BUSCADOR RECOLECTA

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.

The 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 ß 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.

Respiratory infections pose a continuous threat to humans due to their easy dissemination via aerial transmission. As a consequence, they are leading causes of mortality and morbidity worldwide. Lower respiratory tract infections (LRTI) remained the deadliest communicable diseases causing 3 million deaths worldwide in 2016 (1). Similarly, although the number of tuberculosis (TB) deaths tends to decrease, it is still among the top 10 causes of global mortality with a yearly death burden of about 1.6 million (2). The growing emergence of bacterial antibiotic resistance is a major global challenge for the coming years, and several major respiratory pathogens are included in the WHO priority list of bacteria for which new antibiotics are urgently needed (3). In terms of target population, children under the age of five are the most susceptible hosts to a plethora of respiratory pathogens.

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here.

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)., This PhD thesis work has been focused on three independent aspects of the pathoadaptation
by two closely related bacterial species of host-restricted respiratory
colonizing opportunistic pathogens. Bacterial patho-adaptive evolution has been
considered in both experimental (Chapter 2) and within-host (Chapters 1 and 3) settings., Este trabajo de tesis se ha llevado a cabo mediante el disfrute de un contrato
de Formación de Personal Investigador (FPI) con referencia BES-2013-062644
por parte de Javier Moleres Apilluelo, adscrito a los proyectos SAF2012-31166
(enero 2014-diciembre 2015), SAF2015-66520-R (enero 2016-diciembre 2017)
concedidos a la Dra. Junkal Garmendia García., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

La 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., This PhD Thesis work tackled three aspects of the interaction between the colonizing opportunistic pathogen nontypeable Haemophilus influenzae (NTHi) and the human airways, by considering the concepts of phase-variable regulation of pathoadaptive traits (Chapter 1), the importance of molecular systems involved in maintaining the bacterial surface integrity (Chapter 2), and the therapeutic potential of xenohormetic molecules (Chapter 3).
Altogether, this work contributes expanding our understanding on molecular mechanisms of NTHi pathoadaptation regulated by phase variation, provides evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules, and highlights the therapeutic potential of xenohormetic molecules against NTHi infection., Este trabajo de Tesis Doctoral se ha desarrollado mediante el disfrute de contratos adscritos a los proyectos Departamento de Innovación, Empresa y Empleo IIQ14064.RI1 (Gobierno de Navarra); SAF2012-311666 (Ministerio deEconomía y Competitividad), SAF2015-66520-R (Ministerio de Economía y Competitividad), Departamento de Salud 3/2016 (Gobierno de Navarra) y RTI2018-096369-B-I00 (Ministerio de Ciencia, Innovación y Universidades)., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

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., This work has been funded by MINECO grants SAF2012-31166 and SAF2015-66520-R to J.G. and grants CTQ2014-57141-R and CTQ2017-88353-R to S.M.-S.; grant 03/2016 from the Health Department, Regional Government of Navarra, Spain, and SEPAR grant 31/2015 to J.G.; and NIDCD grant 5R01 DC 02148 and NIDDK grant 1U01 DK082316 from the U.S. National Institutes of Health to G.D.E. CIBERES is an initiative from the Instituto de Salud Carlos III (ISCIII), Madrid, Spain. J.M. and L.P.-R. were funded by Ph.D. studentships BES-2013-062644 and BES-2012-053653 from MINECO, Spain. A.F.-C. was funded by a contract from MINECO, reference number 20132RC947. I.R.-A. was funded by a Ph.D. studentship from the Universidad Pública de Navarra, Spain. S.M. is funded by a postdoctoral contract from CIBERES.

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., A.F.C was funded by a contract from Ministerio Economía y Competitividad-MINECO, reference 20132RC947, Spain; I.R.A. is funded by a PhD studentship from Universidad Pública de Navarra, Spain; J.M. was funded by PhD studentship BES-2013-062644 from MINECO; S.M. is funded by a postdoctoral contract from CIBERES; L.C. was funded by a contract from MINECO, reference CS_NAV_IDAB_005, Spain; T.L.B. is the recipient of a PhD fellowship funded by the Department for Employment and Learning (Northern Ireland, UK). This work has been funded by grants from MINECO SAF2012-31166 and SAF2015-66520-R, from Health Department, Regional Govern from Navarra, Spain, reference 03/2016, and from SEPAR 31/2015 to J.G.; and by grant from MINECO DPI2015-64221 to COdS. CIBER is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain.

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., This work was supported by National Institutes of Health Ruth Kirschstein postdoctoral fellowship F32AI084427 (to JCM); a Canadian Institute of Health Research operating grant (to RJR); Genome British Columbia grant SOF122 (to RJR and JCM); the Faculty of Pharmaceutical Sciences, Canadian Foundation for Innovation (to CN); National Institutes of Heath R01 grant DC002873 (to JWSG); and (to JG) grants from Ministerio Economía y Competitividad-MINECO SAF2012-31166 and SAF2015-66520-R, Dpto. Salud Gobierno Navarra 359/2012 and Ministerio de Educación PRX12/00191.

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., IR is funded by a Ph.D. studentship from Universidad Pública de Navarra, Spain; JM is funded by Ph.D. studentship BES-2013-062644 from Ministerio Economía y Competitividad-MINECO, Spain; SM is funded by a postdoctoral contract from CIBER Enfermedades Respiratorias (CIBERES); NL is funded by a contract from Department of Economy, Regional Govern from Navarra, Spain, reference 0011-1307-2015-000037. This work has been funded by grants from MINECO SAF2012-31166 and SAF2015-66520-R, Health Department, Regional Govern from Navarra, Spain, reference 03/2016, and SEPAR 31/2015 to JG. CIBERES is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain. The authors acknowledge support of the publication fee by the CSIC Open Access Publication Support
Initiative through its Unit of Information Resources for Research (URICI).

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here., The authors gratefully acknowledge financial support from the “CIBER
de Enfermedades Respiratorias” (CIBERES), an initiative of
the “Instituto de Salud Carlos III” (ISCIII), Spain. Research
activities in the participating laboratories received further
funding from the following sources: Centro Nacional de
Microbiología, ISCIII, PI15CIII/00024 and MINECO (SAF2015-
67033-R); Centro Nacional de Biotecnología, MINECO
(BFU2014-57797-R); Hospital Universitari Germans Trias
I Pujol, Spanish Society of Pneumology and Thoracic
Surgery (SEPAR 054/2011); Departamento de Bioquímica y
Biología Molecular I, MINECO (SAF2015-65307-R); Centro de
Investigaciones Biológicas, MINECO (SAF2012-39444-C01/02);
Fundación de Investigación Sanitaria de las Islas Baleares,
MINECO (SAF2012-39841); Instituto de Agrobiotecnología,
MINECO (SAF2015-66520-R); Instituto de Química Física
Rocasolano, MINECO (BFU2015-70052-R) and the Marie
Curie Initial Training Network GLYCOPHARM (PITN-GA-
2012-317297). Subprograma Estatal de Formación (BES-2013-
065355).

The 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., N.L.L. was funded by a contract from Department of Economy, Regional Govern from Navarra, Spain, reference 0011–1307; I.R.A. is funded by a PhD studentship from Universidad Pública de Navarra, Spain. This work has been funded by grants from MINECO SAF2012-31166 and SAF2015-66520-R, Health Department, Regional Govern from Navarra, Spain, reference 3/2016, and SEPAR 31/2015 to J.G.

Este trabajo de tesis doctoral se centra en el estudio de las bases moleculares de la infección respiratoria por la bacteria Haemophilus influenzae, miembro del microbioma respiratorio humano y patógeno oportunista de especial relevancia en la progresión de
la Enfermedad Pulmonar Obstructiva Crónica (EPOC)., This PhD thesis has been focused on the study of the molecular basis of respiratory
infection by the bacterium Haemophilus influenzae, a member of the human respiratory
microbiome, but also an opportunistic pathogen of main relevance in the progression of
Chronic Obstructive Pulmonary Disease (COPD)., Este trabajo se ha desarrollado mediante el disfrute de un contrato de Formación de Personal Investigador de la Universidad Pública de Navarra por parte de Irene Rodríguez Arce, adscrito a los proyectos SAF2012-31166 (junio 2014-diciembre 2015), SAF2015-66520-R (enero 2016-junio 2018), y Departamento de Salud Gobierno de Navarra proyecto 3/2016 (diciembre 2016-junio 2018), concedidos a la Dra. Junkal Garmendia García., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here.

Recognition of bacterial surface epitopes by host receptors plays an important role in the infectious process and is intimately associated with bacterial virulence. Delineation of bacteria-host interactions commonly relies on the detection of binding events between purified bacteria- and host-target molecules. In this work, we describe a combined microarray and quartz crystal microbalance (QCM) approach for the analysis of carbohydrate-mediated interactions directly on the bacterial surface, thus preserving the native environment of the bacterial targets. Nontypeable Haemophilus influenzae (NTHi) was selected as a model pathogenic species not displaying a polysaccharide capsule or O-antigen-containing lipopolysaccharide, a trait commonly found in several important respiratory pathogens. Here, we demonstrate the usefulness of NTHi microarrays for exploring the presence of carbohydrate structures on the bacterial surface. Furthermore, the microarray approach is shown to be efficient for detecting strain-selective binding of three innate immune lectins, namely, surfactant protein D, human galectin-8, and Siglec-14, to different NTHi clinical isolates. In parallel, QCM bacteria-chips were developed for the analysis of lectin-binding kinetics and affinity. This novel QCM approach involves capture of NTHi on lectin-derivatized chips followed by formaldehyde fixation, rendering the bacteria an integrated part of the sensor chip, and subsequent binding assays with label-free lectins. The binding parameters obtained for selected NTHi-lectin pairs provide further insights into the interactions occurring at the bacterial surface., We gratefully acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (Grants BFU2012-36825, BFU2015-70052-R, SAF2012-31166, and SAF2015-66520-R), the Department of Health of the Navarra Government (ref 359/2012), the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII), and the Marie Curie Initial Training Networks DYNANO (Grant PITN-GA-2011-289033), GLYCOPHARM (Grant PITN-GA-2012-317297), and WntsApp (GA-No. 608180, FP7-PEOPLE-2013). I.K. and D.P. were funded by Marie Curie contracts from the European Commission., Peer Reviewed

[Abstract] 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 hmw2Aby hmw1Aresulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1Awas 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 hmw1Amight be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1Abacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation., [Author Summary] Many bacteria are naturally competent, actively taking up DNA from their surroundings and incorporating it into their genomes by homologous recombination. This cellular process has had a large impact on the evolution of these species, for example by enabling pathogens to acquire virulence factors and antibiotic resistances from their relatives. But natural competence can also be exploited by researchers to identify the underlying genetic variation responsible for naturally varying phenotypic traits, similar to how eukaryotic geneticists use meiotic recombination during sexual reproduction to create genetically admixed populations. Here we exploited natural competence, phenotypic selection, and deep sequencing to rapidly identify the hmw1 locus as a major contributor to intracellular invasion of airway epithelial cells by the human pathogen Haemophilus influenzae, a trait that likely allows bacterial cells to evade the immune system and therapeutic interventions during chronic infections. Genetic variation in this locus can strongly modulate bacterial intracellular invasion rates, and possession of a certain allele favors adhesion and self-aggregation, which appear to prompt bacteria to invade airway cells as groups, rather than as individuals. Overall, our findings indicate that targeting HMW1 could block the ability of H. influenzae to invade airway cells, which would make antibiotic therapy to treat chronic lung infections more effective. Furthermore, our new approach to identifying the genetic basis of natural phenotypic variation is applicable to a wide-range of phenotypically selectable traits within the widely distributed naturally competent bacterial species, including pathogenesis traits in many human pathogens., This work was supported by National Institutes of Health Ruth Kirschstein postdoctoral fellowship F32AI084427 (to JCM); a Canadian Institute of Health Research operating grant (to RJR); Genome British Columbia grant SOF122 (to RJR and JCM); the Faculty of Pharmaceutical Sciences, Canadian Foundation for Innovation (to CN); National Institutes of Heath R01 grant DC002873 (to JWSG); and (to JG) grants from Ministerio Economía y Competitividad-MINECO SAF2012-31166 and SAF2015-66520-R, Dpto. Salud Gobierno Navarra 359/2012 and Ministerio de Educación PRX12/00191., Peer Reviewed

23 p.-9 fig.-4 tab., 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 9years. 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 DeltafadL strains' niche. These results show how H. influenzae adapts to host-generated inflammatory mediators in the COPD airways., This work has been funded by MINECO grants SAF2012-31166 and SAF2015-66520-R to J.G. and grants CTQ2014-57141-R and CTQ2017-88353-R to S.M.-S.; grant 03/2016 from the Health Department, Regional Government of Navarra, Spain, and SEPAR grant 31/2015 to J.G.; and NIDCD grant 5R01 DC 02148 and NIDDK grant 1U01 DK082316 from the U.S. National Institutes of Health to G.D.E. CIBERES is an initiative from the Instituto de Salud Carlos III (ISCIII), Madrid, Spain. J.M. and L.P.-R. were funded by Ph.D.studentships BES-2013-062644 and BES-2012-053653 from MINECO, Spain., Peer reviewed

The 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., We thank Javier Moleres for his technical support. N.L.L. was funded by a contract from Department of Economy,
Regional Govern from Navarra, Spain, reference 0011–1307; I.R.A. is funded by a PhD studentship from
Universidad Pública de Navarra, Spain. This work has been funded by grants from MINECO SAF2012-31166
and SAF2015-66520-R, Health Department, Regional Govern from Navarra, Spain, reference 3/2016, and SEPAR
31/2015 to J.G. CIBERES is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain., Peer reviewed

Trabajo presentado en el 19th European Carbohydrate Symposium EuroCarb, celebrado en Barcelona (España), del 2 al 6 de julio de 2017, Bacterial glycocalyx components are intimately associated with virulence and commonly used for
strain typification. Up until recently, analysis of their recognition by lectins and antibodies was
commonly performed following isolation of selected constituents from the pathogen surface. Of
note, in these ligand-targeted approaches, the natural presentation and accessibility of glycans along
with possible cooperativity of neighbour molecules are not considered, an aspect particularly
relevant in the case of weak binders. We recently developed a combined approach of static
microarrays and QCM biosensor assays under flow, based on the use of entire bacteria, and
demonstrated its usefulness for detection and real-time monitoring of lectin recognition processes
[1]. We benefitted from the availability of a panel of nontypeable Heamophilus influenzae (strain
NTHi375) mutants, defective for selected lipooligosaccharide (LOS)-specific enzymes, and focused
our studies on the effect of LOS truncation in lectin binding to the bacteria. Intriguingly, LOS
truncation gave rise to distinct binding profiles for Viscum album and Ricinus communis
agglutinins, pointing to the recognition of different Gal-bearing targets. Besides the LOS molecule,
a variety of glycoconjugates can be present at the bacterial surface, also encompassing
glycoproteins of great significance in pathogenesis. In particular, in NTHi375, hmw loci code for
two heavily glycosylated adhesins that are absent in other NTHi strains, as e.g. Rd KW20, a noninvasive laboratory strain. In this work, we pursued the dissection of bacterial surface ligands
recognised by these two lectins. To this aim, we comparatively examined their binding to NTHi375,
an NTHi375Δomp5 mutant suspected to overexpress LOS, Rd KW20, and an isogenic Rd KW20
mutant expressing HMW1, using our combined bacteria-based microarray and QCM approach. In
parallel, recognition of the isolated NTHi375 and Rd KW20 LOSs, whose structures are known to
be different, was also examined using LOS-based microarrays. Furthermore, saturation transfer
difference NMR experiments allowed the identification of the sugar residues involved in the
recognition. Altogether, the results revealed strain- and lectin-specific binding patterns, from the
level of LOS molecules to the intact bacterial surface., This work was supported by the Marie Curie Initial Training Networks DYNANO
(PITN-GA-2011-289033), GLYCOPHARM (PITN-GA-2012-317297) and WntsApp (PITN-GA-2013-
608180), the CIBER of Respiratory Diseases (CIBERES) – an initiative from the Spanish Institute of Health
Carlos III (ISCIII), and the Spanish Ministry of Economy and Competitiveness (grants CTQ2015-64597-C2-
2-P, BFU2012-36825, BFU2015-70052-R, SAF2012-31166 and SAF2015-66520-R). I.K. was funded by a
Marie Curie contract from the European Commission., Peer reviewed

Tesis llevada a cabo para conseguir el grado de Doctor por la Universidad de Pública de Navarra.--2018-12-20.--Sobresaliente cum laudem, [ES] Este trabajo de tesis doctoral se centra en el estudio de las bases moleculares de la infección respiratoria por la bacteria Haemophilus influenzae, miembro del microbioma respiratorio humano y patógeno oportunista de especial relevancia en la progresión de la Enfermedad Pulmonar Obstructiva Crónica (EPOC)., [EN] This PhD thesis has been focused on the study of the molecular basis of respiratory infection by the bacterium Haemophilus influenzae, a member of the human respiratory microbiome, but also an opportunistic pathogen of main relevance in the progression of Chronic Obstructive Pulmonary Disease (COPD)., Este trabajo se ha desarrollado mediante el disfrute de un contrato de Formación de Personal Investigador de la Universidad Pública de Navarra por parte de Irene Rodríguez Arce, adscrito a los proyectos SAF2012-31166 (junio 2014-diciembre 2015), SAF2015-66520-R (enero 2016-junio 2018), y Departamento de Salud Gobierno de Navarra proyecto 3/2016 (diciembre 2016-junio 2018), concedidos a la Dra. Junkal Garmendia García., Peer reviewed

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammation and impaired airway immunity, providing an opportunistic platform for nontypeable <i>Haemophilus influenzae</i> (NTHi) infection. In this context, therapies targeting not only overactive inflammation without significant adverse effects, but also infection are of interest. Increasing evidence suggests that polyphenols, plant secondary metabolites with anti-inflammatory and antimicrobial properties, may be protective. Here, a <i>Cistus salviifolius</i> plant extract containing quercetin, myricetin, and punicalagin was shown to reduce NTHi viability. Analysis of these polyphenols revealed that quercetin has a bactericidal effect on NTHi, does not display synergies, and that bacteria do not seem to develop resistance. Moreover, quercetin lowered NTHi airway epithelial invasion through a mechanism likely involving inhibition of Akt phosphorylation, and reduced the expression of bacterially-induced proinflammatory markers <i>il-8</i>, <i>cxcl-1</i>, <i>il-6</i>, <i>pde4b</i>, and <i>tnfα</i>. We further tested quercetin’s effect on NTHi murine pulmonary infection, showing a moderate reduction in bacterial counts and significantly reduced expression of proinflammatory genes, compared to untreated mice. Quercetin administration during NTHi infection on a zebrafish septicemia infection model system showed a bacterial clearing effect without signs of host toxicity. In conclusion, this study highlights the therapeutic potential of the xenohormetic molecule quercetin against NTHi infection., This work has been funded by grants from MINECO SAF2015-66520-R and RTI2018-096369-B-I00,
PI011 from Economical Development Department, Regional Navarra Govern, Spain, and 31/2015 from SEPAR
to J.G., by grant from MINECO DRTI2018-094494-B-C22 (MCIU/AEI/FEDER, UE) to C.O.S., by grants from
MINECO AGL2015-67995-C3-1-R and RTI2018-096724-B-C21, PROMETEO/2016/006 from Generalitat Valenciana,
and CIBERobn (CB12/03/30038) to V.M, by grant 03/2016 from Health Department, Regional Navarra Govern,
Spain to J.G., C.O.S. and J.L, and by grant CIBERES (CB06/06/1102) to M.M. CIBER is an initiative from Instituto
de Salud Carlos III (ISCIII), Madrid, Spain.

20 pags., 7 figs., 2 tabs., Genetic variants arising from within-patient evolution shed light on bacterial adaptation during chronic infection. Contingency loci generate high levels of genetic variation in bacterial genomes, enabling adaptation to the stringent selective pressures exerted by the host. A significant gap in our understanding of phase-variable contingency loci is the extent of their contribution to natural infections. The human-adapted pathogen nontypeable Haemophilus influenzae (NTHi) causes persistent infections, which contribute to underlying disease progression. The phase-variable high-molecular-weight (HMW) adhesins located on the NTHi surface mediate adherence to respiratory epithelial cells and, depending on the allelic variant, can also confer high epithelial invasiveness or hyperinvasion. In this study, we characterize the dynamics of HMW-mediated hyperinvasion in living cells and identify a specific HMW binding domain shared by hyperinvasive NTHi isolates of distinct pathological origins. Moreover, we observed that HMW expression decreased over time by using a longitudinal set of persistent NTHi strains collected from chronic obstructive pulmonary disease (COPD) patients, resulting from increased numbers of simple-sequence repeats (SSRs) downstream of the functional P2hmw1A promoter, which is the one primarily driving HMW expression. Notably, the increased SSR numbers at the hmw1 promoter region also control a phenotypic switch toward lower bacterial intracellular invasion and higher biofilm formation, likely conferring adaptive advantages during chronic airway infection by NTHi. Overall, we reveal novel molecular mechanisms of NTHi pathoadaptation based on within-patient lifestyle switching controlled by phase variation. IMPORTANCE Human-adapted bacterial pathogens have evolved specific mechanisms to colonize their host niche. Phase variation is a contingency strategy to allow adaptation to changing conditions, as phase-variable bacterial loci rapidly and reversibly switch their expression. Several NTHi adhesins are phase variable. These adhesins are required for colonization but also immunogenic, in such a way that bacteria with lower adhesin levels are better equipped to survive an immune response, making their contribution to natural infections unclear. We show here that the major NTHi adhesin HMW1A displays allelic variation, which can drive a phase-variable epithelial hyperinvasion phenotype. Over time, hmw1A phase variation lowers adhesin expression, which controls an NTHi lifestyle switch from high epithelial invasiveness to lower invasion and higher biofilm formation. This reversible loss of function aligns with the previously stated notion that epithelial infection is essential for NTHi infection establishment, but once established, persistence favors gene inactivation, in this case facilitating biofilm growth., This work has been funded by grants SAF2015-66520-R and RTI2018-096369-B-I00
from MINECO, 875/2019 from SEPAR, and PC150-151-152 from the Gobierno de Navarra
to J.G. and RTI2018-099985-B-I00 to M.M. CIBER is an initiative from the Instituto de
Salud Carlos III (ISCIII), Madrid, Spain. A.T.-A. is funded by a Ministry of Science and
Innovation grant (PID2019-105216GB-I00)

