Study of antibiotic heteoresistance, epigenomic regulation and programmable gene inactivation during Haemophilus influenzae respiratory infection
Authors:   , Gil Campillo Celia 
Published: December 2024

This PhD Thesis addresses three key aspects on the study of respiratory infections caused by the opportunistic pathogen Haemophilus influenzae, by focusing on mechanisms different to antibiotic resistance but associated to therapeutic failure (Chapter 1), on the epigenetic regulation of gene expression (Chapter 2), and on the development of innovative genetic engineering tools for genome-wide gene functional studies in H. influenzae (Chapter 3). H. influenzae, due to its increasing resistance to ampicillin, is included in the World Health Organization’s List of Priority Pathogens for which the development of new antimicrobials is a health priority worldwide. In addition to resistance, there are other mechanisms, such as antibiotic heteroresistance, tolerance or persistence, which are underdetected through standardized procedures in the clinical practice but contribute to therapeutic failure. Carbapenem antibiotics, such as imipenem, are useful for initial empirical treatment of severe infections due to their low toxicity and low resistance rates. H. influenzae resistance to imipenem is rare, although available evidence suggests that heteroresistance levels are underestimated. In Chapter 1, we evaluated the resistance, heteroresistance, and tolerance to imipenem in a collection of clinical strains of H. influenzae isolated from respiratory samples of patients suffering Chronic Obstructive Pulmonary Disease (COPD), and whose genomes had been previously sequenced. Through disc diffusion, Etest, TD-test and population analysis profiling assays, we identified a low level of resistance, absence of tolerance, and a significant proportion of imipenem heteroresistance. Although we sought genomic traits responsible for the observed heteroresistance through: i) analysis of allelic variation in the ftsI, acrA, acrB, and acrR genes, previously associated with heteroresistance; ii) analysis of allelic variation at the genomic level between strains belonging to the same clonal type but phenotypically heterogeneous; and iii) comparative analysis of gene distribution at the genomic level in susceptible and heteroresistant strains, we did not detect significant links between specific genetic traits and the observed heteroresistance. Overall, we show that heteroresistance is a highly prevalent, complex, and multifactorial phenomenon, highlighting the need to implement easy and rapid protocols for its identification in clinical practice. Furthermore, H. influenzae is a well-adapted bacterial pathobiont in humans, causing respiratory infections in immunocompromised patients, including those suffering COPD. The regulatory elements that dictate the survival and adaptation of H. influenzae in the lungs of COPD patients are poorly understood. In γ-proteobacteria, the methylation status of GATC motifs located in regulatory regions modulates RNA polymerase and transcription factor binding, therefore affecting transcription. In bacterial genomes, most GATC motifs are methylated by the Dam methyltransferase, although some GATC sites can remain unmethylated or hemi-methylated if Dam activity is blocked by proteins binding to the DNA. The combination of methylated and hemi- or non-methylated GATC motifs in regulatory or promoter regions may be indicative of epigenetic regulation of gene expression. In Chapter 2, we analyzed the survival of a panel of H. influenzae mutants in a murine model of lung infection through transposon mutagenesis coupled with deep sequencing (Tn-seq), identifying the contribution of the Dam methyltransferase to H. influenzae lung survival. The study of the genome-wide Dam methylation pattern through single-molecule real-time (SMRT) sequencing revealed unmethylated or hemi-methylated GATC motifs in regulatory regions, leading us to identify the first case of phenotypic variation controlled by Dam methylation in an isogenic population of H. influenzae cells. Moreover, inactivation of the dam gene and the study of its effect at the transcriptomic level through RNA sequencing (RNA-seq) and differential gene expression analysis, revealed a new regulatory network where Dam methylation and the transcriptional regulators FNR and Fur coordinately regulate the expression of a panel of genes involved in H. influenzae anaerobic defense against reactive nitrogen species. These results have patho-adaptive value in niches with nitrosative stress and low oxygenation, such as the lower airways of patients suffering COPD. Lastly, H. influenzae was the first free-living organism to have its complete genome sequenced, pioneering the development and use of -omic technologies. Transposon insertion sequencing methods have been useful in conducting gene function studies and identifying essential genes in H. influenzae, although most of them have unknown functions due to the inability to generate mutants. Bacterial gene interference based on clustered, regularly interspaced, short palindromic repeat-CRISPR technology coupled to deep sequencing (CRISPRi-seq), overcomes this limitation by allowing the screening and analysis of essential genes at the genomic scale through gene silencing. In Chapter 3, we developed and validated the utility of an anhydrotetracycline-inducible CRISPRi-seq platform for genome-wide programmable gene silencing in H. influenzae, opening new avenues for the functional understanding of its genome. Together, this PhD Thesis work significantly contributes to our understanding of the molecular mechanisms associated to H. influenzae infection, and presents novel bacterial genomic engineering tools applicable to therapeutic target screening in the development of strategies to improve the clinical management of this pathogen.