Search

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., N.L.-L. is funded by a PhD studentship from Regional Navarra Govern, Spain, reference 0011-1408-2017-000000. C.G.-C. is funded by a PhD studentship from Agencia Española de Investigación (AEI), Spain, reference PRE2019-088382. This work has been funded by grants from MINECO RTI2018-096369-B-I00, 875/2019 from SEPAR, and PC150-151–152 from Gobierno de Navarra to J.G. CIBER is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain.

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].

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., We thank Dr. I. Rodriguez-Arce for technical support. N.L.-L. is funded by a PhD studentship from Regional Navarra Govern, Spain, reference 0011-1408-2017-000000. J.H. is the recipient of an Australian Commonwealth Government Research Training Program Award. This work has been funded by grants from MINECO SAF2015-66520-R and RTI2018-096369-B-I00, from the Health Department, Regional Navarra Govern, Spain, reference 03/2016, from SEPAR 31/2015 to J.G., and from the National Health and Medical Research Council (NHMRC, GNT1043532) to U.K. CIBER is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain.

Resumen del trabajo presentado en el European Respiratory Society International Congress, celebrado en Madrid (España), del 28 de septiembre al 2 de octubre de 2019, 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.

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.

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.