Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-Gram-negative bacterial activity

The present study demonstrates the antibacterial potential of a phage endolysin against Gram-negative pathogens, particularly against multidrug resistant strains of Acinetobacter baumannii. We have cloned, heterologously expressed and characterized a novel endolysin (ABgp46) from Acinetobacter phage vb_AbaP_CEB1 and tested its antibacterial activity against several multidrug-resistant A. baumannii strains. LC-MS revealed that ABgp46 is an N-acetylmuramidase, that is also active over a broad pH range (4.0-10.0) and temperatures up to 50°C. Interestingly, ABgp46 has intrinsic and specific anti-A. baumannii activity, reducing multidrug resistant strains by up to 2 logs within 2 hours. By combining ABgp46 with several organic acids that act as outer membrane permeabilizing agents, it is possible to increase and broaden antibacterial activity to include other Gram-negative bacterial pathogens. In the presence of citric and malic acid, ABgp46 reduces A. baumannii below the detection limit (> 5 log) and more than 4 logs P. aeruginosa and Salmonella Typhimurium strains. Overall, this globular endolysin exhibits a broad and high activity against Gram-negative pathogens, that can be enhanced in presence of citric and malic acid, and be used in human and veterinary medicine.

Chlamydia pneumoniae and atherosclerosis. Identification of bacterial DNA in the arterial wall

OBJECTIVE: The intracellular Gram-negative bacterium Chlamydia pneumoniae has been associated with atherosclerosis. The presence of Chlamydia pneumoniae has been investigated in fragments of the arterial wall with a technique for DNA identification. METHODS: Arterial fragments obtained from vascular surgical procedures in 58 patients were analyzed. From these patients, 39 were males and the mean age was 65±6 years. The polymerase chain reaction was used to identify the bacterial DNA with a pair of primers that codify the major outer membrane protein (MOMP) of Chlamydia pneumoniae. The amplified product was visualized by electrophoresis in the 2% agarose gel stained with ethidium bromide, and it was considered positive when migrating in the band of molecular weight of the positive controls. RESULTS: Seven (12%) out of the 58 patients showed positive results for Chlamydia pneumoniae. CONCLUSION: DNA from Chlamydia pneumoniae was identified in the arterial wall of a substantial number of patients with atherosclerosis. This association, which has already been described in other countries, corroborates the evidence favoring a role played by Chlamydia pneumoniae in atherogenesis.

Participación del tráfico y la distribución de proteínas en la polaridad y el desarrollo de procesos neuronales. Protein sorting and trafficking participation in neuronal processes polarity and development.

El estudio del tráfico intracelular en neuronas ha despertado gran interés en los últimos años, debido a que un gran número de enfermedades neurodegenerativas y neuropsiquiátricas parecen tener origen en en el transporte defectuoso de proteínas en estos tipos celulares. Mediante el uso de técnicas de biología celular y molecular, fuimos capaces de describir una de las vías que regula la fisión de las vesículas que llevan su cargo desde la última cisterna del Aparato de Golgi hacia la superficie celular en células epiteliales no polarizadas. Uno de los componentes clave de esa vía resultó ser la Proteina Kinasa D1 (PKD1), cuya actividad en el Aparato de Golgi es esencial para un normal transporte intracelular. Sorprendentemente, observamos que la PKD1 en neuronas con polaridad establecida no regula la fisión en el Golgi, pero si estaría involucrada en la selectividad y distribución (sorting) de vesículas cuyo cargo debe ser específicamente dirigido a las membranas dendríticas. El bloqueo de la actividad de la PKD1 no solamente cambia el destino final de estos cargos, que son enviados de esta forma a la membrana terminal del axón, sino que también es capaz de inducir defectos en el desarrollo y crecimiento de los procesos dendríticos a largo plazo. En este proyecto estudiaremos de que manera influye la perturbación del sorting, en ausencia de PKD1 activa y de otros componentes que la regulan, en la distribución de receptores de factores neurotróficos y de neurotransmisores glutamatérgicos, y cómo estos cambios en su distribución afectan el número, tamaño, y funcionalidad de los procesos neuronales (axones y dendritas). Estos resultados contribuirán a adquirir mayores conocimientos de los mecanismos dependientes del transporte y sorting de proteínas de membrana que participan en la regulación del crecimiento neuronal, los cuales a su vez aportarán información valiosa en la comprensión de un gran número de enfermedades neurológicas. The study of intracellular trafficking in neurons has arisen a great deal of interest in the last years, since a great number of neurodegenerative and neuropsychiatric disorders seem to be originated in abnormal protein transport in these type of cells. Using cell and molecular biology methodologies, we have been capable of describe one of the pathways that regulate the fission of vesicles that carry their cargo from the last Golgi Apparatus cisternae to the cell surface in non-polarized epithelial cells. One of the key components in this pathway is the Protein Kinase D1 (PKD1), whose activity in the Golgi Apparatus is essential for a normal intracelular transport. Surprisingly, we have observed that PKD1 does not regulate fission in neurons with established polarity, but it would be involved in vesicles' sorting at Golgi, particularly of those that carry specific dendritic cargo. Blocking PKD1 activity changes the final destination of these cargoes, which is now sent to the axons' terminal membranes, and also produces late dendritic development and growing defects. In this project we will study how sorting perturbation in absence of PKD1 and its regulators activities influences selectivity and distribution of neurotrophic and neurotransmitter receptors, and how these sorting changes affect number, size and functionality of neuronal processes (axons and dendrites). These results will help to acquire greater knowledge about transport and sorting mechanisms of neuronal growth regulatory membrane proteins. In addition, these studies will contribute with new valuable information necessary to understand numerous neurological diseases.

