BUSCADOR RECOLECTA

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.

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., J.M. is funded by Ph.D. studentship BES-2013-062644 from the Ministerio de Economía y Competitividad-MINECO, Spain. This work has been
funded by grants from MINECO (SAF2012-31166) and the Departamento Industria Gobierno Navarra (IlQ14064.R12) to J.G., MINECO (AGL2013-45662) to V.A., and EU projects EvoTAR 282004-FP7 and EFFORT 613754-FP7 to B.G.-Z.

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.

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., J.M. is funded by Ph.D. studentship BES-2013-062644 from the Ministerio Economía y Competitividad-MINECO, Spain. This study was funded by grants from ISCIII (PS09/00130), the Ministerio Economía y Competitividad (MINECO SAF2012-31166), and the Departamento de Salud Gobierno Navarra (359/2012) to J.G.

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., This study was supported by ISCIII PS09/00130, Ministerio Economía y Competitividad-MINECO SAF2012-31166, Dpto. Salud Gobierno Navarra 359/2012, CIBERES.

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).

Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults
suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic
respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To
provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent
isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel
electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic
characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance
to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and
inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation
clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent
isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation.
Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed
comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these
three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition
systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter
ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a
repertoire of genetic variants in the context of persistent infection by NTHi., JCM was supported by a postdoctoral fellowship from the U.S. National Institutes of Health (5F32AI084427). This work has been funded by grants from ISCIII PS09/00130, Ministerio Economía y Competitividad (MINECO) SAF2012-31166, Dpto. Salud Gobierno Navarra 359/2012, and Ministerio Educación PRX 12/00191 to JG, PS09/01904 to JL, and Canadian Institutes of Health Research grant to RJR. CIBERES is an initiative from ISCIII, Spain.

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.

Biofilm formation by nontypeable (NT) Haemophilus influenzae remains a controversial topic. Nevertheless, biofilm-like structures
have been observed in the middle-ear mucosa of experimental chinchilla models of otitis media (OM). To date, there have
been no studies of biofilm formation in large collections of clinical isolates. This study aimed to investigate the initial adhesion
to a solid surface and biofilm formation by NT H. influenzae by comparing isolates from healthy carriers, those with noninvasive
respiratory disease, and those with invasive respiratory disease. We used 352 isolates from patients with nonbacteremic
community-acquired pneumonia (NB-CAP), chronic obstructive pulmonary disease (COPD), OM, and invasive disease and a
group of healthy colonized children. We then determined the speed of initial adhesion to a solid surface by the BioFilm ring test
and quantified biofilm formation by crystal violet staining. Isolates from different clinical sources displayed high levels of biofilm
formation on a static solid support after growth for 24 h. We observed clear differences in initial attachment and biofilm
formation depending on the pathology associated with NT H. influenzae isolation, with significantly increased biofilm formation
for NT H. influenzae isolates collected from patients with invasive disease and OM compared with NT H. influenzae isolates
from patients with NB-CAP or COPD and healthy colonized subjects. In all cases, biofilm structures were detached by proteinase
K treatment, suggesting an important role for proteins in the initial adhesion and static biofilm formation measured by crystal
violet staining., This study was supported by grants from the Fondo de Investigaciones
Sanitarias de la Seguridad Social (PI 0901904) and MINECO (SAF2012-
31166) and by CIBER de Enfermedades Respiratorias, CIBERES (CB06/
06/0037), run by the Instituto de Salud Carlos III (ISCIII), Madrid, Spain.
The work with the BioFilm ring test was performed in the framework of a
collaboration with Biofilm Control. C.P. was supported by FPU grant
(Formación de Profesorado Universitario, Ministerio de Educación,
Spain). S.M. was supported by Sara Borrell postdoctoral contract CD10/
00298 from the Instituto de Salud Carlos III (ISCIII), Madrid, Spain.

Originally isolated from a pediatric patient with otitis media, Haemophilus influenzae strain 375 (Hi375) has been extensively studied as a model system for intracellular invasion of airway epithelial cells and other pathogenesis traits. Here, we report its complete genome sequence and methylome., This work was supported by the National Institutes of Health Ruth Kirschstein Postdoctoral Fellowship to J.C.M. and R01 DC0214 to G.D.E., a Canadian Institutes of Health Research grant to R.J.R., and MINECO
SAF2012-31166 and CIBERES funding 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)

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic pathogen that is an important cause of acute exacerbations of
chronic obstructive pulmonary disease (AECOPD). COPD is an inflammatory disease of the airways, and exacerbations are acute
inflammatory events superimposed on this background of chronic inflammation. Azithromycin (AZM) is a macrolide antibiotic
with antibacterial and anti-inflammatory properties and a clinically proven potential for AECOPD prevention and management.
Relationships between AZM efficacy and resistance by NTHI and between bactericidal and immunomodulatory effects on NTHI
respiratory infection have not been addressed. In this study, we employed two pathogenic NTHI strains with different AZM susceptibilities
(NTHI 375 [AZM susceptible] and NTHI 353 [AZM resistant]) to evaluate the prophylactic and therapeutic effects of
AZM on the NTHI-host interplay. At the cellular level, AZM was bactericidal toward intracellular NTHI inside alveolar and
bronchial epithelia and alveolar macrophages, and it enhanced NTHI phagocytosis by the latter cell type. These effects correlated
with the strain MIC of AZM and the antibiotic dose. Additionally, the effect of AZM on NTHI infection was assessed in a mouse
model of pulmonary infection. AZM showed both preventive and therapeutic efficacies by lowering NTHI 375 bacterial counts in
lungs and bronchoalveolar lavage fluid (BALF) and by reducing histopathological inflammatory lesions in the upper and lower
airways of mice. Conversely, AZM did not reduce bacterial loads in animals infected with NTHI 353, in which case a milder antiinflammatory
effect was also observed. Together, the results of this work link the bactericidal and anti-inflammatory effects of
AZM and frame the efficacy of this antibiotic against NTHI respiratory infection., J.M. was funded by Ph.D. studentship BES-2013-062644 from the Ministerio de Economía y Competitividad (MINECO), Spain. This work was
funded by grants from MINECO (grant SAF2012-31166) and the Departamento
Salud Gobierno Navarra (grant 359/2012) to J.G.

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.

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., J.M. is funded by Ph.D. studentship BES-2013-062644 from the Ministerio Economía y Competitividad-MINECO, Spain. This study was funded by grants from ISCIII (PS09/00130), the Ministerio Economía y Competitividad (MINECO SAF2012-31166), and the Departamento de Salud Gobierno Navarra (359/2012) to J.G. CIBERES is an initiative from ISCIII, Spain., Peer Reviewed

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic pathogen that is an important cause of acute exacerbations of chronic obstructive pulmonary disease (AECOPD). COPD is an inflammatory disease of the airways, and exacerbations are acute inflammatory events superimposed on this background of chronic inflammation. Azithromycin (AZM) is a macrolide antibiotic with antibacterial and anti-inflammatory properties and a clinically proven potential for AECOPD prevention and management. Relationships between AZM efficacy and resistance by NTHI and between bactericidal and immunomodulatory effects on NTHI respiratory infection have not been addressed. In this study, we employed two pathogenic NTHI strains with different AZM sus- ceptibilities (NTHI 375 [AZM susceptible] and NTHI 353 [AZM resistant]) to evaluate the prophylactic and therapeutic effects of AZM on the NTHI-host interplay. At the cellular level, AZM was bactericidal toward intracellular NTHI inside alveolar and bronchial epithelia and alveolar macrophages, and it enhanced NTHI phagocytosis by the latter cell type. These effects correlated with the strain MIC of AZM and the antibiotic dose. Additionally, the effect of AZM on NTHI infection was assessed in a mouse model of pulmonary infection. AZM showed both preventive and therapeutic efficacies by lowering NTHI 375 bacterial counts in lungs and bronchoalveolar lavage fluid (BALF) and by reducing histopathological inflammatory lesions in the upper and lower airways of mice. Conversely, AZM did not reduce bacterial loads in animals infected with NTHI 353, in which case a milder anti- inflammatory effect was also observed. Together, the results of this work link the bactericidal and anti-inflammatory effects of AZM and frame the efficacy of this antibiotic against NTHI respiratory infection.

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.