Person: Fernández Calvet, Ariadna
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Fernández Calvet
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Ariadna
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0000-0002-3340-703X
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Publication 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 PublikoaTracking 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.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 In vitro modeling of polyclonal infection dynamics within the human airways by Haemophilus influenzae differential fluorescent labeling(American Society for Microbiology, 2023) Rapún Araiz, Beatriz; Sorzabal-Bellido, Ioritz; Asensio López, Javier; Lázaro-Díez, María; Ariz Galilea, Mikel; Sobejano de la Merced, Carlos; Euba, Begoña; Fernández Calvet, Ariadna; Cortés Domínguez, Iván; Burgui Erice, Saioa; Toledo Arana, Alejandro; Ortiz de Solórzano, Carlos; Garmendia García, Juncal; Ingeniería Eléctrica, Electrónica y de Comunicación; Ingeniaritza Elektrikoa, Elektronikoa eta Telekomunikazio Ingeniaritza; Institute of Smart Cities - ISCStandardized clinical procedures for antibiotic administration rely on pathogen identification and antibiotic susceptibility testing, often performed on single-colony bacterial isolates. For respiratory pathogens, this could be questionable, as chronic patients may be persistently colonized by multiple clones or lineages from the same bacterial pathogen species. Indeed, multiple strains of nontypeable Haemophilus influenzae, with different antibiotic susceptibility profiles, can be co-isolated from cystic fibrosis and chronic obstructive pulmonary disease sputum specimens. Despite this clinical evidence, we lack information about the dynamics of H. influenzae polyclonal infections, which limits the optimization of therapeutics. Here, we present the engineering and validation of a plasmid toolkit (pTBH, toolbox for Haemophilus), with standardized modules consisting of six reporter genes for fluorescent or bioluminescent labeling of H. influenzae. This plasmid set was independently introduced in a panel of genomically and phenotypically different H. influenzae strains, and two of them were used as a proof of principle to analyze mixed biofilm growth architecture and antibiotic efficacy, and to visualize the dynamics of alveolar epithelial co-infection. The mixed biofilms showed a bilayer architecture, and antibiotic efficacy correlated with the antibiotic susceptibility of the respective single-species strains. Furthermore, differential kinetics of bacterial intracellular location within subcellular acidic compartments were quantified upon co-infection of cultured airway epithelial cells. Overall, we present a panel of novel plasmid tools and quantitative image analysis methods with the potential to be used in a whole range of bacterial host species, assay types, and¿or conditions and generate meaningful information for clinically relevant settings.Publication Open Access Mecanismos moleculares de adaptación del patógeno respiratorio Haemophilus influenzae y desarrollo de nuevos antimicrobianos(2020) Fernández Calvet, Ariadna; Garmendia García, Juncal; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Gobierno de Navarra / Nafarroako GobernuaLa tesis doctoral aborda tres aspectos de la interacción entre el patógeno oportunista colonizador Haemophilus influenzae no tipificable (HiNT) y el sistema respiratorio humano, considerando la regulación patoadaptativa por variación de fase (Capítulo 1), la importancia del mantenimiento de la integridad superficial bacteriana (Capítulo 2), y el potencial terapéutico de moléculas xenohorméticas (Capítulo 3). En conjunto, este trabajo amplía nuestro conocimiento sobre los mecanismos moleculares de patoadaptación respiratoria de HiNT, proporciona evidencias sobre el papel de VacJ/MlaA en la modulación de la supervivencia bacteriana en las vías respiratorias, y señala el potencial terapéutico de moléculas xenohorméticas.Publication Open Access Imipenem heteroresistance but not tolerance in Haemophilus influenzae during chronic lung infection associated with chronic obstructive pulmonary disease(Frontiers Media, 2023) Gil Campillo, Celia; González-Díaz, Aida; Rapún Araiz, Beatriz; Iriarte-Elizaintzin, Oihane; Elizalde Gutiérrez, Iris; Fernández Calvet, Ariadna; Lázaro-Díez, María; Martí, Sara; Garmendia García, Juncal; Ciencias; Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMABAntibiotic resistance is a major Public Health challenge worldwide. Mechanisms other than resistance are described as contributors to therapeutic failure. These include heteroresistance and tolerance, which escape the standardized procedures used for antibiotic treatment decision-making as they do not involve changes in minimal inhibitory concentration (MIC). Haemophilus influenzae causes chronic respiratory infection and is associated with exacerbations suffered by chronic obstructive pulmonary disease (COPD) patients. Although resistance to imipenem is rare in this bacterial species, heteroresistance has been reported, and antibiotic tolerance cannot be excluded. Moreover, development of antibiotic heteroresistance or tolerance during within-host H. influenzae pathoadaptive evolution is currently unknown. In this study, we assessed imipenem resistance, heteroresistance and tolerance in a previously sequenced longitudinal collection of H. influenzae COPD respiratory isolates. The use of Etest, disc diffusion, population analysis profiling, tolerance disc (TD)-test methods, and susceptibility breakpoint criteria when available, showed a significant proportion of imipenem heteroresistance with differences in terms of degree among strains, absence of imipenem tolerance, and no specific trends among serial and clonally related strains could be established. Analysis of allelic variation in the ftsI, acrA, acrB, and acrR genes rendered a panel of polymorphisms only found in heteroresistant strains, but gene expression and genome-wide analyses did not show clear genetic traits linked to heteroresistance. In summary, a significant proportion of imipenem heteroresistance was observed among H. influenzae strains isolated from COPD respiratory samples over time. These data should be useful for making more accurate clinical recommendations to COPD patients.Publication 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 PublikoaAirway 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.Publication 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, 359/2012Many 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.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.