Ectodysplasin A1 promotes placodal cell fate during early morphogenesis of ectodermal appendages.

Organs developing as appendages of the ectoderm are initiated from epithelial thickenings called placodes. Their formation is regulated by interactions between the ectoderm and underlying mesenchyme, and several signalling molecules have been implicated as activators or inhibitors of placode formation. Ectodysplasin (Eda) is a unique signalling molecule in the tumour necrosis factor family that, together with its receptor Edar, is necessary for normal development of ectodermal organs both in humans and mice. We have shown previously that overexpression of the Eda-A1 isoform in transgenic mice stimulates the formation of several ectodermal organs. In the present study, we have analysed the formation and morphology of placodes using in vivo and in vitro models in which both the timing and amount of Eda-A1 applied could be varied. The hair and tooth placodes of K14-Eda-A1 transgenic embryos were enlarged, and extra placodes developed from the dental lamina and mammary line. Exposure of embryonic skin to Eda-A1 recombinant protein in vitro stimulated the growth and fusion of placodes. However, it did not accelerate the initiation of the first wave of hair follicles giving rise to the guard hairs. Hence, the function of Eda-A1 appears to be downstream of the primary inductive signal required for placode initiation during skin patterning. Analysis of BrdU incorporation indicated that the formation of the epithelial thickening in early placodes does not involve increased cell proliferation and also that the positive effect of Eda-A1 on placode expansion is not a result of increased cell proliferation. Taken together, our results suggest that Eda-A1 signalling promotes placodal cell fate during early development of ectodermal organs.

Notch1 and T-cell development: insights from conditional knockout mice.

Notch proteins influence cell-fate decisions in many developmental systems. Gain-of-function studies have suggested a crucial role for Notch1 signaling at several stages during lymphocyte development, including the B/T, alphabeta/gammadelta and CD4/CD8 lineage choices. Here, we critically re-evaluate these conclusions in the light of recent studies that describe inducible and tissue-specific targeting of the Notch1 gene.

Pathfinding of corticothalamic axons relies on a rendezvous with thalamic projections.

Major outputs of the neocortex are conveyed by corticothalamic axons (CTAs), which form reciprocal connections with thalamocortical axons, and corticosubcerebral axons (CSAs) headed to more caudal parts of the nervous system. Previous findings establish that transcriptional programs define cortical neuron identity and suggest that CTAs and thalamic axons may guide each other, but the mechanisms governing CTA versus CSA pathfinding remain elusive. Here, we show that thalamocortical axons are required to guide pioneer CTAs away from a default CSA-like trajectory. This process relies on a hold in the progression of cortical axons, or waiting period, during which thalamic projections navigate toward cortical axons. At the molecular level, Sema3E/PlexinD1 signaling in pioneer cortical neurons mediates a "waiting signal" required to orchestrate the mandatory meeting with reciprocal thalamic axons. Our study reveals that temporal control of axonal progression contributes to spatial pathfinding of cortical projections and opens perspectives on brain wiring.

Nanosensors for cancer detection.

Cancer is a major burden in today's society and one of the leading causes of death in industrialised countries. Various avenues for the detection of cancer exist, most of which rely on standard methods, such as histology, ELISA, and PCR. Here we put the focus on nanomechanical biosensors derived from atomic force microscopy cantilevers. The versatility of this novel technology has been demonstrated in different applications and in some ways surpasses current technologies, such as microarray, quartz crystal microbalance and surface plasmon resonance. The technology enables label free biomarker detection without the necessity of target amplification in a total cellular background, such as BRAF mutation analysis in malignant melanoma. A unique application of the cantilever array format is the analysis of conformational dynamics of membrane proteins associated to surface stress changes. Another development is characterisation of exhaled breath which allows assessment of a patient's condition in a non-invasive manner.

Notch signaling in T- and B-cell development.

The Notch family of evolutionarily conserved proteins regulates a broad spectrum of cell-fate decisions and differentiation processes during fetal and post-natal development. The best characterized role of Notch signaling during mammalian hematopoiesis and lymphopoiesis is the essential function of the Notch1 receptor in T-cell lineage commitment. More recent studies have addressed the roles of other Notch receptors and ligands, as well as their downstream targets, revealing additional novel functions of Notch signaling in intra-thymic T-cell development, B-cell development and peripheral T-cell function.

Simultaneous CD8+ T cell responses to multiple tumor antigen epitopes in a multipeptide melanoma vaccine.

The recent identification and molecular characterization of tumor-associated antigens recognized by tumor-reactive CD8+ T lymphocytes has led to the development of antigen-specific immunotherapy of cancer. Among other approaches, clinical studies have been initiated to assess the in vivo immunogenicity of tumor antigen-derived peptides in cancer patients. In this study, we have analyzed the CD8+ T cell response of an ocular melanoma patient to a vaccine composed of four different tumor antigen-derived peptides administered simultaneously in incomplete Freund's adjuvant (IFA). Peptide NY-ESO-1(157-165) was remarkably immunogenic and induced a CD8+ T cell response detectable ex vivo at an early time point of the vaccination protocol. A CD8+ T cell response to the peptide analog Melan-A(26-35 A27L) was also detectable ex vivo at a later time point, whereas CD8+ T cells specific for peptide tyrosinase(368-376) were detected only after in vitro peptide stimulation. No detectable CD8+ T cell response to peptide gp100(457-466) was observed. Vaccine-induced CD8+ T cell responses declined rapidly after the initial response but increased again after further peptide injections. In addition, tumor antigen-specific CD8+ T cells were isolated from a vaccine injection site biopsy sample. Importantly, vaccine-induced CD8+ T cells specifically lysed tumor cells expressing the corresponding antigen. Together, these data demonstrate that simultaneous immunization with multiple tumor antigen-derived peptides can result in the elicitation of multiepitope-directed CD8+ T cell responses that are reactive against antigen-expressing tumors and able to infiltrate antigen-containing peripheral sites.

MARCKS-related protein (MRP) is a substrate for the Leishmania major surface protease leishmanolysin (gp63).

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in diverse cell types. Activation of murine macrophages by cytokines increases MRP expression, but infection with Leishmania promastigotes during activation results in MRP depletion. We therefore examined the effect of Leishmania major LV39 on recombinant MRP. Both live promastigotes and a soluble fraction of LV39 lysates degraded MRP to yield lower molecular weight fragments. Degradation was independent of MRP myristoylation and was inhibited by protein kinase C-dependent phosphorylation of MRP. MRP was similarly degraded by purified leishmanolysin (gp63), a Leishmania surface metalloprotease. Degradation was evident at low enzyme/substrate ratios, over a broad pH range, and was inhibited by 1,10-phenanthroline and by a hydroxamate dipeptide inhibitor of leishmanolysin. Using mass spectrometric analysis, cleavage was shown to occur within the effector domain of MRP between Ser(92) and Phe(93), in accordance with the substrate specificity of leishmanolysin. Moreover, an MRP construct in which the effector domain had been deleted was resistant to cleavage. Thus, Leishmania infection may result in leishmanolysin-dependent hydrolysis of MRP, a major protein kinase C substrate in macrophages.

The endodermis--development and differentiation of the plant's inner skin.

Controlling external compound entrance is essential for plant survival. To set up an efficient and selective sorting of nutrients, free diffusion via the apoplast in vascular plants is blocked at the level of the endodermis. Although we have learned a lot about endodermal specification in the last years, information regarding its differentiation is still very limited. A differentiated endodermal cell can be defined by the presence of the "Casparian strip" (CS), a cell wall modification described first by Robert Caspary in 1865. While the anatomical description of CS in many vascular plants has been very detailed, we still lack molecular information about the establishment of the Casparian strips and their actual function in roots. The recent isolation of a novel protein family, the CASPs, that localizes precisely to a domain of the plasma membrane underneath the CS represents an excellent point of entry to explore CS function and formation. In addition, it has been shown that the endodermis contains transporters that are localized to either the central (stele-facing) or peripheral (soil-facing) plasma membranes. These features suggest that the endodermis functions as a polar plant epithelium.

Arenavirus Nucleoprotein Targets Interferon Regulatory Factor-Activating Kinase IKKε.

Arenaviruses perturb innate antiviral defense by blocking induction of type I interferon (IFN) production. Accordingly, the arenavirus nucleoprotein (NP) was shown to block activation and nuclear translocation of interferon regulatory factor 3 (IRF3) in response to virus infection. Here, we sought to identify cellular factors involved in innate antiviral signaling targeted by arenavirus NP. Consistent with previous studies, infection with the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) prevented phosphorylation of IRF3 in response to infection with Sendai virus, a strong inducer of the retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS) pathway of innate antiviral signaling. Using a combination of coimmunoprecipitation and confocal microscopy, we found that LCMV NP associates with the IκB kinase (IKK)-related kinase IKKε but that, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKKε interaction was highly conserved among arenaviruses from different clades. In LCMV-infected cells, IKKε colocalized with NP but not with MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKKε (IKBKE) and blocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our data identify IKKε as a novel target of arenavirus NP. Engagement of NP seems to sequester IKKε in an inactive complex. Considering the important functions of IKKε in innate antiviral immunity and other cellular processes, the NP-IKKε interaction likely plays a crucial role in arenavirus-host interaction.

Normal hemopoiesis and lymphopoiesis in the combined absence of numb and numblike.

The mammalian ortholog of the conserved Drosophila adaptor protein Numb (Nb) and its homolog Numblike (Nbl) modulate neuronal cell fate determination at least in part by antagonizing Notch signaling. Because the Notch pathway has been implicated in regulating hemopoietic stem cell self-renewal and T cell fate specification in mammals, we investigated the role of Nb and Nbl in hemopoiesis using conditional gene targeting. Surprisingly simultaneous deletion of both Nb and Nbl in murine bone marrow precursors did not affect the ability of stem cells to self-renew or to give rise to differentiated myeloid or lymphoid progeny, even under competitive conditions in mixed chimeras. Furthermore, T cell fate specification and intrathymic T cell development were unaffected in the combined absence of Nb and Nbl. Collectively our data indicate that the Nb family of adaptor proteins is dispensable for hemopoiesis and lymphopoiesis in mice, despite their proposed role in neuronal stem cell development.

Mutations in the cdc10 start gene of Schizosaccharomyces pombe implicate the region of homology between cdc10 and SWI6 as important for p85cdc10 function.

The cdc10 gene of the fission yeast Schizosaccharomyces pombe is required for traverse of start and commitment to the mitotic cell division cycle rather than other fates. The product of the gene, p85cdc10, is a component of a factor that is thought to be involved in regulating the transcription of genes that are required for DNA synthesis. In order to define regions of the p85cdc10 protein that are important for its function a fine structure genetic map of the cdc10 gene was derived and the sequences of 13 cdc10ts mutants determined. The 13 mutants tested define eight alleles. Eleven of the mutants are located in the region that contains the two copies of the cdc10/SWI6 repeat motif, implicating it as important for p85cdc10 function.

Is the thymus a target organ in infectious diseases?

The thymus is a central lymphoid organ, in wich T cell precursors differentiale and generate most of the so-called T cell reprtoire. Along with a variety of acute infectious diseases, we and others determined important changes in both microenvironmental and lymphoid compartments of the organ. For example, one major and common feature observed in acute viral, bacterial and parasitic diseases, is a depletion of cortical thymocytes, mostly those bearing the CD4-CD8 double positive phenotype. This occurs simmultaneously to the relative enrichment in medullary CD4 or CD8 single positive cells, expressing high densities of the CD3 complex. Additionally we noticed a variety of changes in the thymic microenvironment (and particularly is epithelial component), comprising abnormal location of thymic epithelial cell subsets as well has a denser Ia-bearing cellular network. Moreover, the extracellular matrix network was altered with an intralobular increase of basement membrane proteins that positively correlated with the degree of thymocyte death. Lastly, anti-thymic cell antibodies were detected in both human and animal models of infectious diseases, and in some of them a phenomenon of molecular mimicry could be evidenced. Taken together, the data receiwed herein clearly show that the thymus should be regarded as a target in infectious diseases.