Estudio de la nucleasa EndoG en el protozoo "Leishmania infantum": caracterización e implicaciones en la supervivencia y muerte del parásito

Texto en español y resumen, conclusiones y artículos en inglésEn los últimos años, ha sido demostrado y aceptado que organismos unicelulares como los parasitos del género Leishmania son susceptibles de sufrir procesos de muerte celular que recuerdan a la apoptosis descrita en metazoos, y que pueden ser inducidos por una amplia variedad de estímulos. Sin embargo, los mecanismos moleculares que participan y regulan este proceso todavía son bastante desconocidos y se han descrito pobremente. Además, las moléculas que podrían participar en dichos procesos todavía no han sido identificadas. Debido a que la degradación del ADN es probablemente el evento más caracterizado durante el proceso similar a apoptosis que tiene lugar en Leishmania, nos hemos centrado en identificar a la nucleasa responsable de dicha degradación. Los resultados presentados en esta tesis demuestran que los promastigotes de Leishmania infantum expresan una nucleasa similar a las Endonucleasas G de eucariotas superiores que, de acuerdo con la predicción para su estructura, pertenece a la superfamilia de nucleasas con dominio ββα de unión a metal. El pH óptimo y los requerimientos catiónicos de la EndoG de L. infantum recuerdan a los determinados previamente para otras EndoGs. Además, la nucleasa del parásito procesa cada hebra de ADN de forma independiente, al igual que se ha descrito para sus homólogas en otros organismos, y muestra, al igual que la nucleasa Nuc1p de levaduras, actividad endo-exonucleasa. La actividad de la proteína recombinante puede ser además inhibida por la acción del ácido aurín-tricarboxílico (ATA), que es un clásico inhibidor de nucleasas, y por el agente reductor β-mercaptoetanol. Este último resultado se debe probablemente a la presencia de un puente disulfuro entre los residuos de cisteína 100 y 116. La actividad de esta nucleasa se puede inhibir también por la acción del análogo de timidina LEI-49, que muestra una mayor actividad anti-Leishmania frente a parásitos que sobre-expresan EndoG que frente a parásitos silvestres. La EndoG de L. infantum contiene un péptido señal que causa su translocación a la mitocondria, donde se localiza en condiciones normales de crecimiento. Sin embargo, ante un estímulo de muerte como el tratamiento de los parásitos con edelfosina o miltefosina, la nucleasa es liberada de la mitocondria y se transloca hasta el núcleo, donde interviene en la degradación del ADN que tiene lugar durante el proceso apoptótico. Nuestros resultados también demuestran que la sobre-expresión de la proteína en parásitos tratados con edelfosina o miltefosina, causa un incremento significativo en el porcentaje de parásitos TUNEL positivos, pero no conlleva ningún efecto en condiciones normales de crecimiento. Por otro lado, parásitos que expresan niveles de proteína más bajos de lo normal crecen en menor medida que los parásitos silvestres y son más resistentes ante un estímulo de muerte, produciéndose una disminución del porcentaje de parásitos TUNEL positivos tras tratamiento con edelfosina con respecto a los parásitos silvestres. Análisis mutacionales revelan que los aminoácidos presentes en el centro catalítico de la nucleasa tienen diversa relevancia en su actividad catalítica. Así, mientras que la H214 y la R212 parecen fundamentales para la actividad de la enzima debido al papel que desempeñan en el ataque nucleófilo y en el reconocimiento de sustrato, respectivamente, la S211 no parece ser fundamental para la correcta arquitectura del sitio catalítico. Finalmente, hemos identificado un dominio putativo de auto-inhibición en la EndoG de L. infantum, que podría estar implicado en la regulación de la actividad catalítica de la misma

Estudio de la nucleasa EndoG en el protozoo "Leishmania infantum": caracterización e implicaciones en la supervivencia y muerte del parásito

