Mechanical Identities of RNA and DNA Double Helices Unveiled at the Single-Molecule Level

[EN] Double-stranded (ds) RNA is the genetic material of a variety of viruses and has been recently recognized as a relevant molecule in cells for its regulatory role. Despite that the elastic response of dsDNA has been thoroughly characterized in recent years in single-molecule stretching experiments, an equivalent study with dsRNA is still lacking. Here, we have engineered long dsRNA molecules for their individual characterization contrasting information with dsDNA molecules of the same sequence. It is known that dsRNA is an A-form molecule unlike dsDNA, which exhibits B-form in physiological conditions. These structural types are distinguished at the single-molecule level with atomic force microscopy (AFM) and are the basis to understand their different elastic response. Force¿extension curves of dsRNA with optical and magnetic tweezers manifest two main regimes of elasticity, an entropic regime whose end is marked by the A-form contour- length and an intrinsic regime that ends in a low-cooperative overstretching transition in which the molecule extends to 1.7 times its A-form contour-length. DsRNA does not switch between the A and B conformations in the presence of force. Finally, dsRNA presents both a lower stretch modulus and overstretching transition force than dsDNA, whereas the electrostatic and intrinsic contributions to the persistence length are larger.This work was supported by grants from the Spanish Ministry of Science and Innovation (BFU2011-29038 and BFU2010-15703) and the Comunidad de Madrid (S2009/MAT/1507). IRA.-G. acknowledges a Ramon y Cajal contract from the Spanish Ministry of Science and Innovation (RYC-2007-01765). Work in the F.M.-H. laboratory was supported by a Starting Grant from the European Research Council (no. 206117) and a grant from the Spanish Ministry of Science and Innovation (FIS2011-24638). We thank M. S. Dillingham for kindly providing the pSP73-JY0 plasmid, M. Menendez for access to a spectropolarimeter, A. Monserrate for polylysine-AFM control experiments, and B. Ibarra for fruitful discussions.Herrero-Galán, E.; Fuentes-Perez. M.E.; Carrasco, C.; Valpuesta, J.; Carrascosa, J.; Moreno-Herrero, F.; Arias-Gonzalez, JR. (2013). Mechanical Identities of RNA and DNA Double Helices Unveiled at the Single-Molecule Level. Journal of the American Chemical Society. 135(1):122-131. https://doi.org/10.1021/ja3054755S122131135

Selection of Salmonella enterica Serovar Typhi Genes Involved during Interaction with Human Macrophages by Screening of a Transposon Mutant Library

The human-adapted Salmonella enterica serovar Typhi (S. Typhi) causes a systemic infection known as typhoid fever. This disease relies on the ability of the bacterium to survive within macrophages. In order to identify genes involved during interaction with macrophages, a pool of approximately 105 transposon mutants of S. Typhi was subjected to three serial passages of 24 hours through human macrophages. Mutants recovered from infected macrophages (output) were compared to the initial pool (input) and those significantly underrepresented resulted in the identification of 130 genes encoding for cell membrane components, fimbriae, flagella, regulatory processes, pathogenesis, and many genes of unknown function. Defined deletions in 28 genes or gene clusters were created and mutants were evaluated in competitive and individual infection assays for uptake and intracellular survival during interaction with human macrophages. Overall, 26 mutants had defects in the competitive assay and 14 mutants had defects in the individual assay. Twelve mutants had defects in both assays, including acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, SPI-4, STY1867-68, and STY2346. The complementation of several mutants by expression of plasmid-borne wild-type genes or gene clusters reversed defects, confirming that the phenotypic impairments within macrophages were gene-specific. In this study, 35 novel phenotypes of either uptake or intracellular survival in macrophages were associated with Salmonella genes. Moreover, these results reveal several genes encoding molecular mechanisms not previously known to be involved in systemic infection by human-adapted typhoidal Salmonella that will need to be elucidated

Antimicrobial susceptibility trends and evolution of isolates with extended spectrum ß-lactamases among gram-negative organisms recovered during the SMART study in Spain (2011-2015)

Introducción. El estudio SMART (Study for Monitoring Antimicrobial Resistance Trends) monitoriza la sensibilidad antimicrobiana y las ß-lactamasas de espectro extendido (BLEE) en bacilos gramnegativos obtenidos de infecciones intraabdominales (IIA). Material y Métodos. Se ha analizado la sensibilidad antimicrobiana (microdilución estándar, criterios EUCAST) y las BLEE (detección fenotípica) de 5.343 aislados de IIA en 11 centros del programa SMART-España durante 2011-2015 en comparación con 2002-2010. Resultados. Escherichia coli, el microorganismo más prevalente, disminuyó significativamente en las IIA de origen comunitario (60, 9% 2002-2010 vs. 56, 1% 2011- 2015, P=0, 0003). Fue seguido en prevalencia por Klebsiella pneumoniae que aumentó tanto en IIA comunitaria (8, 9% vs. 10, 8%, P=0, 016) como nosocomial (9, 2% vs. 10, 8%, P=0, 029) y por P. aeruginosa que aumentó en la IIA comunitaria (5, 6% vs. 8, 0%, P=0, 0003). Las BLEE fueron más prevalentes en la IIA nosocomial por K. pneumoniae (16, 3%) que por E. coli (9, 5%), siendo más frecuentes en pacientes de mayor edad (>60 años). Considerando todas las Enterobacteriaceae, ertapenem (92, 3- 100%) y amikacina (95, 5%-100%) fueron los antimicrobianos más activos. La sensibilidad a ertapenem, al contrario que a amoxicilina-clavulánico o piperacilina-tazobactam, se mantuvo sin cambios en E. coli con (98, 8%) y sin BLEE (100%). Su sensibilidad disminuyó en BLEE-K. pneumoniae (74, 7%) pero fue mayor que la de amoxicilina-clavulánico (14, 0%) o piperacilina-tazobactam (24, 0%). Es de resaltar que esta actividad se mantuvo >60% en los aislados con BLEE resistentes a amoxicilina-clavulánico, piperacilina-tazobactam o fluoroquinolonas. Conclusiones. El estudio SMART-España sustenta las guías actuales que incluyen al ertapenem como tratamiento empírico en la IIA leve-moderada comunitaria, en particular con BLEE. Estas recomendaciones precisaran actualizarse con la reciente introducción de nuevos antimicrobianos. Introduction. The SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study monitors antimicrobial susceptibility and extended spectrum ß-lactamases (ESBLs) in Gram-negative bacilli recovered from intra-abdominal infections (IAI). Material and methods. Antimicrobial susceptibility of 5, 343 isolates from IAI recovered in 11 centres during the 2011-2015 SMART-Spain program was analysed by standard microdilution (EUCAST criteria) and compared with that from 2002-2010. ESBLs were phenotypically detected. Results. Escherichia coli, the most common isolate, significantly decreased in community acquired IAI (60.9% 2002-2010 vs. 56.1% 2011-2015, P=0.0003). It was followed in prevalence by Klebsiella pneumoniae that increased both in the community (8.9% vs. 10.8%, P=0.016) and nosocomial (9.2% vs. 10.8%, P=0.029) IAI and P. aeruginosa, which significantly increased in community acquired IAI (5.6% vs. 8.0%, P=0.0003). ESBLs were more prevalent in K. pneumoniae (16.3%) than in E. coli (9.5%) of nosocomial origin and were more frequently isolated from elderly patients (>60 years). Considering all Enterobacteriaceae, ertapenem (92.3-100%) and amikacin (95.5%-100%) were the most active antimicrobials. Ertapenem activity, unlike amoxicillin-clavulanate or piperacillin-tazobactam, remained virtually unchanged in ESBL (100%) and non-ESBL (98.8%) E. coli producers. Its activity decreased in ESBL-K. pneumoniae (74.7%) but was higher than that of amoxicillin-clavulanate (14.0%) and piperacillin-tazobactam (24.0%). Interestingly, ertapenem susceptibility was maintained in >60% of ESBL isolates that were resistant to amoxicillin-clavulanate, piperacillin-tazobactam or fluoroquinolones. Conclusions. SMART-Spain results support current guidelines which include ertapenem as empiric treatment in mild-moderate community-acquired IAI, particularly with ESBL producers. These recommendations will need to be updated with the recently introduction of new antimicrobials