Heterogeneidad fenotípica en el Sistema de Secreción tipo III de Pseudomonas syringae durante la interacción con la planta

La heterogeneidad fenotípica es un fenómeno que se ha descrito en poblaciones bacterianas de diversas especies. Un patrón de expresión génica heterogéneo puede llegar a volverse bimodal en ambientes homogéneos, proceso conocido como bistabilidad. El desarrollo de métodos de análisis de células individuales, como la microscopía confocal, la citometría o la microfluídica, ha llevado a la identificación de nuevos ejemplos de variación fenotípica y de biestabilidad. La relevancia de estos procesos se ha demostrado en patógenos humanos y de animales. No obstante, se conoce muy poco sobre la relevancia de este tipo de procesos en el proceso de adaptación a huéspedes no animales. P. syringae es una bacteria patógena de plantas con un amplio rango de hospedador, existiendo más de 50 patovares. La virulencia de Pseudomonas syringae dependiende del Sistema de Secreción Tipo III (T3SS) y de los efectores tipo III (T3E). Mediante fusiones transcripcionales a proteínas fluorescentes generadas en el genoma de P. syringae pv. phaseolicola, y el uso de microscopía de fluorescencia y citometría de flujo, nuestro laboratorio ha demostrado que la expresión del T3SS y de T3E es heterogénea en el interior de la planta y biestable en medio mínimo. En este trabajo presentamos los resultados obtenidos en nuestro análisis del impacto de la expresión heterogénea del T3SS para la adaptación a la planta, que incluyen la evaluación de la viabilidad de las variantes (T3SSON/T3SSOFF) en el apoplasto de la hoja, y de la dinámica de expresión en la población en diferentes escenarios de activación de defensa en la planta.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Metilasas y metilación en la regulación génica de Pseudomonas syringae

La metilación del ADN llevada a cabo por metilasas no asociadas a enzimas de restricción, conocidas como metilasas huérfanas, es un mecanismo epigenético ampliamente extendido en bacterias y archeas. La especificidad de motivos y los patrones de metilación de las metilasas huérfanas sugieren su participación en la regulación génica y en la replicación del ADN [1]. En E. coli y en Salmonella, este mecanismo se ha asociado a la regulación del inicio de la replicación, la reparación de errores del ADN y la transcripción. En poblaciones clonales de dichas especies, la metilación del ADN también está implicada en la aparición de heterogeneidad fenotípica, con una fuerte implicación en virulencia [2]. Sin embargo, poco se sabe del papel de la metilación en Pseudomonas syringae. P. syringae es una importante bacteria fitopatógena, responsable de una gran variedad de enfermedades de plantas y con un alto impacto en la producción agrícola a nivel mundial. Esta bacteria también se usa frecuentemente como modelo para el estudio de las interacciones planta-patógeno [3]. Con el objetivo de descifrar la importancia de la metilación en P. syringae, nuestro grupo decidió abordar la caracterización de este mecanismo epigenético en dicho patógeno. Para ello, se han estudiado las metilasas identificadas en P. syringae y se ha establecido su metiloma. Con respecto al metiloma, hemos identificado diversos motivos de metilación.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Phenotypically heterogeneous loci in the plant pathogen Pseudomonas syringae.

Phenotypic heterogeneity usually refers to the co-existance of different phenotypes within a population. Phenotypic differences may arise through genetic variation (genomic rearrangements or mutations) or through the response to differences in the stimuli as encountered within the microenvironment. But also, sometimes, the sources of variation may not be deterministic, i.e. directly related to stimuli, but a consequence of molecular noise in gene expression and/or a programmed event under genetic or epigenetic control. A particular example of phenotypic heterogeneity is bistability. Bistability occurs when a bacterial clonal population splits into two subpopulations showing distinct phenotypes. Phenotypic heterogeneity can be beneficial in fluctuating environments by allowing some individuals within the clonal population to survive sudden changes (risk-spreading). It can also benefit the entire population through cooperation between individuals displaying phenotypic differences (division of labour). These processes and their biological relevance have been described in some animal pathogens, but little is known about them in plant-pathosystems. Pseudomonas syringae is a plant-pathogenic bacterium whose virulence depends on the expression of a type III secretion system (T3SS). Our team has previously reported that T3SS expression is bistable under inducing conditions, generating two subpopulations (T3SSON/T3SSOFF) that show differences in virulence. We have identified other loci including genes related to motility, biofilm formation and DNA methylation that also display phenotypic heterogeneity to very different degrees and with different dynamics and are at different stages on their molecular and biological characterization. Our latest advances on this front will be presented and discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Methylases and methylation in gene regulation in Pseudomonas syringae

DNA methylation carried out by methyltransferases not associated with restriction enzymes, known as orphan methyltransferases, is an epigenetic mechanism widely spread in bacteria and archea. These orphan methylases show motif specificities and methylation patterns that support their function in gene regulation and DNA replication [1]. In E. coli and Salmonella, this mechanism has been related to the regulation of the initiation of replication, DNA mismatch repair and transcription. DNA methylation is also involved in generating phenotypic heterogeneity within clonal populations in these species, with strong implications for virulence [2]. However, little is known about the role of methylation in Pseudomonas syringae. P. syringae is a relevant phytopathogenic bacteria, responsible for a great variety of plant diseases and with a huge impact in crop production worlwide. This bacteria is also used as a model for the study of plant-pathogen interactions [3]. In order to unveil the importance of methylation in P. syringae, we decided to address the characterization of this epigenetic process in this pathogenic bacteria. For that, we have studied the methylases identified in P. syringae, and established its methylome. Regarding the methylome, we have identified several methylation motifs.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech