Estudio de infección policlonal, vida extracelular y adaptación metabólica de Haemophilus influenzae en el sistema respiratorio humano
Authors:   , Asensio López Javier 
Published: December 2024

This PhD Thesis work addresses three aspects of the interaction between the nontypeable Haemophilus influenzae (NTHi) and the human respiratory system, considering the development of genetic tools for differential labelling and their application to the study of polyclonal infections (Chapter 1), the anti-biofilm therapeutic potential of cinnamaldehydederived molecules (Chapter 2), and the role of tryptophan and its derivatives in modulating this host-pathogen interaction (Chapter 3). Standardized clinical procedures for the treatment of bacterial infections are based on pathogen identification and determination of its antibiotic susceptibility, often performed on bacteria isolated from a single colony. In the case of respiratory pathogens, this practice may be questionable since chronic patients can be simultaneously colonized by different strains of the same pathogen, with distinct genotypic and phenotypic characteristics that can lead to different antibiotic susceptibility profiles. This heterogeneity has been detected in H. influenzae isolates obtained from respiratory samples of chronic respiratory patients, whose differential antibiotic susceptibility profiles can lead to therapeutic failure in clinical practice. In Chapter 1 of this work, we generated a collection of module-based plasmids that allow differential labelling of H. influenzae strains. Using this differential labelling, we studied the formation of mixed biofilms by two genetically distinct H. influenzae strains, with different biofilm-forming capacities and antibiotic susceptibility, proving selective antibiotic action in these polyclonal biofilms. Additionally, we studied the dynamics of epithelial co-infection by two H. influenzae strains, revealing the intracellular localization of strains with heterogeneous invasive capacities. This panel of genetic tools paves the way for polyclonal and polymicrobial studies of the different H. influenzae lifestyles. On the other hand, chronic respiratory diseases such as Chronic Obstructive Pulmonary Disease (COPD) promote the pulmonary access of opportunistic pathogens like H. influenzae, which persist by forming biofilms, a protective lifestyle that contributes to chronicity of infection and facilitates the emergence of antibiotic resistances. H. influenzae is included in the World Health Organization List of Priority Pathogens due to its resistance to ampicillin, making it a priority to both develop new antimicrobials and standardize methods for evaluating the efficacy of anti-biofilm drugs. In Chapter 2 of this work, we present a panel of protocols for systematic analysis of anti-biofilm drug effect, customized to H. influenzae biofilms. As proof of concept, we evaluated the antimicrobial effect of cinnamaldehyde-derived molecules against H. influenzae strains with different biofilmforming capacities. We observed that (E)-Trans-2-nonenal and (E)-3-Decen-2-one inhibit the formation and eradicate pre-formed biofilms by H. influenzae. Additionally, the low toxicity of (E)-Trans-2-nonenal led to its selection for encapsulation in polymeric nanoparticles, which allow for greater solubility, maintain the anti-biofilm effect, and may be useful for the controlled and safe release of drugs. Lastly, H. influenzae has patho-adaptation mechanisms including the evasion of nutritional immunity. Tryptophan (Trp) is an essential metabolite for this bacterium, which is able to synthesize its own Trp and to acquire exogenous Trp through at least two transport systems. On the other hand, the host restricts Trp availability through the action of the enzyme indoleamine 2,3-dioxygenase (IDO), which catabolizes Trp generating kynurenines that, along with other Trp derivatives, act as ligands for the aryl hydrocarbon receptor (AhR). IDO and AhR are involved in controlling the immune response and maintaining pulmonary homeostasis. In Chapter 3 of this work, we analyzed the effect of exogenous Trp availability on the physiology of H. influenzae, as well as the importance of Trp synthesis in respiratory infection by this pathogen. Furthermore, we examine how the availability of Trp in the respiratory system influences this host-pathogen interaction, showing that Trp and its derivatives modulate the expression of IDO, AhR, and their effectors, thereby modulating the host's immune response to H. influenzae infection. Altogether, the work carried out during this PhD Thesis represents a significant step forward in the understanding of the molecular basis of the interaction between H. influenzae and the human airways, which may be useful in the development of strategies to improve its clinical management.