Person: López López, Nahikari
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López López
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Nahikari
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Publication Open Access Inactivation of the Thymidylate synthase thyA in non-typeable Haemophilus influenzae modulates antibiotic resistance and has a strong impact on its interplay with the host airways(Frontiers Media, 2017) Rodríguez Arce, Irene; Martí, Sara; Euba, Begoña; Fernández Calvet, Ariadna; Moleres Apilluelo, Javier; López López, Nahikari; Barberán, Montserrat; Ramos Vivas, José; Tubau, Fe; Losa, Carmen; Ardanuy, Carmen; Leiva, José; Yuste, José R.; Garmendia García, Juncal; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa; Gobierno de Navarra / Nafarroako GobernuaAntibacterial treatment with cotrimoxazol (TxS), a combination of trimethoprim and sulfamethoxazole, generates resistance by, among others, acquisition of thymidine auxotrophy associated with mutations in the thymidylate synthase gene thyA, which can modify the biology of infection. The opportunistic pathogen non-typeable Haemophilus influenzae (NTHi) is frequently encountered in the lower airways of chronic obstructive pulmonary disease (COPD) patients, and associated with acute exacerbation of COPD symptoms. Increasing resistance of NTHi to TxS limits its suitability as initial antibacterial against COPD exacerbation, although its relationship with thymidine auxotrophy is unknown. In this study, the analysis of 2,542 NTHi isolates recovered at Bellvitge University Hospital (Spain) in the period 2010–2014 revealed 119 strains forming slow-growing colonies on the thymidine low concentration medium Mueller Hinton Fastidious, including one strain isolated from a COPD patient undergoing TxS therapy that was a reversible thymidine auxotroph. To assess the impact of thymidine auxotrophy in the NTHi-host interplay during respiratory infection, thyA mutants were generated in both the clinical isolate NTHi375 and the reference strain RdKW20. Inactivation of the thyA gene increased TxS resistance, but also promoted morphological changes consistent with elongation and impaired bacterial division, which altered H. influenzae self-aggregation, phosphorylcholine level, C3b deposition, and airway epithelial infection patterns. Availability of external thymidine contributed to overcome such auxotrophy and TxS effect, potentially facilitated by the nucleoside transporter nupC. Although, thyA inactivation resulted in bacterial attenuation in a lung infection mouse model, it also rendered a lower clearance upon a TxS challenge in vivo. Thus, our results show that thymidine auxotrophy modulates both the NTHi host airway interplay and antibiotic resistance, which should be considered at the clinical setting for the consequences of TxS administration.Publication Open Access Eliminación del gen marcador de selección en plantas transplastómicas mediante la expresión transitoria de la recombinasa CRE(2011) López López, Nahikari; Veramendi Charola, Jon; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa; Producción Agraria; Nekazaritza EkoizpenaLa formación de plantas transgénicas o transplastómicas requiere de la incorporación al genoma, ya sea al nuclear o al plastidial, del gen de interés acompañado de un gen marcador de selección que, generalmente, confiere resistencia a un antibiótico. Una vez que se ha obtenido la planta transgénica o transplastómica, es aconsejable eliminar el gen marcador de selección de la planta debido a la carga genética que supone para las células pero sobre todo al riesgo para la salud humana y animal que implica la posible incorporación de este gen de resistencia a un antibiótico a bacterias patógenas pues puede disminuir la eficacia de los antibióticos. En este trabajo se utilizaron dos variedades transplastómicas de Nicotiana tabacum (tabaco). Estas variedades son: “ Virginia Gold ” y “ Havana 503 - B”. Llevan incorporados el gen “tiorredoxina” (“TRX”) y el gen marcador aadA que confiere resistencia al antibiótico espectinomicina y estreptomicina. Se propone un método para la eliminación de este gen marcador que consiste en la expresión transitoria de la recombinasa CRE la cual reconoce las secuencias lox flanqueadoras del gen aadA y suprime el fragmento de ADN que está en medio de estas dos secuencias. Se utilizó la agroinfiltración como método para incorporar la recombinasa CRE en el interior del tejido vegetal y que ésta se exprese de forma transitoria, sin introducirse en el núcleo