Comparative and Functional Genomics of Legionella Identified Eukaryotic Like Proteins as Key Players in Host–Pathogen Interactions

Although best known for its ability to cause severe pneumonia in people whose immune defenses are weakened, Legionella pneumophila and Legionella longbeachae are two species of a large genus of bacteria that are ubiquitous in nature, where they parasitize protozoa. Adaptation to the host environment and exploitation of host cell functions are critical for the success of these intracellular pathogens. The establishment and publication of the complete genome sequences of L. pneumophila and L. longbeachae isolates paved the way for major breakthroughs in understanding the biology of these organisms. In this review we present the knowledge gained from the analyses and comparison of the complete genome sequences of different L. pneumophila and L. longbeachae strains. Emphasis is given on putative virulence and Legionella life cycle related functions, such as the identification of an extended array of eukaryotic like proteins, many of which have been shown to modulate host cell functions to the pathogen’s advantage. Surprisingly, many of the eukaryotic domain proteins identified in L. pneumophila as well as many substrates of the Dot/Icm type IV secretion system essential for intracellular replication are different between these two species, although they cause the same disease. Finally, evolutionary aspects regarding the eukaryotic like proteins in Legionella are discussed

Growth response of Saccharomyces cerevisiae strains to stressors associated to the vine cycle

Saccharomyces cerevisiae isolates from grapes, soil, vine bark and buds collected at seven phenological stages of an annual growth cycle, were molecular typed by Microsatellite Multiplex PCR. Subsequently 30 S. cerevisiae genotypes were selected and the effect of vineyard environmental stressors, in both sublethal upper and lower levels, on their growth parameters was evaluated. The effect of low and high temperature (7–40 ◦C), pH (2.5–8.0), glucose concentration (3.0–300.0 g/L), nitrogen concentration (0.008–8.0 g/L), and copper presence (24 mg/L) were modelled individually using the reparametrized Gompertz equation. Multivariate ANOVA and Generalized Procrustes Analysis were used to determine the environmental stressor’s influence over the lag phase (λ) and the maximum specific growth rate (μmax). Both parameters were significantly affected by the S. cerevisiae genotype, the treatments, and the interaction between them. Despite a generalized reduction in μmax and a variable answer in λ, the 30 S. cerevisiae genotypes were able to overcome all the treatments. Extreme glucose limitation, copper presence and low temperature had the highest impact over the growth parameters. Interestingly, ten genotypes mostly distributed in the vineyard were the least affected, suggesting a greater acclimatization fitness and the possibility to persist in the changing conditions of the vine annual cycle.EEA MendozaFil: Gonzalez, Magali Lucia Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gonzalez, Magali Lucia Rosa. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Valero, Eva. Universidad Pablo de Olavide. Departamento de Biología Molecular e Ingeniería Bioquímica; EspañaFil: Chimeno, Selva Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Garrido Fernandez, Antonio. Universidad Pablo de Olavide. Departamento de Biotecnología de Alimentos, Instituto de la Grasa (IG); España. Consejo Superior de Investigaciones Científicas (CSIC); EspañaFil: Rodriguez Gomez, Francisco. Universidad Pablo de Olavide. Departamento de Biotecnología de Alimentos, Instituto de la Grasa (IG); España. Consejo Superior de Investigaciones Científicas (CSIC); EspañaFil: Rojo, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Rojo, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Paolinelli, Marcos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Arroyo Lopez, Francisco Noe. Universidad Pablo de Olavide. Departamento de Biotecnología de Alimentos, Instituto de la Grasa (IG); España. Consejo Superior de Investigaciones Científicas (CSIC); EspañaFil: Combina, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Mercado, Laura Analia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; Argentin

Analysis of the Legionella longbeachae Genome and Transcriptome Uncovers Unique Strategies to Cause Legionnaires' Disease

