kinase inhibitors as new anti-chlamydial agents

Chlamydia trachomatis (Ctr) es la causa bacteriana de enfermedad de transmisión sexual más frecuente en el mundo. En la mujer, Ctr provoca desde infecciones agudas como cervicitis, endometritis, salpingitis hasta complicaciones frecuentes y graves como enfermedad inflamatoria pélvica, dolor crónico, abortos espontáneos e infertilidad. La aparición de resistencia a antibióticos como rifamicinas, macrólidos, quinolonas, y azitromicina, y la generación de bacterias aberrantes persistentes en presencia de antibióticos β-lactámicos, requiere el desarrollo de nuevas herramientas farmacológicas. Dado que Chlamydia trachomatis es una bacteria intracelular obligada, su interacción con la célula hospedadora es fundamental para el inicio y establecimiento de la infección. En este proyecto proponemos analizar la utilidad de inhibidores de kinasas, particularmente de la vía Akt/AS160/Rab, como agentes anti-clamidiales. La bacteria utiliza esta vía de señalización intracelular para capturar los esfingolípidos sintetizados en el aparato de Golgi, que son necesarios para el desarrollo y la replicación bacteriana. Analizaremos la capacidad de los inhibidores de kinasas para impedir la invasión, limitar el desarrollo de la infección, evitar la cronificación y mejorar el curso clínico, disminuyendo la inflamación tisular asociada. Se espera que los resultados obtenidos en el transcurso del presente proyecto conduzcan a la generación de nuevas alternativas farmacológicas, diferentes a los antibióticos actuales, que sean más eficientes como agentes anti-clamidiales, particularmente cuando existe antibiotico-resistencia o las bacterias ingresan en un estado persistente que permanece por años quiescente dentro de las células infectadas.Chlamydia trachomatis (Ct) is the most common bacterial cause of sexually transmitted infections in the world. In women, Ct causes from acute infections such as cervicitis, endometritis, salpingitis to frequent and serious complications such as pelvic inflammatory disease, chronic pain, spontaneous abortions, and infertility. The emergence of resistance to antibiotics such as rifamycins, macrolides, quinolones, and azithromycin, and the generation of persistent aberrant bacteria in the presence of β-lactam antibiotics, requires the development of new pharmacological tools. Since Chlamydia trachomatis is an obligate intracellular bacterium, its interaction with the host cell is fundamental for the initiation and establishment of the infection. In this project, we propose to analyze the usefulness of kinase inhibitors, particularly of the Akt / AS160 / Rab pathway, as anti-chlamydial agents. The bacterium uses this intracellular signaling pathway to capture the sphingolipids synthesized in the Golgi apparatus, which are necessary for bacterial development and replication. We will analyze the ability of kinase inhibitors to prevent invasion, limit the development of infection, prevent chronic course and improve the clinical outcome, decreasing associated tissue inflammation. It is expected that the results obtained in this project lead to the generation of new pharmacological alternatives, different from current antibiotics, that are more efficient as anti-chlamydial agents, particularly when there is antibiotic resistance or the bacteria enter a persistent state and remain for years quiescent inside the infected cells

Akt/AS160 Signaling Pathway Inhibition Impairs Infection by Decreasing Rab14-Controlled Sphingolipids Delivery to Chlamydial Inclusions