celular. Se probaron cuatro condiciones diferentes variando la presión de vacío (2 Torr ó 10 Torr) y el tiempo que Agrobacterium estuvo en contacto con los explantos vegetales después de la agroinfiltración (2 ó 4 días). Las plantas obtenidas tras la agroinfiltración se analizaron mediante PCR para chequear la presencia del aadA y las negativas para este gen se analizaron mediante Southern blot. Además, paralelamente se colocaron fragmentos de hoja de las plantas que salieron negativas para la PCR en un medio de cultivo que contenía espectinomicina para comprobar la sensibilidad/resistencia a dicho antibiótico. Aunque algunas de las plantas salieron negativas en la PCR y presentaron sensibilidad a la espectinomicina, el Southern blot confirmó que todas ellas eran portadoras del gen marcador de selección aadA .Publication Open Access Resveratrol therapeutics combines both antimicrobial and immunomodulatory properties against respiratory infection by nontypeable Haemophilus influenzae(Springer Nature, 2017) Euba, Begoña; López López, Nahikari; Rodríguez Arce, Irene; Fernández Calvet, Ariadna; Barberán, Montserrat; Caturla, Nuria; Martí, Sara; Díez Martínez, Roberto; Garmendia García, Juncal; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaThe respiratory pathogen nontypeable Haemophilus influenzae (NTHi) is an important cause of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) that requires efficient treatments. A previous screening for host genes differentially expressed upon NTHi infection identified sirtuin-1, which encodes a NAD-dependent deacetylase protective against emphysema and is activated by resveratrol. This polyphenol concomitantly reduces NTHi viability, therefore highlighting its therapeutic potential against NTHi infection at the COPD airway. In this study, resveratrol antimicrobial effect on NTHi was shown to be bacteriostatic and did not induce resistance development in vitro. Analysis of modulatory properties on the NTHi-host airway epithelial interplay showed that resveratrol modulates bacterial invasion but not subcellular location, reduces inflammation without targeting phosphodiesterase 4B gene expression, and dampens β defensin-2 gene expression in infected cells. Moreover, resveratrol therapeutics against NTHi was evaluated in vivo on mouse respiratory and zebrafish septicemia infection model systems, showing to decrease NTHi viability in a dose-dependent manner and reduce airway inflammation upon infection, and to have a significant bacterial clearing effect without signs of host toxicity, respectively. This study presents resveratrol as a therapeutic of particular translational significance due to the attractiveness of targeting both infection and overactive inflammation at the COPD airway.Publication Open Access A simple experimental approach to simulate drought stress conditions in agar Petri dishes: characterization of the response in Medicago truncatula plantlets(2015) López López, Nahikari; González García, Esther; Seminario Huárriz, Amaia; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola TeknikoaDrought stress is the major factor in the reduction of crop yields causing significant economic losses. However, this stress is difficult to standardize under controlled conditions for research purposes. For molecular biology studies, which usually need highly standardized protocols for screening of large populations, PEG, mannitol or NaCl are employed to apply water deficit conditions. However, these osmolites do not properly simulate drought stress effects. In the present study, a simple experimental approach employing different agar concentration to simulate drought stress conditions in Petri dishes is tested with Medicago truncatula seedlings. The response of root morphology, respiration and drought stress markers is characterized under mild (≈ -1.0 MPa) and moderate drought (≈ -1,5MPa). Mild drought provoked an increase of root length that was not observed under moderate drought. Root area, volume and diameter decrease progressively with the increase of drought severity. Respiration rate was only significantly affected under moderate drought conditions, without changes in cytochrome and alternative pathways capacity. According to this, sucrose, malate and -ketoglutarate content decreased concomitantly with the increase of stress severity. Typical drought stress markers, as proline and amino acids, were also accumulated. Overall, these results validate this simple protocol to study drought stress response of seedlings under controlled conditions.