Chronic obstructive pulmonary disease (COPD) patients undergo infectious exacerbations whose frequency identifies a clinically meaningful phenotype. Mouse models have been mostly used to separately study both COPD and the infectious processes, but a reliable model of the COPD frequent exacerbator phenotype is still lacking. Accordingly, we first established a model of single bacterial exacerbation by nontypeable Haemophilus influenzae (NTHi) infection on mice with emphysema-like lesions. We characterized this single exacerbation model combining both noninvasive in vivo imaging and ex vivo techniques, obtaining longitudinal information about bacterial load and the extent of the developing lesions and host responses. Bacterial load disappeared 48 hours post-infection (hpi). However, lung recovery, measured using tests of pulmonary function and the disappearance of lung inflammation as revealed by micro-computed X-ray tomography, was delayed until 3 weeks post-infection (wpi). Then, to emulate the frequent exacerbator phenotype, we performed two recurrent episodes of NTHi infection on the emphysematous murine lung. Consistent with the amplified infectious insult, bacterial load reduction was now observed 96 hpi, and lung function recovery and disappearance of lesions on anatomical lung images did not happen until 12 wpi. Finally, as a proof of principle of the use of the model, we showed that azithromycin successfully cleared the recurrent infection, confirming this macrolide utility to ameliorate infectious exacerbation. In conclusion, we present a mouse model of recurrent bacterial infection of the emphysematous lung, aimed to facilitate investigating the COPD frequent exacerbator phenotype by providing complementary, dynamic information of both infectious and inflammatory processes., This work was supported by the Departamento de
Universidad, Innovación y Transformación Digital,
Gobierno de Navarra [PC150-151-152]; Ministerio de
Ciencia, Innovación y Universidades (MICIU), Gobierno de
España [RTI2018-096369-B-I00]; MICIU, Gobierno de
España [RED2018-102469-T]; MICIU , Gobierno de España
[SAF2015-66520-R]; MICIU , Gobierno de España [RTI2018-
094494-B-C222]; Departamento de Salud, Gobierno de
Navarra [03/2016]; Sociedad Española de Neumología
y Cirugía Torácica [31/2015].

Respiratory infections pose a continuous threat to humans due to their easy dissemination via aerial transmission. As a consequence, they are leading causes of mortality and morbidity worldwide. Lower respiratory tract infections (LRTI) remained the deadliest communicable diseases causing 3 million deaths worldwide in 2016 (1). Similarly, although the number of tuberculosis (TB) deaths tends to decrease, it is still among the top 10 causes of global mortality with a yearly death burden of about 1.6 million (2). The growing emergence of bacterial antibiotic resistance is a major global challenge for the coming years, and several major respiratory pathogens are included in the WHO priority list of bacteria for which new antibiotics are urgently needed (3). In terms of target population, children under the age of five are the most susceptible hosts to a plethora of respiratory pathogens. The elderly, and immunocompromised respiratory patients suffering from cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), bronchiectasis, neutrophilic asthma, or silicosis are also highly targeted by respiratory pathogens, which often accelerates the fatal progression of the underlying chronic disease. Accordingly, understanding microbial pathogenicity and host immunity against respiratory infections is essential for the rational development of new and more effective therapeutics., This work was supported by grants from MINECO
SAF2015-66520-R and RTI2018-096369-B-I00, from
Health Department, Regional Govern from Navarra,
Spain, reference 03/2016, and from SEPAR 31/2015 to JG,
and also from MINECO BFU2015-72190-EXP to JG-A.
CIBER is an initiative from Instituto de Salud Carlos
III (ISCIII), Madrid.