Person:
Moleres Apilluelo, Javier

Last Name

Moleres Apilluelo

First Name

Javier

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Instituto de Agrobiotecnología (IdAB)

ORCID

0000-0002-2749-6988

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810165

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Now showing 1 - 10 of 11

Open Access
Antagonistic pleiotropy in the bifunctional surface protein fadl (OmpP1) during adaptation of Haemophilus influenzae to chronic lung infection associated with chronic obstructive pulmonary disease
(American Society for Microbiology, 2018) Moleres Apilluelo, Javier; Fernández Calvet, Ariadna; Ehrlich, Rachel L.; Martí, Sara; Pérez Regidor, Lucía; Euba, Begoña; Rodríguez Arce, Irene; Balashov, Sergey; Cuevas, Ester; Liñares, Josefina; Ardanuy, Carmen; Martín Santamaría, Sonsoles; Ehrlich, Garth D.; Mell, Joshua Chang; Garmendia García, Juncal; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Tracking bacterial evolution during chronic infection provides insights into how host selection pressures shape bacterial genomes. The human-restricted opportunistic pathogen nontypeable Haemophilus influenzae (NTHi) infects the lower airways of patients suffering chronic obstructive pulmonary disease (COPD) and contributes to disease progression. To identify bacterial genetic variation associated with bacterial adaptation to the COPD lung, we sequenced the genomes of 92 isolates collected from the sputum of 13 COPD patients over 1 to 9 years. Individuals were colonized by distinct clonal types (CTs) over time, but the same CT was often reisolated at a later time or found in different patients. Although genomes from the same CT were nearly identical, intra-CT variation due to mutation and recombination occurred. Recurrent mutations in several genes were likely involved in COPD lung adaptation. Notably, nearly a third of CTs were polymorphic for null alleles of ompP1 (also called fadL), which encodes a bifunctional membrane protein that both binds the human carcinoembryonic antigen-related cell adhesion molecule 1 (hCEACAM1) receptor and imports long-chain fatty acids (LCFAs). Our computational studies provide plausible three-dimensional models for FadL’s interaction with hCEACAM1 and LCFA binding. We show that recurrent fadL mutations are likely a case of antagonistic pleiotropy, since loss of FadL reduces NTHi’s ability to infect epithelia but also increases its resistance to bactericidal LCFAs enriched within the COPD lung. Supporting this interpretation, truncated fadL alleles are common in publicly available NTHi genomes isolated from the lower airway tract but rare in others. These results shed light on molecular mechanisms of bacterial pathoadaptation and guide future research toward developing novel COPD therapeutics. IMPORTANCE Nontypeable Haemophilus influenzae is an important pathogen in patients with chronic obstructive pulmonary disease (COPD). To elucidate the bacterial pathways undergoing in vivo evolutionary adaptation, we compared bacterial genomes collected over time from 13 COPD patients and identified recurrent genetic changes arising in independent bacterial lineages colonizing different patients. Besides finding changes in phase-variable genes, we found recurrent loss-of-function mutations in the ompP1 (fadL) gene. We show that loss of OmpP1/FadL function reduces this bacterium’s ability to infect cells via the hCEACAM1 epithelial receptor but also increases its resistance to bactericidal fatty acids enriched within the COPD lung, suggesting a case of antagonistic pleiotropy that restricts ΔfadL strains’ niche. These results show how H. influenzae adapts to host-generated inflammatory mediators in the COPD airways.
Open Access
Genome expression profiling-based identification and administration efficacy of host-directed antimicrobial drugs against respiratory infection by nontypeable Haemophilus influenzae
(American Society for Microbiology, 2015) Euba, Begoña; Moleres Apilluelo, Javier; Segura, Víctor; Viadas Martínez, Cristina; Morey Sancho, Pau; Moranta, David; Leiva, José; Torres, Juan de; Bengoechea Alonso, José Antonio; Garmendia García, Juncal; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
Therapies that are safe, effective, and not vulnerable to developing resistance are highly desirable to counteract bacterial infections. Host-directed therapeutics is an antimicrobial approach alternative to conventional antibiotics based on perturbing host pathways subverted by pathogens during their life cycle by using host-directed drugs. In this study, we identified and evaluated the efficacy of a panel of host-directed drugs against respiratory infection by nontypeable Haemophilus influenzae (NTHi). NTHi is an opportunistic pathogen that is an important cause of exacerbation of chronic obstructive pulmonary disease (COPD). We screened for host genes differentially expressed upon infection by the clinical isolate NTHi375 by analyzing cell whole-genome expression profiling and identified a repertoire of host target candidates that were pharmacologically modulated. Based on the proposed relationship between NTHi intracellular location and persistence, we hypothesized that drugs perturbing host pathways used by NTHi to enter epithelial cells could have antimicrobial potential against NTHi infection. Interfering drugs were tested for their effects on bacterial and cellular viability, on NTHi-epithelial cell interplay, and on mouse pulmonary infection. Glucocorticoids and statins lacked in vitro and/or in vivo efficacy. Conversely, the sirtuin-1 activator resveratrol showed a bactericidal effect against NTHi, and the PDE4 inhibitor rolipram showed therapeutic efficacy by lowering NTHi375 counts intracellularly and in the lungs of infected mice. PDE4 inhibition is currently prescribed in COPD, and resveratrol is an attractive geroprotector for COPD treatment. Together, these results expand our knowledge of NTHi-triggered host subversion and frame the antimicrobial potential of rolipram and resveratrol against NTHi respiratory infection.
Open Access
Patho-adaptive evolution of Haemophilus spp. bacterial respiratory colonizing opportunistic pathogens
(2018) Moleres Apilluelo, Javier; Garmendia García, Juncal; Producción Agraria; Nekazaritza Ekoizpena
Esta tesis doctoral se ha centrado en tres aspectos de la pato-adaptación de dos especies bacterianas relacionadas filogenéticamente, que colonizan de forma asintomática y también son patógenos oportunistas en los sistemas respiratorios porcino y humano, respectivamente. Para ello, hemos analizado rasgos de evolución patoadaptativa bacteriana en un modelo de trabajo experimental (Capítulo 2) y de forma natural dentro del hospedador (Capítulos 1 y 3).
Open Access
Modulation of Haemophilus influenzae interaction with hydrophobic molecules by the VacJ/MlaA lipoprotein impacts strongly on its interplay with the airways
(Springer, 2018) Fernández Calvet, Ariadna; Rodríguez Arce, Irene; Almagro Zabalza, Goizeder; Moleres Apilluelo, Javier; Caballero Coronado, Lucía; Garmendia García, Juncal; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, 03/2016; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Airway infection by nontypeable Haemophilus influenzae (NTHi) associates to chronic obstructive pulmonary disease (COPD) exacerbation and asthma neutrophilic airway inflammation. Lipids are key inflammatory mediators in these disease conditions and consequently, NTHi may encounter free fatty acids during airway persistence. However, molecular information on the interplay NTHi-free fatty acids is limited, and we lack evidence on the importance of such interaction to infection. Maintenance of the outer membrane lipid asymmetry may play an essential role in NTHi barrier function and interaction with hydrophobic molecules. VacJ/MlaA-MlaBCDEF prevents phospholipid accumulation at the bacterial surface, being the only system involved in maintaining membrane asymmetry identified in NTHi. We assessed the relationship among the NTHi VacJ/MlaA outer membrane lipoprotein, bacterial and exogenous fatty acids, and respiratory infection. The vacJ/mlaA gene inactivation increased NTHi fatty acid and phospholipid global content and fatty acyl specific species, which in turn increased bacterial susceptibility to hydrophobic antimicrobials, decreased NTHi epithelial infection, and increased clearance during pulmonary infection in mice with both normal lung function and emphysema, maybe related to their shared lung fatty acid profiles. Altogether, we provide evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules.
Open Access
Novel blaROB-1-bearing plasmid conferring resistance to β-lactams in Haemophilus parasuis isolates from healthy weaning pigs
(American Society for Microbiology, 2015) Moleres Apilluelo, Javier; Santos López, Alfonso; Lázaro, Isidro; Labairu, Javier; Prat, Cristina; Ardanuy, Carmen; González Zorn, Bruno; Aragon, Virginia; Garmendia García, Juncal; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Haemophilus parasuis, the causative agent of Glässer’s disease, is one of the early colonizers of the nasal mucosa of piglets. It is prevalent in swine herds, and lesions associated with disease are fibrinous polyserositis and bronchopneumonia. Antibiotics are commonly used in disease control, and resistance to several antibiotics has been described in H. parasuis. Prediction of H. parasuis virulence is currently limited by our scarce understanding of its pathogenicity. Some genes have been associated with H. parasuis virulence, such as lsgB and group 1 vtaA, while biofilm growth has been associated with nonvirulent strains. In this study, 86 H. parasuis nasal isolates from farms that had not had a case of disease for more than 10 years were obtained by sampling piglets at weaning. Isolates were studied by enterobacterial repetitive intergenic consensus PCR and determination of the presence of lsgB and group 1 vtaA, biofilm formation, inflammatory cell response, and resistance to antibiotics. As part of the diversity encountered, a novel 2,661-bp plasmid, named pJMA-1, bearing the blaROB-1 β-lactamase was detected in eight colonizing strains. pJMA-1 was shown to share a backbone with other small plasmids described in the Pasteurellaceae, to be 100% stable, and to have a lower biological cost than the previously described plasmid pB1000. pJMA-1 was also found in nine H. parasuis nasal strains from a separate collection, but it was not detected in isolates from the lesions of animals with Glässer’s disease or in nontypeable Haemophilus influenzae isolates. Altogether, we show that commensal H. parasuis isolates represent a reservoir of β-lactam resistance genes which can be transferred to pathogens or other bacteria.
