CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism.This work was funded by the Ministerio de Economía y Competitividad (BIO2011-24417)

Enzibióticos bactericidas mejorados frente a neumococo y otras bacterias

La presente invención se encuadra dentro del campo de la biotecnología. En la presente invención se presenta una secuencia polipeptídica derivada del módulo de unión a pared del enzima lítica del fago Cp7 (Cpl-7), que permite la construcción de nuevas enzimas líticas con actividad bactericida mejorada y amplio espectro. Igualmente, en esta invención se incluyen enzimas quiméricas que contienen dicho módulo de unión a pared mejorado y se dan ejemplos de su actividad frente a especies Gram-positivas y Gram-negativas.Peer reviewedConsejo Superior de Investigaciones Científicas, CIBER Enfermedades Respiratorias (CIBERES)A2 Solicitud de patente sin informe sobre el estado de la técnic

Substrate recognition and catalysis by LytB, a pneumococcal peptidoglycan hydrolase involved in virulence

17 p.-7 fig.-2 tab. Rico-Lastres, Palma et al.Streptococcus pneumoniae is a major cause of life-threatening diseases worldwide. Here we provide an in-depth functional characterization of LytB, the peptidoglycan hydrolase responsible for physical separation of daughter cells. Identified herein as an N-acetylglucosaminidase, LytB is involved also in colonization and invasion of the nasopharynx, biofilm formation and evasion of host immunity as previously demonstrated. We have shown that LytB cleaves the GlcNAc-β-(1,4)-MurNAc glycosidic bond of peptidoglycan building units. The hydrolysis occurs at sites with fully acetylated GlcNAc moieties, with preference for uncross-linked muropeptides. The necessity of GlcN acetylation and the presence of a single acidic moiety (Glu585) essential for catalysis strongly suggest a substrate-assisted mechanism with anchimeric assistance of the acetamido group of GlcNAc moieties. Additionally, modelling of the catalytic region bound to a hexasaccharide tripentapeptide provided insights into substrate-binding subsites and peptidoglycan recognition. Besides, cell-wall digestion products and solubilisation rates might indicate a tight control of LytB activity to prevent unrestrained breakdown of the cell wall. Choline-independent localization at the poles of the cell, mediated by the choline-binding domain, peptidoglycan modification, and choline-mediated (lipo) teichoic-acid attachment contribute to the high selectivity of LytB. Moreover, so far unknown chitin hydrolase and glycosyltransferase activities were detected using GlcNAc oligomers as substrate.Research was funded by grants from the Ministerio de Ciencia e Innovación (MICINN) and the Ministerio de Economía y Competitividad (MINECO) to P. García (SAF2009-10824 and SAF2012-39444-C02-01) and M. Menéndez (BFU2009-10052 and BFU2012-36825), the Consejería de Educación de la Comunidad de Madrid (S2010/BMD/2457) to M. Menéndez. The work in the United Kingdom and the US was supported by grants from the BBSRC (BB/G015902/1) to W. Vollmer and from the National Institutes of Health (GM61629) to S. Mobashery. Additional funding was provided by the CIBER de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III (ISCIII). Palma Rico-Lastres and Roberto Díez-Martínez were the recipients of fellowships from the MICINN (FPI program).Peer reviewe

Ecoestabilidad femenina y malnutrición severa infantil: Evidencia a partir de intervenciones de ayuda humanitaria de Acción Contra el Hambre en países africanos, Asiáticos y Latinoamericanos

Introducción: Evidencias previas han reportado diferencias en la condición nutricional de niños y niñas pertenecientes a la misma comunidad y sometidos a idénticas condiciones de privación alimentaria. El objetivo del presente trabajo es analizar las diferencias sexuales en prevalencia de malnutrición severa en menores de 5 años, sometidos a situación de crisis alimentaria. Métodos: Se analizaron datos recogidos en intervenciones de ayuda humanitaria llevadas a cabo por Acción Contra el Hambre entre 2002 y 2010 en 24 países. Dichas intervenciones se efectuaron en poblaciones de Africa, Latinoamérica y Asia que se encontraban en situación de grave crisis alimentaria. La muestra se compone de un total de 367.258 menores (186,156 niños y 181,102 niñas) con edad (E) entre 6 y 59 meses. Se midió el peso (P) y la talla (T) siguiendo la metodología SMART y se estimó la prevalencia de severo bajo peso (P/E <-3DE), desnutrición aguda severa (P/T <-3DE) y desnutrición crónica severa (T/E <-3DE) de acuerdo a los estándares de la OMS. Resultados: Para el total de la muestra, la proporción de niños con severo bajo peso fue de 9,8% en comparación al 7,3% de niñas (p <0.001). La desnutrición aguda severa afectó al 3,9% de los niños frente al 2,5% de las niñas (p <0.001). Las diferencias también fueron notables en la des-nutrición crónica: el 19, 5% de los niños frente al 15% de las niñas (p<0,001) presentaron crecimiento retardado. Conclusiones: Los resultados avalan la idea de la deno-minada eco-estabilidad femenina, de acuerdo a la cual, las mujeres serian menos sensibles a los factores externos que modulan el desarrollo ontogénico, mientras los varones se ve-rían más negativamente afectados por las agresiones medio-ambientales

The Genotype of the Donor for the (GT)n Polymorphism in the Promoter/Enhancer of FOXP3 Is Associated with the Development of Severe Acute GVHD but Does Not Affect the GVL Effect after Myeloablative HLA-Identical Allogeneic Stem Cell Transplantation

The FOXP3 gene encodes for a protein (Foxp3) involved in the development and functional activity of regulatory T cells (CD4+/CD25+/Foxp3+), which exert regulatory and suppressive roles over the immune system. After allogeneic stem cell transplantation, regulatory T cells are known to mitigate graft versus host disease while probably maintaining a graft versus leukemia effect. Short alleles (≤(GT)15) for the (GT)n polymorphism in the promoter/enhancer of FOXP3 are associated with a higher expression of FOXP3, and hypothetically with an increase of regulatory T cell activity. This polymorphism has been related to the development of auto- or alloimmune conditions including type 1 diabetes or graft rejection in renal transplant recipients. However, its impact in the allo-transplant setting has not been analyzed. In the present study, which includes 252 myeloablative HLA-identical allo-transplants, multivariate analysis revealed a lower incidence of grade III-IV acute graft versus host disease (GVHD) in patients transplanted from donors harboring short alleles (OR = 0.26, CI 0.08-0.82, p = 0.021); without affecting chronic GVHD or graft versus leukemia effect, since cumulative incidence of relapse, event free survival and overall survival rates are similar in both groups of patients