Texto en español y resumen, conclusiones y artículos en inglésEn los últimos años, ha sido demostrado y aceptado que organismos unicelulares como los parasitos del género Leishmania son susceptibles de sufrir procesos de muerte celular que recuerdan a la apoptosis descrita en metazoos, y que pueden ser inducidos por una amplia variedad de estímulos. Sin embargo, los mecanismos moleculares que participan y regulan este proceso todavía son bastante desconocidos y se han descrito pobremente. Además, las moléculas que podrían participar en dichos procesos todavía no han sido identificadas. Debido a que la degradación del ADN es probablemente el evento más caracterizado durante el proceso similar a apoptosis que tiene lugar en Leishmania, nos hemos centrado en identificar a la nucleasa responsable de dicha degradación. Los resultados presentados en esta tesis demuestran que los promastigotes de Leishmania infantum expresan una nucleasa similar a las Endonucleasas G de eucariotas superiores que, de acuerdo con la predicción para su estructura, pertenece a la superfamilia de nucleasas con dominio ββα de unión a metal. El pH óptimo y los requerimientos catiónicos de la EndoG de L. infantum recuerdan a los determinados previamente para otras EndoGs. Además, la nucleasa del parásito procesa cada hebra de ADN de forma independiente, al igual que se ha descrito para sus homólogas en otros organismos, y muestra, al igual que la nucleasa Nuc1p de levaduras, actividad endo-exonucleasa. La actividad de la proteína recombinante puede ser además inhibida por la acción del ácido aurín-tricarboxílico (ATA), que es un clásico inhibidor de nucleasas, y por el agente reductor β-mercaptoetanol. Este último resultado se debe probablemente a la presencia de un puente disulfuro entre los residuos de cisteína 100 y 116. La actividad de esta nucleasa se puede inhibir también por la acción del análogo de timidina LEI-49, que muestra una mayor actividad anti-Leishmania frente a parásitos que sobre-expresan EndoG que frente a parásitos silvestres. La EndoG de L. infantum contiene un péptido señal que causa su translocación a la mitocondria, donde se localiza en condiciones normales de crecimiento. Sin embargo, ante un estímulo de muerte como el tratamiento de los parásitos con edelfosina o miltefosina, la nucleasa es liberada de la mitocondria y se transloca hasta el núcleo, donde interviene en la degradación del ADN que tiene lugar durante el proceso apoptótico. Nuestros resultados también demuestran que la sobre-expresión de la proteína en parásitos tratados con edelfosina o miltefosina, causa un incremento significativo en el porcentaje de parásitos TUNEL positivos, pero no conlleva ningún efecto en condiciones normales de crecimiento. Por otro lado, parásitos que expresan niveles de proteína más bajos de lo normal crecen en menor medida que los parásitos silvestres y son más resistentes ante un estímulo de muerte, produciéndose una disminución del porcentaje de parásitos TUNEL positivos tras tratamiento con edelfosina con respecto a los parásitos silvestres. Análisis mutacionales revelan que los aminoácidos presentes en el centro catalítico de la nucleasa tienen diversa relevancia en su actividad catalítica. Así, mientras que la H214 y la R212 parecen fundamentales para la actividad de la enzima debido al papel que desempeñan en el ataque nucleófilo y en el reconocimiento de sustrato, respectivamente, la S211 no parece ser fundamental para la correcta arquitectura del sitio catalítico. Finalmente, hemos identificado un dominio putativo de auto-inhibición en la EndoG de L. infantum, que podría estar implicado en la regulación de la actividad catalítica de la misma

Identification of a proteasome-targeting arylsulfonamide with potential for the treatment of Chagas' disease

Phenotypic screening identified an arylsulfonamide compound with activity against Trypanosoma cruzi, the causative agent of Chagas’ disease. Comprehensive mode of action studies revealed that this compound primarily targets the T. cruzi proteasome, binding at the interface between β4 and β5 subunits that catalyze chymotrypsin-like activity. A mutation in the β5 subunit of the proteasome was associated with resistance to compound 1, while overexpression of this mutated subunit also reduced susceptibility to compound 1. Further genetically engineered and in vitro-selected clones resistant to proteasome inhibitors known to bind at the β4/β5 interface were cross-resistant to compound 1. Ubiquitinated proteins were additionally found to accumulate in compound 1-treated epimastigotes. Finally, thermal proteome profiling identified malic enzyme as a secondary target of compound 1, although malic enzyme inhibition was not found to drive potency. These studies identify a novel pharmacophore capable of inhibiting the T. cruzi proteasome that may be exploitable for anti-chagasic drug discovery

Repositioning of a diaminothiazole series confirmed to target the cyclin-dependent kinase CRK12 for use in the treatment of African animal trypanosomiasis

African animal trypanosomiasis or nagana, caused principally by infection of the protozoan parasites Trypanosoma congolense and Trypanosoma vivax, is a major problem in cattle and other livestocks in sub-Saharan Africa. Current treatments are threatened by the emergence of drug resistance and there is an urgent need for new, effective drugs. Here, we report the repositioning of a compound series initially developed for the treatment of human African trypanosomiasis. A medicinal chemistry program, focused on deriving more soluble analogues, led to development of a lead compound capable of curing cattle infected with both T. congolense and T. vivax via intravenous dosing. Further optimization has the potential to yield a single-dose intramuscular treatment for this disease. Comprehensive mode of action studies revealed that the molecular target of this promising compound and related analogues is the cyclin-dependent kinase CRK12

Micromón València (Universitat de València)

En Julio de 2017 se creó la red SWI@Spain, auspiciada por el grupo de Docencia y Difusión de la Microbiología (DDM) de la Sociedad Española de Microbiología (SEM), para desarrollar la iniciativa internacional Small World Initiative (SWI) en la península ibérica. En la Universitat de València (UV) se constituyó entonces el grupo de Innovación Docente en Microbiología (IDM) para implementar el proyecto a nivel local. Avalados por el Servei de Formació Permanent i Innovació Educativa (SFPIE) de la UV, el grupo ha llevado a cabo diferentes iniciativas relacionadas con el objetivo fundamental del proyecto: divulgar la problemática actual relacionada con el uso inadecuado de antibióticos, el incremento de bacterias resistentes a éstos y la necesidad de encontrar nuevas moléculas con actividad antibacteriana para combatir las infecciones que provocan

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Spatiotemporal Characteristics of the Largest HIV-1 CRF02_AG Outbreak in Spain: Evidence for Onward Transmissions

Background and Aim: The circulating recombinant form 02_AG (CRF02_AG) is the predominant clade among the human immunodeficiency virus type-1 (HIV-1) non-Bs with a prevalence of 5.97% (95% Confidence Interval-CI: 5.41–6.57%) across Spain. Our aim was to estimate the levels of regional clustering for CRF02_AG and the spatiotemporal characteristics of the largest CRF02_AG subepidemic in Spain.Methods: We studied 396 CRF02_AG sequences obtained from HIV-1 diagnosed patients during 2000–2014 from 10 autonomous communities of Spain. Phylogenetic analysis was performed on the 391 CRF02_AG sequences along with all globally sampled CRF02_AG sequences (N = 3,302) as references. Phylodynamic and phylogeographic analysis was performed to the largest CRF02_AG monophyletic cluster by a Bayesian method in BEAST v1.8.0 and by reconstructing ancestral states using the criterion of parsimony in Mesquite v3.4, respectively.Results: The HIV-1 CRF02_AG prevalence differed across Spanish autonomous communities we sampled from (p &lt; 0.001). Phylogenetic analysis revealed that 52.7% of the CRF02_AG sequences formed 56 monophyletic clusters, with a range of 2–79 sequences. The CRF02_AG regional dispersal differed across Spain (p = 0.003), as suggested by monophyletic clustering. For the largest monophyletic cluster (subepidemic) (N = 79), 49.4% of the clustered sequences originated from Madrid, while most sequences (51.9%) had been obtained from men having sex with men (MSM). Molecular clock analysis suggested that the origin (tMRCA) of the CRF02_AG subepidemic was in 2002 (median estimate; 95% Highest Posterior Density-HPD interval: 1999–2004). Additionally, we found significant clustering within the CRF02_AG subepidemic according to the ethnic origin.Conclusion: CRF02_AG has been introduced as a result of multiple introductions in Spain, following regional dispersal in several cases. We showed that CRF02_AG transmissions were mostly due to regional dispersal in Spain. The hot-spot for the largest CRF02_AG regional subepidemic in Spain was in Madrid associated with MSM transmission risk group. The existence of subepidemics suggest that several spillovers occurred from Madrid to other areas. CRF02_AG sequences from Hispanics were clustered in a separate subclade suggesting no linkage between the local and Hispanic subepidemics

Monkeypox virus genomic accordion strategies

The 2023 monkeypox (mpox) epidemic was caused by a subclade IIb descendant of a monkeypox virus (MPXV) lineage traced back to Nigeria in 1971. Person-to-person transmission appears higher than for clade I or subclade IIa MPXV, possibly caused by genomic changes in subclade IIb MPXV. Key genomic changes could occur in the genome's low-complexity regions (LCRs), which are challenging to sequence and are often dismissed as uninformative. Here, using a combination of highly sensitive techniques, we determine a high-quality MPXV genome sequence of a representative of the current epidemic with LCRs resolved at unprecedented accuracy. This reveals significant variation in short tandem repeats within LCRs. We demonstrate that LCR entropy in the MPXV genome is significantly higher than that of single-nucleotide polymorphisms (SNPs) and that LCRs are not randomly distributed. In silico analyses indicate that expression, translation, stability, or function of MPXV orthologous poxvirus genes (OPGs), including OPG153, OPG204, and OPG208, could be affected in a manner consistent with the established "genomic accordion" evolutionary strategies of orthopoxviruses. We posit that genomic studies focusing on phenotypic MPXV differences should consider LCR variability.We would like to thank the work of the Rapid Response Unit of the National Center for Microbiology, especially MªJosé Buitrago, and Cristobal Belda, ISCIII General Director. We also thank Anya Crane (Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases, National Institutes of Health) for critically editing the manuscript and Jiro Wada (Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases, National Institutes of Health) for helping with figure preparation. The work for this study performed at Instituto de Salud Carlos III was partially funded by Acción Estratégica “Impacto clínico y microbiológico del brote por el virus de la viruela del mono en pacientes en España (2022): proyecto multicéntrico MONKPOX-ESP22” (CIBERINFEC) (M.P.S.S.). The work for this study done at the Icahn School of Medicine at Mount Sinai Department of Microbiology as part of Global Health Emerging Pathogen Institute activities was funded by institutional funds (G.P.) from the Icahn School of Medicine at Mount Sinai Department of Microbiology in support of Global Health Emerging Pathogen Institute activities. This work was also supported in part through Laulima Govern ment Solutions, LLC, prime contract with the U.S. National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272201800013C. J.H.K. performed this work as an employee of Tunnell Government Services (TGS), a subcontractor of Laulima Government Solutions, LLC, under Contract No. HHSN272201800013C. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of Health and Human Services or of the institutions and companies affiliated with the authors, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.S

Repositioning of a Diaminothiazole Series Confirmed to Target the Cyclin-Dependent Kinase CRK12 for Use in the Treatment of African Animal Trypanosomiasis

Coordinates for the homology model of T. brucei CRK12 with compound 2 docked into the ATP site, as shown in Figure 5 within the article