Legionella pneumophila and L. longbeachae are two species of a large genus of bacteria that are ubiquitous in nature. L. pneumophila is mainly found in natural and artificial water circuits while L. longbeachae is mainly present in soil. Under the appropriate conditions both species are human pathogens, capable of causing a severe form of pneumonia termed Legionnaires' disease. Here we report the sequencing and analysis of four L. longbeachae genomes, one complete genome sequence of L. longbeachae strain NSW150 serogroup (Sg) 1, and three draft genome sequences another belonging to Sg1 and two to Sg2. The genome organization and gene content of the four L. longbeachae genomes are highly conserved, indicating strong pressure for niche adaptation. Analysis and comparison of L. longbeachae strain NSW150 with L. pneumophila revealed common but also unexpected features specific to this pathogen. The interaction with host cells shows distinct features from L. pneumophila, as L. longbeachae possesses a unique repertoire of putative Dot/Icm type IV secretion system substrates, eukaryotic-like and eukaryotic domain proteins, and encodes additional secretion systems. However, analysis of the ability of a dotA mutant of L. longbeachae NSW150 to replicate in the Acanthamoeba castellanii and in a mouse lung infection model showed that the Dot/Icm type IV secretion system is also essential for the virulence of L. longbeachae. In contrast to L. pneumophila, L. longbeachae does not encode flagella, thereby providing a possible explanation for differences in mouse susceptibility to infection between the two pathogens. Furthermore, transcriptome analysis revealed that L. longbeachae has a less pronounced biphasic life cycle as compared to L. pneumophila, and genome analysis and electron microscopy suggested that L. longbeachae is encapsulated. These species-specific differences may account for the different environmental niches and disease epidemiology of these two Legionella species

More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells.

The genus Legionella comprises 65 species, among which Legionella pneumophila is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80 Legionella genomes spanning 58 species. Uniquely, an immense repository of 18,000 secreted proteins encoding 137 different eukaryotic-like domains and over 200 eukaryotic-like proteins is paired with a highly conserved type IV secretion system (T4SS). Specifically, we show that eukaryotic Rho- and Rab-GTPase domains are found nearly exclusively in eukaryotes and Legionella Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F-box, U-box, and SET domains were present in >70% of all species, suggesting that manipulation of host signal transduction, protein turnover, and chromatin modification pathways are fundamental intracellular replication strategies for legionellae. In contrast, the Sec-7 domain was restricted to L. pneumophila and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly, this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis, indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by interdomain gene transfer. Furthermore, we revealed the surprising extent to which legionellae have coopted genes and thus cellular functions from their eukaryotic hosts, providing an understanding of how dynamic reshuffling and gene acquisition have led to the emergence of major human pathogens

Complementary pre-screening strategies to uncover hidden prodromal and mild Alzheimer's disease : Results from the MOPEAD project

The Models of Patient Engagement for Alzheimer's Disease (MOPEAD) project was conceived to explore innovative complementary strategies to uncover hidden prodromal and mild Alzheimer's disease (AD) dementia cases and to raise awareness both in the general public and among health professionals about the importance of early diagnosis. Four different strategies or RUNs were used: (a) a web-based (WB) prescreening tool, (2) an open house initiative (OHI), (3) a primary care-based protocol for early detection of cognitive decline (PC), and (4) a tertiary care-based pre-screening at diabetologist clinics (DC). A total of 1129 patients at high risk of having prodromal AD or dementia were identified of 2847 pre-screened individuals (39.7%). The corresponding proportion for the different initiatives were 36.8% (WB), 35.6% (OHI), 44.4% (PC), and 58.3% (DC). These four complementary pre-screening strategies were useful for identifying individuals at high risk of having prodromal or mild AD

La variación en los parámetros de crecimiento de cepas Saccharomyces cerevisiae en respuesta a condiciones de estrés presentes durante el ciclo fenológico de la vid evidencia una aclimatación al nicho de viñedo

PosterLa producción de vid y la elaboración de vino constituyen dos actividades de amplia tradición e impacto económico en Mendoza, Argentina. La Zona Alta del Río Mendoza (ZARM) es la principal región vitícola donde se cultiva el Malbec, vino emblemático de Argentina. Saccharomyces cerevisiae es la principal responsable de la fermentación alcohólica del vino y en general, se acepta que el viñedo es el hábitat natural de las levaduras enológicas y que las uvas son la principal fuente de ellas. No obstante, la vid es una especie de hoja caduca y por ello necesita una secuencia específica de condiciones ambientales para renovar todos sus tejidos a través de un ciclo de crecimiento anual. Entonces, ¿qué sucede con las poblaciones de S. cerevisiae en los viñedos durante el ciclo de crecimiento anual de la vid? Esta pregunta plantea la posibilidad de que S. cerevisiae esté adaptada a hábitats ecológicos del viñedo distintos de las uvas, y que estos reservorios puedan constituir un refugio para las levaduras cuando la fruta no está disponible. Asimismo, dado que la tolerancia o resistencia al estrés ambiental tiene componentes tanto genéticos como ecológicos, es necesario estudiar el impacto de factores relacionados con las condiciones estacionales del ciclo anual de la vid para confirmar la adaptación de las cepas de S. cerevisiae a los nichos ecológicos del viñedo. El objetivo del presente estudio fue evaluar el efecto de los cambios estacionales del ciclo anual de la vid sobre el crecimiento de Saccharomyces cerevisiae. Se consideraron 10 condiciones de estrés subletal que reflejaran los cambios ambientales y del sustrato que se presentan en el viñedo. Se evaluó el efecto de: temperatura (7-40 °C), pH (2.5-8.0), concentración de glucosa (3-300 g. L-1), concentración de nitrógeno (0.008-8 g. L-1) y presencia de cobre (Cu2+,24 mg. L-1) sobre la cinética de crecimiento de un conjunto representativo de 30 cepas de S. cerevisiae, previamente aisladas de diferentes nichos ecológicos de un viñedo Malbec durante un ciclo completo de crecimiento de la vid. Los parámetros de crecimiento, fase de latencia, velocidad máxima y máximo asintótico de crecimiento, se estimaron a partir de las curvas de densidad óptica obtenidas con el espectrofotómetro Bioscreen C. El análisis estadístico se realizó mediante ANOVA multivariado (prueba de comparación post-hoc Fisher-LSD) y también se aplicó el Análisis Procrustes Generalizado para evaluar la interacción cepa-condiciones de crecimiento. Los resultados mostraron que el conjunto de cepas de S. cerevisiae caracterizadas fueron capaces de crecer cuando fueron sometidas a los tratamientos que simulaban los cambios ambientales estacionales en el ecosistema del viñedo. La limitación extrema de glucosa, la presencia de cobre y la baja temperatura fueron los factores con mayor impacto en el crecimiento de S. cerevisiae. Se verificaron respuestas de crecimiento homogéneas o variables según el estresor evaluado. Sin embargo, 10 cepas altamente aisladas en el viñedo fueron menos afectadas por todos los factores ensayados, lo que sugiere su aclimatación y capacidad para sobrevivir en las condiciones cambiantes del ciclo anual de la vid.EEA MendozaFil: Gonzalez, Magali Lucia Rosa. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Chimeno, Selva Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Valero, E. Universidad Pablo de Olavide. Departamento de Biología Molecular e Ingeniería Bioquímica; EspañaFil: Arroyo Lopez, F. N. Consejo Superior de Investigaciones Científicas (CSIC). Instituto de la Grasa; EspañaFil: Garrido Fernandez, A. Consejo Superior de Investigaciones Científicas (CSIC). Instituto de la Grasa; España. Universidad Pablo de Olavide. Departamento de Biotecnología de Alimentos; EspañaFil: Rodriguez Gomez, F. Consejo Superior de Investigaciones Científicas (CSIC). Instituto de la Grasa; España. Universidad Pablo de Olavide. Departamento de Biotecnología de Alimentos; EspañaFil: Sturm, Maria Elena. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Rojo, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Combina, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Mercado, Laura Analia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; Argentin

Activation of Type 1 Cannabinoid Receptor (CB1R) promotes neurogenesis in murine subventricular zone cell cultures

The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here, we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal, proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive), neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells, as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs, an effect blocked by Notch pathway inhibition. Moreover, R-m-AEA treatment for 48 h, increased proliferation as assessed by BrdU incorporation assay, an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly, stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation), at 7 days, as shown by counting the number of NeuN-positive neurons in the cultures. Moreover, by monitoring intracellular calcium concentrations ([Ca2+](i)) in single cells following KCl and histamine stimuli, a method that allows the functional evaluation of neuronal differentiation, we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251, for 7 days, thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation, R-m-AEA also increased neurite growth, as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together, these results demonstrate that CB1R activation induces proliferation, self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures.Fundacao para a Ciencia e a Tecnologia - Portugal [POCTI/SAU-NEU/68465/2006, PTDC/SAU-NEU/104415/2008, PTDC/SAU-NEU/101783/2008, POCTI/SAU-NEU/110838/2009]; Fundacao Calouste Gulbenkian [96542]; Fundacao para a Ciencia e Tecnologiainfo:eu-repo/semantics/publishedVersio

Multiple major disease-associated clones of Legionella pneumophila have emerged recently and independently.

Legionella pneumophila is an environmental bacterium and the leading cause of Legionnaires' disease. Just five sequence types (ST), from more than 2000 currently described, cause nearly half of disease cases in northwest Europe. Here, we report the sequence and analyses of 364 L. pneumophila genomes, including 337 from the five disease-associated STs and 27 representative of the species diversity. Phylogenetic analyses revealed that the five STs have independent origins within a highly diverse species. The number of de novo mutations is extremely low with maximum pairwise single-nucleotide polymorphisms (SNPs) ranging from 19 (ST47) to 127 (ST1), which suggests emergences within the last century. Isolates sampled geographically far apart differ by only a few SNPs, demonstrating rapid dissemination. These five STs have been recombining recently, leading to a shared pool of allelic variants potentially contributing to their increased disease propensity. The oldest clone, ST1, has spread globally; between 1940 and 2000, four new clones have emerged in Europe, which show long-distance, rapid dispersal. That a large proportion of clinical cases is caused by recently emerged and internationally dispersed clones, linked by convergent evolution, is surprising for an environmental bacterium traditionally considered to be an opportunistic pathogen. To simultaneously explain recent emergence, rapid spread and increased disease association, we hypothesize that these STs have adapted to new man-made environmental niches, which may be linked by human infection and transmission

Response trajectories of gambling severity after cognitive behavioral therapy in young-adult pathological gamblers

Background and aims: the significant increase in the prevalence of gambling disorder (GD) among young adults in recent years has attracted interest in determining therapeutic efficiency in this sector of the population. The aim of this work was to estimate the response trajectories of gambling severity during the six-month follow-up after a cognitive behavioral therapy (CBT) program in young adult patients and to identify the main variables associated with each trajectory. Methods: the sample included n = 192 patients, aged 19-35 years old, seeking treatment for GD. Response trajectories were identified through latent class growth analysis. Results: three trajectories emerged: T1 (n = 118, 61.5%), composed of patients with severe GD at pre-treatment and good evolution to recovery; T2 (n = 62, 32.3%), with patients with moderate-high GD affectation at baseline and good evolution to recovery; and T3 (n = 12, 6.3%), with participants with severe baseline GD severity and poor evolution after CBT (Abbott, 2019). The highest risk of poor therapeutic outcomes was related to lower social index positions, high emotional distress, high scores in harm avoidance and low scores in self-directedness. Discussion and conclusions: differences in the response trajectories at short-term follow-up after CBT reveal heterogeneity in the samples including young and young-adult GD patients. Patients' phenotype at baseline should be considered when developing efficient, person-centered intervention programs, which should comprise strategies aimed at increasing emotional regulation capacities, self-esteem and self-efficacy, with the aim of avoiding relapses in the medium-long term after therapy

The influence of chronological age on cognitive biases and impulsivity levels in male patients with gambling disorder

Altres ajuts: Delegación del Gobierno para el Plan Nacional sobre Drogas: 2017I067 i 2019I47Due to the contribution of age to the etiology of gambling disorder (GD), there is a need to assess the moderator effect of the aging process with other features that are highly related with the clinical profile. The objective of this study is to examine the role of the chronological age into the relationships between cognitive biases, impulsivity levels and gambling preference with the GD profile during adulthood. Sample included n = 209 patients aged 18-77 years-old recruited from a Pathological Gambling Outpatients Unit. Orthogonal contrasts explored polynomial patterns in data, and path analysis implemented through structural equation modeling assessed the underlying mechanisms between the study variables. Compared to middle-age patients, younger and older age groups reported more impairing irrational beliefs (P = 0.005 for interpretative control and P = 0.043 for interpretative bias). A linear trend showed that as people get older sensation seeking (P = 0.006) and inability to stop gambling (P = 0.018) increase. Path analysis showed a direct effect between the cognitive bias and measures of gambling severity (standardized effects [SE] between 0.12 and 0.17) and a direct effect between impulsivity levels and cumulated debts due to gambling (SE = 0.22). Screening tools and intervention plans should consider the aging process. Specific programs should be developed for younger and older age groups, since these are highly vulnerable to the consequences of gambling activities and impairment levels of impulsivity and cognitive biases