Chlamydia trachomatis, an obligate intracellular bacterium, intercepts different trafficking pathways of the host cell to acquire essential lipids for its survival and replication, particularly from the Golgi apparatus via a Rab14-mediated transport. Molecular mechanisms underlying how these bacteria manipulate intracellular transport are a matter of intense study. Here, we show that C. trachomatis utilizes Akt/AS160 signaling pathway to promote sphingolipids delivery to the chlamydial inclusion through Rab14-controlled vesicular transport. C. trachomatis provokes Akt phosphorylation along its entire developmental life cycle and recruits phosphorylated Akt (pAkt) to the inclusion membrane. As a consequence, Akt Substrate of 160 kDa (AS160), also known as TBC1D4, a GTPase Activating Protein (GAP) for Rab14, is phosphorylated and therefore inactivated. Phosphorylated AS160 (pAS160) loses its ability to promote GTP hydrolysis, favoring Rab14 binding to GTP. Akt inhibition by an allosteric isoform-specific Akt inhibitor (iAkt) prevents AS160 phosphorylation and reduces Rab14 recruitment to chlamydial inclusions. iAkt further impairs sphingolipids acquisition by C. trachomatis-inclusion and provokes lipid retention at the Golgi apparatus. Consequently, treatment with iAkt decreases chlamydial inclusion size, bacterial multiplication, and infectivity in a dose-dependent manner. Similar results were found in AS160-depleted cells. By electron microscopy, we observed that iAkt generates abnormal bacterial forms as those reported after sphingolipids deprivation or Rab14 silencing. Taken together, our findings indicate that targeting the Akt/AS160/Rab14 axis could constitute a novel strategy to limit chlamydial infections, mainly for those caused by antibiotic-resistant bacteria

Circulating galectin-1 delineates response to bevacizumab in melanoma patients and reprograms endothelial cell biology

Blockade of vascular endothelial growth factor (VEGF) signaling with bevacizumab, a humanized anti-VEGF monoclonal antibody (mAb), or with receptor tyrosine kinase inhibitors, has improved progression-free survival and, in some indications, overall survival across several types of cancers by interrupting tumor angiogenesis. However, the clinical benefit conferred by these therapies is variable, and tumors from treated patients eventually reinitiate growth. Previously we demonstrated, in mouse tumor models, that galectin-1 (Gal1), an endogenous glycan-binding protein, preserves angiogenesis in anti-VEGF–resistant tumors by co-opting the VEGF receptor (VEGFR)2 signaling pathway in the absence of VEGF. However, the relevance of these findings in clinical settings is uncertain. Here, we explored, in a cohort of melanoma patients from AVAST-M, a multicenter, open-label, randomized controlled phase 3 trial of adjuvant bevacizumab versus standard surveillance, the role of circulating plasma Gal1 as part of a compensatory mechanism that orchestrates endothelial cell programs in bevacizumab-treated melanoma patients. We found that increasing Gal1 levels over time in patients in the bevacizumab arm, but not in the observation arm, significantly increased their risks of recurrence and death. Remarkably, plasma Gal1 was functionally active as it was able to reprogram endothelial cell biology, promoting migration, tubulogenesis, and VEGFR2 phosphorylation. These effects were prevented by blockade of Gal1 using a newly developed fully human anti-Gal1 neutralizing mAb. Thus, using samples from a large-scale clinical trial from stage II and III melanoma patients, we validated the clinical relevance of Gal1 as a potential mechanism of resistance to bevacizumab treatment

Grape pomace extract supplementation activates FNDC5/irisin in muscle and promotes white adipose browning in rats fed a high-fat diet

Irisin is a myokine regulated by peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) in the exercising skeletal muscle and released into the bloodstream after cleavage of FNDC5. Circulating irisin can up-regulate UCP-1 expression in white adipose tissue (WAT) promoting the formation of brown-like adipocytes. The aim of this study was to evaluate if supplementation with a grape pomace extract (GPE) could activate the FNDC5/irisin pathway via PGC-1α in rats fed a high fat diet (HFD). For this purpose we characterized the activation of: i. the FNDC5/irisin pathway and AMPK in skeletal muscle and ii. proteins involved in the formation of brown-like cells in epididymal WAT (eWAT). Consumption of the GPE activated the FNDC5/irisin pathway, increased AMPK phosphorylation in skeletal muscle and enhanced irisin plasma levels. In eWAT, the GPE increased the level of proteins involved in WAT browning, i.e. PGC-1α, PPARγ, PRDM16 and UCP-1. The GPE also prevented HFD-induced adipocyte hypertrophy and systemic insulin resistance. Consistently, in L6 myotubes, (?)-epicatechin (EC), a flavonoid abundant in the GPE, prevented palmitate-mediated downregulation of FNDC5/irisin protein expression and secretion, in part via PGC-1α activation. Consumption of the GPE, a winemaking residue rich in bioactive compounds, could be a beneficial strategy to counteract the adverse effects of Western style diets through the promotion of WAT browning.Fil: Rodriguez Lanzi, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Perdicaro, Diahann Jeanette. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Gambarte Tudela, Julián. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Muscia Saez, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; ArgentinaFil: Fontana, Ariel Ramón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Oteiza, Patricia Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of California; Estados UnidosFil: Vazquez, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; Argentin

Rab39a and Rab39b Display Different Intracellular Distribution and Function in Sphingolipids and Phospholipids Transport

Rab GTPases define the identity and destiny of vesicles. Some of these small GTPases present isoforms that are expressed differentially along developmental stages or in a tissue-specific manner, hence comparative analysis is difficult to achieve. Here, we describe the intracellular distribution and function in lipid transport of the poorly characterized Rab39 isoforms using typical cell biology experimental tools and new ones developed in our laboratory. We show that, despite their amino acid sequence similarity, Rab39a and Rab39b display non-overlapping intracellular distribution. Rab39a localizes in the late endocytic pathway, mainly at multivesicular bodies. In contrast, Rab39b distributes in the secretory network, at the endoplasmic reticulum/cis-Golgi interface. Therefore, Rab39a controls trafficking of lipids (sphingomyelin and phospholipids) segregated at multivesicular bodies, whereas Rab39b transports sphingolipids biosynthesized at the endoplasmic reticulum-Golgi factory. Interestingly, lyso bis-phosphatidic acid is exclusively transported by Rab39a, indicating that both isoforms do not exert identical functions in lipid transport. Conveniently, the requirement of eukaryotic lipids by the intracellular pathogen Chlamydia trachomatis rendered useful for dissecting and distinguishing Rab39a- and Rab39b-controlled trafficking pathways. Our findings provide comparative insights about the different subcellular distribution and function in lipid transport of the two Rab39 isoforms

Galectin-1 impacts on glucose homeostasis by modulating pancreatic insulin release

Type-2 diabetes mellitus (T2DM) is an expanding global health problem, involving defective insulin secretion by pancreatic β-cells and peripheral insulin resistance, leading to impaired glucose regulation. Galectin-1, an endogenous lectin with affinity for N-acetyllactosamine (LacNAc)-containing glycans, has emerged as a regulator of inflammatory and metabolic disorders. However, the role of galectin-1 in glucose homeostasis and pancreatic β-cell function, independently of hypercaloric diets, has not been explored. Here, we identified a phenotype compatible with T2DM, involving alterations in glucose metabolism and pancreatic insulin release, in female but not male mice lacking galectin-1 (Lgals1-/-). Compared with age-matched controls, Lgals1-/female mice exhibited higher body weight and increased food intake ad libitum as well as after fasting and acute re-feeding. Although fasted serum insulin levels and insulin sensitivity were similar in both genotypes, Lgals1-/- female mice presented altered glucose tolerance and higher basal glucose levels depending on the fasting period. Insulin response to glucose overload was impaired, while pancreatic insulin content was enhanced in the absence of galectin-1. Accordingly, recombinant galectin-1 enhanced glucose-stimulated insulin release in vitro. Our study identifies a role for galectin-1 in regulating glucose metabolism through modulation of pancreatic insulin secretion, highlighting novel opportunities to control T2DM.Fil: Sundblad, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Garcia Tornadu, Isabel Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Ornstein, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Martínez Allo, Verónica Candela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Lorenzo, Rodrigo Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad Nacional de Tierra del Fuego, Antártida e Islas del Atlántico Sur. Instituto de Ciencias Polares, Recursos Naturales y Ambiente. Departamento de Biología; ArgentinaFil: Gatto, Sabrina Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Morales, Rosa María. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Gambarte Tudela, Julián A. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Manselle Cocco, Montana Nicolle. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Croci Russo, Diego Omar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Becu, Damasia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentin