Implicación del metabolismo bacteriano en la interacción de Haemophilus influenzae con el sistema respiratorio humano: bases moleculares y explotación terapéutica
Authors:   , López López Nahikari 
Published: March 2022

This PhD Thesis work addresses the role of three aspects of bacterial metabolism (purine synthesis, glucose catabolism, fatty acid synthesis) in the interaction between nontypeable Haemophilus influenzae (NTHi) and the human airways. Global gene expression analysis, gene inactivation and phenotypic characterization in vitro and in vivo, computational modeling, medical chemistry, and antimicrobial evaluation at the preclinical level, led us to study pathogen and host transcriptional profiles during respiratory infection (Chapter 2), the contribution of glucose catabolism to H. influenzae pathogenesis (Chapter 3), and the antimicrobial potential of inhibiting this bacterial fatty acid biosynthesis pathway (Chapter 4). H. influenzae was the first free-living organism whose genome was fully sequenced, thus pioneering in the development and use of -omics techniques. Chapter 1 of this work reviewed the contribution of -omic approaches including genomics, transcriptomics, proteomics and metabolomics, to the study of this host-pathogen interplay. In Chapter 2, we carried out an in vivo multi-omic study, using dual RNA-seq and Tn-seq during murine respiratory infection by NTHi. Differential gene expression profiling of bacteria grown in vitro compared to those recovered from murine bronchoalveolar lavage fluid samples showed overexpression of genes that encode enzymes involved in purine and amino acids synthesis, as well as of genes encoding part of the bacterial natural competence machinery. The increase of glucose levels in the respiratory tract of patients suffering chronic respiratory diseases facilitates the proliferation of pathogens able to metabolize this sugar. NTHi catabolizes glucose through respiration-assisted fermentation involving the excretion of acetate, formate, and succinate. In Chapter 3 of this work, we designed, generated, and characterized a panel of mutant strains that did not produce acetate, formate, or succinate by inactivating the ackA, pflA, and frdA genes, respectively. Inactivation of the ackA gene limited acetate production and bacterial growth, and stimulated both anaerobic lactate production and bacterial attenuation in vivo. The excreted acetate stimulated the expression of pro-inflammatory genes by cultured respiratory epithelial cells, which suggests that glucose catabolism contributes not only to the growth of NTHi but also to immunomodulation within the human respiratory system. The H. influenzae resistance to β-lactam antibiotics led to its inclusion in the list of bacterial pathogens for which the WHO considers a priority the search and development of new antimicrobials. In Chapter 4 of this work, we developed and validated a H. influenzae genome-scale metabolic model, which we used as an in silico screening tool to identify bacterial essential genes suitable as therapeutic targets. This model predicted the essentiality of a large number of genes involved in lipid synthesis. We focused on the enzyme FabH, which catalyzes the decarboxylative condensation of malonyl-ACP and acyl-CoA in the initiation of fatty acid biosynthesis. Computational modeling showed the suitability of the interaction of the chemical inhibitor 1- (5- (2-Fluoro-5- (hydroxymethyl) phenyl) pyridin-2-yl) piperidine-4-acetic acid with FabH. Likewise, this inhibitor reduced bacterial viability in a dose-dependent manner. The inhibitory effect observed was variable among clinical isolates carrying different allelic variants of the fabH gene, and independent of this gene expression. The inhibitor did not generate synergies, did not favor the development of resistance, and did not alter the dynamics of epithelial infection by NTHi. Notably, this chemical inhibitor showed a protective effect against NTHi infection in vivo. Altogether, this PhD Thesis work provides novel knowledge on the role of bacterial metabolism in the NTHi-human respiratory system interplay, intended to be useful in the development of antiinfective strategies that will improve the clinical management of infectious diseases associated to this pathogen.