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Genomic diversity of nontypeable H. influenzae strains confers phenotypic heterogeneity. Multiple strains of H. influenzae can be simultaneously isolated from clinical specimens, but we lack detailed information about polyclonal infection dynamics by this pathogen. A long-term barrier to our understanding of this host-pathogen interplay is the lack of genetic tools for strain engineering and differential labeling. Here, we present a novel plasmid toolkit named pTBH (toolbox for Haemophilus), with standardized modules for fluorescent or bioluminescent labeling, adapted to H. influenzae requirements but designed to be versatile so it can be utilized in other bacterial species. We present detailed experimental and quantitative image analysis methods, together with proof-of-principle examples, and show the ample possibilities of 3D microscopy, combined with quantitative image analysis, to model H. influenzae polyclonal infection lifestyles and unravel the co-habitation and co-infection dynamics of this respiratory pathogen.

Haemophilus influenzae is a human-adapted bacterial pathogen that causes airway infections. Bacterial and host elements associated with the fitness of H. influenzae within the host lung are not well understood. Here, we exploited the strength of in vivo-omic analyses to study host-microbe interactions during infection. We used in vivo transcriptome sequencing (RNA-seq) for genome-wide profiling of both host and bacterial gene expression during mouse lung infection. Profiling of murine lung gene expression upon infection showed upregulation of lung inflammatory response and ribosomal organization genes, and downregulation of cell adhesion and cytoskeleton genes. Transcriptomic analysis of bacteria recovered from bronchoalveolar lavage fluid samples from infected mice showed a significant metabolic rewiring during infection, which was highly different from that obtained upon bacterial in vitro growth in an artificial sputum medium suitable for H. influenzae. In vivo RNA-seq revealed upregulation of bacterial de novo purine biosynthesis, genes involved in non-aromatic amino acid biosynthesis, and part of the natural competence machinery. In contrast, the expression of genes involved in fatty acid and cell wall synthesis and lipooligosaccharide decoration was downregulated. Correlations between upregulated gene expression and mutant attenuation in vivo were established, as observed upon purH gene inactivation leading to purine auxotrophy. Likewise, the purine analogs 6-thioguanine and 6-mercaptopurine reduced H. influenzae viability in a dose-dependent manner. These data expand our understanding of H. influenzae requirements during infection. In particular, H. influenzae exploits purine nucleotide synthesis as a fitness determinant, raising the possibility of purine synthesis as an anti-H. influenzae target. IMPORTANCE In vivo-omic strategies offer great opportunities for increased understanding of host-pathogen interplay and for identification of therapeutic targets. H

Patients with chronic obstructive pulmonary disease (COPD) benefit from the immunomodulatory effect of azithromycin, but long-term administration may alter colonizing bacteria. Our goal was to identify changes in Haemophilus influenzae and Haemophilus parainfluenzae during azithromycin treatment. Fifteen patients were followed while receiving prolonged azithromycin treatment (Hospital Universitari de Bellvitge, Spain). Four patients (P02, P08, P11, and P13) were persistently colonized by H. influenzae for at least 3 months and two (P04 and P11) by H. parainfluenzae. Isolates from these patients (53 H. influenzae and 18 H. parainfluenzae) were included to identify, by whole-genome sequencing, antimicrobial resistance changes and genetic variation accumulated during persistent colonization. All persistent lineages isolated before treatment were azithromycin-susceptible but developed resistance within the first months, apart from those belonging to P02, who discontinued the treatment. H. influenzae isolates from P08-ST107 acquired mutations in 23S rRNA, and those from P11-ST2480 and P13-ST165 had changes in L4 and L22. In H. parainfluenzae, P04 persistent isolates acquired changes in rlmC, and P11 carried genes encoding MefE/MsrD efflux pumps in an integrative conjugative element, which was also identified in H. influenzae P11-ST147. Other genetic variation occurred in genes associated with cell wall and inorganic ion metabolism. Persistent H. influenzae strains all showed changes in licA and hgpB genes. Other genes (lex1, lic3A, hgpC, and fadL) had variation in multiple lineages. Furthermore, persistent strains showed loss, acquisition, or genetic changes in prophage-associated regions. Long-term azithromycin therapy results in macrolide resistance, as well as genetic changes that likely favor bacterial adaptation during persistent respiratory colonization. IMPORTANCE The immunomodulatory properties of azithromycin reduce the frequency of exacerbations and improve the

Haemophilus influenzae is a human-adapted bacterial pathogen causing airway infections. We used RNAseq and Tnseq to profile bacterial genome-wide gene expression and mutant fitness during lung infection. Integration of these data sets drawed a comprehensive landscape in terms of bacterial metabolic requirements during infection. Genes involved in tryptophan and serine biosynthesis were identified, and auxotrophy of these aminoacids dampened in vivo bacterial fitness. Moreover, transcriptome analyses showed a likely long 3¿UTR overlapping event in the mtr-sdaCA region, where mtr encodes a tryptophan transporter, sdaC a L-serine transporter, and sdaA a L-serine desaminase. Biological significance and contribution of this tryptophan-serine metabolic axis to H. influenzae infection is being elucidated by combining transport/metabolism mutant phenotyping, analysis of regulatory elements, and evaluation of natural antibiotics as potential inhibitors of tryptophan synthesis. Our results shed light on these aminoacids contribution to H. influenzae infection, and their potential as a source of therapeutic targets.

Introduction Haemophilus influenzae is a human-adapted pathogen causing chronic lower airway infections and recurrent exacerbations in chronic obstructive pulmonary disease (COPD) patients. Unraveling H. influenzae virulence mechanisms will result in identifying targets for drug development. By using transposon insertion sequencing (Tnseq), we screened bacterial genes required for infection in a murine model of airway infection, identified and validated the methyltransferase Dam. Objectives To study the role of Dam GATC methylation in the regulation of H. influenzae gene expression, and the contribution of such epigenetic regulation to this host-pathogen interplay. Methods We followed two complementary approaches: (i) RNA sequencing (RNA-seq) to profile differential gene expression when comparing WT and dam mutant strains. (ii) methylome analysis in a panel of H. influenzae PacBio clinical strain genomes to screen hypo/hemi-methylated GATC sites in non-coding regions, as potential elements of epigenetic regulation of gene expression. Results The oxygen sensitive fumarate and nitrate reductase (FNR) encoding gene, together with the FNR regulon genes ytfE, dmsA and cydD, were overexpressed upon dam inactivation. Further analysis identified GATC motifs in the fnr, dmsA and cydD promoter regions, and Dam methylation of these sites was confirmed. Conversely, methylome analyses recurrently showed GATC hypo/hemi-methylation in a region containing two GATC motifs upstream of the high temperature protein G (htpG) encoding gene, the proximal one overlapping with a putative FNR binding site. Analysis of such GATC sites revealed possible phenotypic heterogeneity in the above mentioned proximal motif, further tested by ad hoc generation of fluorescent reporter strains for single-cell analyses. Conclusions Together, our results shed light on Dam methyltransferase contribution to H. influenzae pulmonary infection, highlight epigenetic regulation of the H. influenzae FNR regulon an

Cepas no tipificables de la bacteria Gram negativa Haemophilus influenzae (NTHi) son patógenos oportunistas frecuentemente aisladas de muestras respiratorias de pacientes que sufren enfermedad pulmonar obstructiva crónica (EPOC), en fases de estabilidad clínica y durante exacerbaciones. Existe una elevada variabilidad genética entre aislados de NTHi, lo que favorece su persistencia en las vías respiratorias bajas al mismo tiempo que dificulta su erradicación. Este Trabajo Fin de Grado aborda tres aspectos independientes en el marco de la interacción hospedador-patógeno entre H. influenzae y sistema respiratorio humano. En primer lugar, en el contexto de la vía de síntesis de purinas de novo como posible diana terapéutica, se ha generado una cepa mutante mediante inactivación del gen purH en la cepa RdKW20. A continuación, se ha analizado su auxotrofía, observando un crecimiento limitado en medio mínimo sin inosina. En segundo lugar, mediante el ensayo cuantitativo de Análisis de Perfiles de Población se ha determinado la existencia de heterorresistencia de distinta intensidad a imipenem entre aislados respiratorios de NTHi. Este fenómeno, no detectable en la clínica al no exhibir un cambio en la CMI tras realización de ensayo de microdilución, puede contribuir a fallo terapéutico. En tercer lugar, se ha contribuido a poner a punto un ensayo de tipo Serum Bactericidal Assay (SBA) para estudio del posible efecto heterólogo de la vacunación antineumocócica en la viabilidad de H. influenzae, generando un primer panel de datos preliminares.

Antibiotic resistance is a serious global public health threat, particularly important in the treatment of lower respiratory tract infections (LRTI)[1]. Its clinical relevance has increased during the ongoing COVID-19 pandemic due to secondary bacterial infections in severe hospitalized cases requiring mechanical ventilation or assisted respiration[2]. Antibiotic resistance induces therapeutic failure on respiratory infectious diseases, which aims urgent development of innovative antimicrobials alternative to conventional antibiotics[3]. A main bacterial pathogen causing LRTI is Haemophilus influenzae, resistant to ampicillin, which is especially relevant in chronic obstructive pulmonary disorder (COPD) and forms biofilms that favors chronic infection and antibiotic resistance[4]. Nowadays there are no anti-biofilm therapies available against H. influenzae. Conversely, the formation, maturation and dispersion of biofilms is regulated by intra- and intercellular signaling systems. Autoinducers play an important role in the intercellular signaling, or quorum sensing. In the case of H. influenzae, autoinducer-2 (AI.2, dihydroxypentanedione-DPD) mediated signaling plays a role in biofilm formation[5]. Based on these needs and evidence, polymeric nanocarriers based on an AI-2 autoinducer analog (trans-2-nonenal) have been developed. Polymeric nanoparticles based on the widely used poly-lactic-co-glycolic-acid (PLGA) have been designed and optimized enclosing trans-2-nonenal as active molecule by simple emulsion-solvent evaporation method. Different parameters of the synthetic procedure have been studied, such as drug/polymer ratio, volume, and composition of aqueous and organic phases, as well as sonication and evaporation times. The physico-chemical properties of the nanocarriers have been analyzed through dynamic light scattering, differential scanning calorimetry and scanning electron microscopy. Encapsulation efficiency and controlled drug release kinetics at 4ºC, RT

Nontypeable Haemophilus infuenzae (NTHi) is a human-adapted bacterial pathogen causing airway infections. NTHi is an intracellular facultative pathogen, capable of both forming bioflms and invading airway epithelial cells, a combination of traits likely contributing to persistence. Standardised clinical procedures for antibiotic administration rely on pathogen identifcation and antibiotic susceptibility testing, often performed on single-colony bacterial isolates. For respiratory pathogens, this could be questionable as chronic patients may be persistently colonised by multiple clones. Moreover, diferences in the susceptibility of the diferent strains present within the same isolate have been reported for a variety of antibiotics. In vitro polyclonal infections can be assessed at the single cell level using bacterial strains engineered for diferential labelling. However, appropriate genetic tools amenable for H. infuenzae are currently limited. Here, we designed a novel plasmid toolkit named pTBH (toolbox for Haemophilus) for engineering H. infuenzae, with functional components (cargo module, two selectable markers, and a replication origin) assembled in modular combinations to create standardised and versatile plasmids. Using this structure, six reporter genes for fuorescent or bioluminescent labelling, expressed from a constitutive promoter, were independently assembled. This plasmid set was introduced into two phenotypically divergent H. infuenzae strains, and the engineered strains were used to analyse mixed bioflm growth architecture, antibiotic efcacy, and to visualise the dynamics of alveolar epithelial co-infection. The mixed bioflms showed a bilayer architecture and selective antibiotic efcacy associated to the respective susceptibility profles of their members. Furthermore, diferential kinetics of bacterial intracellular location within subcellular acidic compartments was quantifed upon co-infection of cultured airway epithelial cells by quantitative image

Antibiotic resistance is a major Public Health challenge worldwide. Mechanisms other than resistance are described as contributors to therapeutic failure. These include heteroresistance and tolerance, which escape the standardized procedures used for antibiotic treatment decision-making as they do not involve changes in minimal inhibitory concentration (MIC). Haemophilus influenzae causes chronic respiratory infection and is associated with exacerbations suffered by chronic obstructive pulmonary disease (COPD) patients. Although resistance to imipenem is rare in this bacterial species, heteroresistance has been reported, and antibiotic tolerance cannot be excluded. Moreover, development of antibiotic heteroresistance or tolerance during within-host H. influenzae pathoadaptive evolution is currently unknown. In this study, we assessed imipenem resistance, heteroresistance and tolerance in a previously sequenced longitudinal collection of H. influenzae COPD respiratory isolates. The use of Etest, disc diffusion, population analysis profiling, tolerance disc (TD)-test methods, and susceptibility breakpoint criteria when available, showed a significant proportion of imipenem heteroresistance with differences in terms of degree among strains, absence of imipenem tolerance, and no specific trends among serial and clonally related strains could be established. Analysis of allelic variation in the ftsI, acrA, acrB, and acrR genes rendered a panel of polymorphisms only found in heteroresistant strains, but gene expression and genome-wide analyses did not show clear genetic traits linked to heteroresistance. In summary, a significant proportion of imipenem heteroresistance was observed among H. influenzae strains isolated from COPD respiratory samples over time. These data should be useful for making more accurate clinical recommendations to COPD patients.

Once relegated to the supply of energy and biosynthetic precursors, it is now indubitable that metabolism mediates most of physiological processes. In the context of bacterial–host interactions where virulence is the outcome (commonly termed bacterial pathogenesis) metabolism expands far beyond its canonical role in bacterial proliferation. In addition to all sorts of recognized molecular determinants or virulence factors (toxins, flagella, translocated effectors, adhesins, invasins, etc.), bacterial pathogens are equipped with specific metabolic traits to circumvent immune defenses and antimicrobial killing, thus facilitating colonization and proliferation within their hosts. As the implementation of high-throughput technologies elevates the pathogenesis field to the era of big data, it concurrently creates considerable challenges for our ability to interpret large data sets and identify factors that impact infectious processes. Metabolic modelling is emerging as a powerful tool allowing the integration and coherent organization of large data sets into the context of biological networks providing non-intuitive insights on biological systems that experimental analysis alone cannot provide. Here, we take a snapshot of the recent understanding of bacterial metabolism and the bacterial–host metabolic interplay during infection, and highlight key outcomes and challenges for the practical implementation of bacterial metabolic modelling computational tools in the pathogenesis field (summarized in Fig. 1).

Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes infections mainly in the upper and lower respiratory tract. The bacterium is associated with bronchitis and exacerbations in patients suffering from chronic obstructive pulmonary disease and frequently causes acute otitis media in preschool children. We have previously demonstrated that the binding of C4b binding protein (C4BP) is important for NTHi complement evasion. In this study, we identified outer membrane protein 5 (P5) of NTHi as a novel ligand of C4BP. Importantly, we observed significantly lower C4BP binding and decreased serum resistance in P5-deficient NTHi mutants. Surface expression of recombinant P5 on Escherichia coli conferred C4BP binding and consequently increased serum resistance. Moreover, P5 expression was positively correlated with C4BP binding in a series of clinical isolates. We revealed higher levels of P5 surface expression and consequently more C4BP binding in isolates from the lower respiratory tract of chronic obstructive pulmonary disease patients and tonsil specimens compared with isolates from the upper respiratory tract and the bloodstream (invasive strains). Our results highlight P5 as an important protein for protecting NTHi against complement-mediated killing.

Haemophilus influenzae has contributed to key bacterial genome sequencing hallmarks, as being not only the first bacterium to be genome-sequenced, but also starring the first genome-wide analysis of chromosomes directly transformed with DNA from a divergent genotype, and pioneering Tn-seq methodologies. Over the years, the phenomenal and constantly evolving development of –omic technologies applied to a whole range of biological questions of clinical relevance in the H. influenzae-host interplay, has greatly moved forward our understanding of this human-adapted pathogen, responsible for multiple acute and chronic infections of the respiratory tract. In this way, essential genes, virulence factors, pathoadaptive traits, and multi-layer gene expression regulatory networks with both genomic and epigenomic complexity levels are being elucidated. Likewise, the unstoppable increasing whole genome sequencing information underpinning H. influenzae great genomic plasticity, mainly when referring to non-capsulated strains, poses major challenges to understand the genomic basis of clinically relevant phenotypes and even more, to clearly highlight potential targets of clinical interest for diagnostic, therapeutic or vaccine development. We review here how genomic, transcriptomic, proteomic and metabolomic-based approaches are great contributors to our current understanding of the interactions between H. influenzae and the human airways, and point possible strategies to maximize their usefulness in the context of biomedical research and clinical needs on this human-adapted bacterial pathogen.

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.

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 sw

Bacterial surfaces are decorated with carbohydrate structures that may serve as ligands for host receptors. Based on their ability to recognize specific sugar epitopes, plant lectins are extensively used for bacteria typing. We previously observed that the galactose-specific agglutinins from Ricinus communis (RCA) and Viscum album (VAA) exhibited differential binding to nontypeable Haemophilus influenzae (NTHi) clinical isolates, their binding being distinctly affected by truncation of the lipooligosaccharide (LOS). Here, we examined their binding to the structurally similar LOS molecules isolated from strains NTHi375 and RdKW20, using microarray binding assays, saturation transfer difference NMR, and molecular dynamics simulations. RCA bound the LOSRdKW20 glycoform displaying terminal Gal beta(1,4) Glc beta, whereas VAA recognized the Gala(1,4) Gal beta(1,4) Glc beta epitope in LOSNTHi375 but not in LOSRdKW20, unveiling a different presentation. Binding assays to whole bacterial cells were consistent with LOSNTHi375 serving as ligand for VAA, and also suggested recognition of the glycoprotein HMW1. Regarding RCA, comparable binding to NTHi375 and RdKW20 cells was observed. Interestingly, an increase in LOSNTHi375 abundance or expression of HMW1 in RdKW20 impaired RCA binding. Overall, the results revealed that, besides the LOS, other carbohydrate structures on the bacterial surface serve as lectin ligands, and highlighted the impact of the specific display of cell surface components on lectin binding.

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 elucidat

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammatory responses and impaired airway immunity, which provides an opportunistic platform for nontypeable Haemophilus influenzae (NTHi) infection. Clinical evidence supports that the COPD airways present increased concentrations of glucose, which may facilitate proliferation of pathogenic bacteria able to use glucose as a carbon source. NTHi metabolizes glucose through respiration-assisted fermentation, leading to the excretion of acetate, formate, and succinate. We hypothesized that such specialized glucose catabolism may be a pathoadaptive trait playing a pivotal role in the NTHi airway infection. To find out whether this is true, we engineered and characterized bacterial mutant strains impaired to produce acetate, formate, or succinate by inactivating the ackA, pflA, and frdA genes, respectively. While the inactivation of the pflA and frdA genes only had minimal physiological effects, the inactivation of the ackA gene affected acetate production and led to reduced bacterial growth, production of lactate under low oxygen tension, and bacterial attenuation in vivo. Moreover, bacterially produced acetate was able to stimulate the expression of inflammatory genes by cultured airway epithelial cells. These results back the notion that the COPD lung supports NTHi growth on glucose, enabling production of fermentative end products acting as immunometabolites at the site of infection. Thus, glucose catabolism may contribute not only to NTHi growth but also to bacterially driven airway inflammation. This information has important implications for developing nonantibiotic antimicrobials, given that airway glucose homeostasis modifying drugs could help prevent microbial infections associated with chronic lung disease.

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.

Many chronic pulmonary diseases have aspects of their pathogenesis influenced by pathogens. Haemophilus influenzae (Hi) infection and chronic obstructive pulmonary disease (COPD) are an example of such an association where a bidirectional relationship between COPD and the bacteria impacts pathogenesis. The COPD airways present increased concentrations of glucose driving proliferation of bacteria able to use glucose as a carbon source. Hi uses a specialized type of glucose catabolism termed respiration assisted fermentation with a respiratory chain that alleviates redox imbalances due to incomplete glucose oxidation. We hypothesise that Hi glucose catabolism is a pathoadaptative trait playing a pivotal role in COPD infection. To find out whether this is true, we engineered bacterial mutant strains unable to produce acetate, formate or succinate, main products of Hi glucose metabolism, by inactivation of the ackA, pflA and frdA genes. Mutant phenotyping was performed by analysis of bacterial morphology, proliferation, fatty acid content, metabolic end products, gene expression profiling, and murine lung infection. Inactivation of the ackA gene impaired acetate production leading to slow bacterial growth, increased myristic and decreased palmitoleic acid content, and production of lactate under low oxygen tension. The mutation also caused bacterial attenuation in vivo. This information has important implications for developing non-antibiotic therapeutics against bacterial infections, given that airway glucose homeostasis modifying drugs could help preventing chronic lung disease exacerbations.

Objectives: To compare the determinants of trimethoprim-sulfamethoxazole resistance with established susceptibility values for fastidious Haemophilus spp., to provide recommendations for optimal trimethoprim-sulfamethoxazole measurement. Methods: We collected 50 strains each of Haemophilus influenzae and Haemophilus parainfluenzae at Bellvitge University Hospital. Trimethoprim-sulfamethoxazole susceptibility was tested by microdilution, E-test and disc diffusion using both Mueller–Hinton fastidious (MH-F) medium and Haemophilus test medium (HTM) following EUCAST and CLSI criteria, respectively. Mutations in folA, folP and additional determinants of resistance were identified in whole-genome-sequenced isolates. Results: Strains presented generally higher rates of trimethoprim-sulfamethoxazole resistance when grown on HTM than on MH-F, independent of the methodology used (average MIC 2.6-fold higher in H. influenzae and 1.2-fold higher in H. parainfluenzae). The main resistance-related determinants were as follows: I95L and F154S/V in folA; 3- and 15-bp insertions and substitutions in folP; acquisition of sul genes; and FolA overproduction potentially linked to mutations in -35 and -10 promoter motifs. Of note, 2 of 19 H. influenzae strains (10.5%) and 9 of 33 H. parainfluenzae strains (27.3%) with mutations and assigned as resistant by microdilution were inaccurately considered susceptible by disc diffusion. This misinterpretation was resolved by raising the clinical resistance breakpoint of the EUCAST guidelines to ≤30 mm. Conclusions: Given the routine use of disc diffusion, a significant number of strains could potentially be miscategorized as susceptible to trimethoprim-sulfamethoxazole despite having resistance-related mutations. A simple modification to the current clinical resistance breakpoint given by the EUCAST guideline for MH-F ensures correct interpretation and correlation with the reference standard method of microdilution.

Nutrient iron sequestration is the most significant form of nutritional immunity and causes bacterial pathogens to evolve strategies of host iron scavenging. Cigarette smoking contains iron particulates altering lung and systemic iron homeostasis, which may enhance colonization in the lungs of patients suffering chronic obstructive pulmonary disease (COPD) by opportunistic pathogens such as nontypeable. NTHi is a heme auxotroph, and the NTHi genome contains multiple heme acquisition systems whose role in pulmonary infection requires a global understanding. In this study, we determined the relative contribution to NTHi airway infection of the four heme-acquisition systems HxuCBA, PE, SapABCDFZ, and HbpA-DppBCDF that are located at the bacterial outer membrane or the periplasm. Our computational studies provided plausible 3D models for HbpA, SapA, PE, and HxuA interactions with heme. Generation and characterization of single mutants in the hxuCBA, hpe, sapA, and hbpA genes provided evidence for participation in heme binding-storage and inter-bacterial donation. The hxuA, sapA, hbpA, and hpe genes showed differential expression and responded to heme. Moreover, HxuCBA, PE, SapABCDFZ, and HbpA-DppBCDF presented moonlighting properties related to resistance to antimicrobial peptides or glutathione import, together likely contributing to the NTHi-host airway interplay, as observed upon cultured airway epithelia and in vivo lung infection. The observed multi-functionality was shown to be system-specific, thus limiting redundancy. Together, we provide evidence for heme uptake systems as bacterial factors that act in a coordinated and multi-functional manner to subvert nutritional- and other sources of host innate immunity during NTHi airway infection.

[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).

[ES] 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)., [EN] 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.

Bacterial surfaces are decorated with a diversity of carbohydrate structures that play important roles in the bacteria–host relationships. They may offer protection against host defense mechanisms, elicit strong antigenic responses, or serve as ligands for host receptors, including lectins of the innate immune system. Binding by these lectins may trigger defense responses or, alternatively, promote attachment, thereby enhancing infection. The outcome will depend on the particular bacterial surface landscape, which may substantially differ among species and strains. In this chapter, we describe two novel methods for exploring interactions directly on the bacterial surface, based on the generation of bacterial microarrays and quartz crystal microbalance (QCM) sensor chips. Bacterial microarrays enable profiling of accessible carbohydrate structures and screening of their recognition by host receptors, also providing information on binding avidity, while the QCM approach allows determination of binding affinity and kinetics. In both cases, the chief element is the use of entire bacterial cells, so that recognition of the bacterial glycan epitopes is explored in their natural environment.

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.