Open Access
Relative contribution of P5 and hap surface proteins to nontypable haemophilus influenzae interplay with the host upper and lower airways
(Public Library of Science, 2015) Euba, Begoña; Moleres Apilluelo, Javier; Viadas Martínez, Cristina; Ruiz de los Mozos Aliaga, Igor; Valle Turrillas, Jaione; Bengoechea Alonso, José Antonio; Garmendia García, Juncal; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, 359/2012
Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection.We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20.We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.
Open Access
Transformed recombinant enrichment profiling rapidly identifies HMW1 as an intracellular invasion locus in Haemophilus influenzae
(Public Library of Science, 2016) Mell, Joshua Chang; Viadas Martínez, Cristina; Moleres Apilluelo, Javier; Sinha, Sunita; Fernández Calvet, Ariadna; Porsch, Eric A.; St. Geme, Joseph W.; Nislow, Corey; Redfield, Rosemary J.; Garmendia García, Juncal; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, 359/2012
Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe “transformed recombinant enrichment profiling” (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen Haemophilus influenzae, a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the hmw1 operon from a clinical isolate (86- 028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing hmw1 and its paralog hmw2) was transformed by the same donor, allelic replacement of hmw2AHi375 by hmw1A86-028NP resulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1A86-028NP was confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by hmw1A86-028NP might be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1A86- 028NP bacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation.
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 Gobernua
Antibacterial 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.
Open Access
Transformación genética de cebada con At-SSIV y análisis de su efecto en el metabolismo del almidón y biomasa
(2011) Moleres Apilluelo, Javier; Bahaji, Abdellatif; Baroja Fernández, Edurne; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa
El almidón es el principal carbohidrato de reserva en plantas. Este metabolito es empleado por la planta para su crecimiento y desarrollo durante la ausencia de luz. Este valioso polisacárido es acumulado en órganos heterotróficos, tales como semillas de cereales, raíces de cassava y tubérculos de patata. El almidón representa la fuente más importante de carbohidratos en la dieta humana y posee muchas aplicaciones tanto comerciales como industriales (Delcour et al., 2010). Se han llevado a cabo muchas investigaciones a través de la ingeniería genética con el fin de mejorar el rendimiento y calidad de almidón, respondiendo a la demanda del sector industrial. Estudios recientes han demostrado que la almidón sintasa clase IV (starch synthase class IV, SSIV) de Arabidopsis Thaliana está envuelta en la iniciación de gránulos de almidón en hojas ya que el doble mutante en los genes SSIII y SSIV mostró un menor contenido de almidón las mismas (Szydlowski et al., 2009). Más significativo resulta el hecho de que la sobreexpresión de At-SSIV en Arabidopsis induce la acumulación de almidón en hojas y aumenta el contenido de almidón en tubérculos de patata (Gámez-Arjona et al., 2011). Con el objeto de caracterizar el papel de SSIV en la producción de biomasa y almidón tanto en órganos autótrofos como en heterótrofos, se generarán líneas de cebada (Golden Promise c.v.) que sobre-expresan SSIV y SSIV fusionada a la proteína verde fluorescente (SSIV-GFP). La transformación genética de la cebada fue llevada a cabo siguiendo el método descrito por Hansel et al. (2004) mediante la cepa de Agrobacterium Tumefaciens AGL1, utilizando los vectores binarios, PBract214-AtSSIV y PBract214-AtSSIV-GFP. Primeramente se seleccionarán líneas transgénicas de cebada mediante germinación en medio selectivo y análisis por PCR. Seguidamente se realizarán análisis por western blot para comprobar la expresión de SSIV y SSIV-GFP. Por último, se cuantificará el contenido en almidón y la biomasa en semillas y hojas de las líneas transgénicas de cebada seleccionadas.
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Estudio y análisis de costes de producción de explotaciones de tomate ecológico bajo invernaderos en Almería
(2009) Moleres Apilluelo, Javier; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa; Gestión de Empresas; Enpresen Kudeaketa