protein to aggregate spontaneously. EF-Tu present in inclusion bodies was not active in GDP binding. Purified protein showed a similar charge heterogeneity as EF-Tu isolated from the mycelium of S. aureofaciens and all of the isoforms reacted with EF-Tu antibodies. All isoforms also reacted with monoclonal antibodies against O-phosphoserine and O-phosphothreonine.

Antibody blocks acquisition of bacterial colonization through agglutination.

Invasive infection often begins with asymptomatic colonization of mucosal surfaces. A murine model of bacterial colonization with Streptococcus pneumoniae was used to study the mechanism for mucosal protection by immunoglobulin. In previously colonized immune mice, bacteria were rapidly sequestered within large aggregates in the nasal lumen. To further examine the role of bacterial agglutination in protection by specific antibodies, mice were passively immunized with immunoglobulin G (IgG) purified from antipneumococcal sera or pneumococcal type-specific monoclonal human IgA (hIgA1 or hIgA2). Systemically delivered IgG accessed the mucosal surface and blocked acquisition of colonization and transmission between littermates. Optimal protection by IgG was independent of Fc fragment and complement and, therefore, did not involve an opsonophagocytic mechanism. Enzymatic digestion or reduction of IgG before administration showed that protection required divalent binding that maintained its agglutinating effect. Divalent hIgA1 is cleaved by the pneumococcal member of a family of bacterial proteases that generate monovalent Fabα fragments. Thus, passive immunization with hIgA1 blocked colonization by an IgA1-protease-deficient mutant (agglutinated) but not the protease-producing wild-type parent (not agglutinated), whereas protease-resistant hIgA2 agglutinated and blocked colonization by both. Our findings highlight the importance of agglutinating antibodies in mucosal defense and reveal how successful pathogens evade this effect.

Antibiotic resistance gene cassettes derived from the omega interposon for use in E. coli and Streptomyces.

Three antibiotic resistance gene cassettes, derived from the omega interposon (Prentki and Krisch (1984) Gene 29, 303-313) were constructed. These cassettes carry different antibiotic resistance genes, conferring resistance to geneticin, hygromycin or viomycin, flanked by short inverted repeats containing transcription and translation termination signals and synthetic polylinkers. These cassettes were designated omega aac, omega hyg and omega vph. Resistance phenotypes conferred by these constructions are selectable in E. coli and Streptomyces. These cassettes can be used for insertional mutagenesis or for vector construction.

Susceptibility of ribosomes of the tetracycline-producing strain of Streptomyces aureofaciens to tetracyclines.

Ribosomes from cells of Streptomyces aureofaciens producing tetracycline antibiotics (Tc-ribosomes) differ in electrophoretic mobility of ribosomal proteins S2, S10 and L19 from those of the same strain, where the production of tetracyclines was suppressed by changed cultivation conditions (C-ribosomes). Purified tight vacant couples C- and Tc-ribosomes are equally active in the translation of poly(U). Both types of S. aureofaciens ribosomes are more sensitive to tetracycline and chlortetracycline than ribosomes of Escherichia coli in the Phe-tRNA binding and the translation of poly(U).

Verification of electron beam therapy with conventional and storage phosphor images: preliminary experience.

Portal verification images were generated by the photon contamination in electron beams produced by a linear accelerator during treatment of patients receiving high-energy electron radiation therapy. Both conventional and storage phosphor methods yielded projection radiographs in which anatomy of the irradiated and surrounding tissue was demonstrated. Exposed phantoms were used to confirm that the images represent a true projection of the radiation field. A preliminary series of 22 cases was evaluated by two radiotherapists and judged subjectively to be of clinical value. Geometric error, or more importantly, the lack thereof, during high-energy electron treatments was easily confirmed with this method. In three cases, the treatment protocol was corrected based on the images obtained. Because the readout process of storage phosphor images allows for gain adjustments and post-processing, the images obtained with this method were found to delineate anatomy in the treated and surrounding tissues somewhat more consistently than could conventional images.

Natural and acquired macrolide resistance in mycobacteria.

The genus Mycobacterium contains two of the most important human pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, the etiologic agents of tuberculosis and leprosy, respectively. Other mycobacteria are mostly saprophytic organisms, living in soil and water, but some of them can cause opportunistic infections. The increasing incidence of tuberculosis as well as infections with non-tuberculous mycobacteria (NTM) in AIDS patients has renewed interest in molecular mechanisms of drug resistance in these pathogens. Mycobacteria show a high degree of intrinsic resistance to most common antibiotics. For instance, species from the M. tuberculosis complex (MTC) are intrinsically resistant to macrolides. Nevertheless, some semi-synthetic macrolides as the erythromycin derivatives clarithromycin, azithromycin and most recently the ketolides, are active against NTM, particularly Mycobacterium avium, and some of them are widely used for infection treatment. However, shortly after the introduction of these new drugs, resistant strains appeared due to mutations in the macrolide target, the ribosome. The mycobacterial cell wall with its specific composition and structure is considered to be a major factor in promoting the natural resistance of mycobacteria to various antibiotics. However, to explain the difference in macrolide sensitivity between the MTC and NTM, the synergistic contribution of a specific resistance mechanism might be required, in addition to possible differences in cell wall permeability. This mini-review summarizes the current knowledge on the natural and acquired macrolide resistance in mycobacteria, gives an overview of potential mechanisms implicated in the intrinsic resistance and brings recent data concerning a macrolide resistance determinant in the MTC.