Canè, Martina (2023) Identification of potential persistence mechanisms engaged by the pathogen Moraxella catarrhalis using human in vitro respiratory models. [Tesi di dottorato]

Anteprima

Testo Cane_Martina_35.pdf Download (3MB) | Anteprima

Abstract

Moraxella catarrhalis (Mcat) is a Gram-negative diplococcus bacterium that colonizes the upper and lower respiratory tracts in human. Although it has long been considered a commensal bacterium, Mcat has been recognized as one of the causative agents of otitis media (OM) in children and exacerbation of chronic obstructive pulmonary disease (COPD) in adults where it forms polymicrobial communities with Nontypeable Heamophilus Influenzae (NTHi). The widespread resistance to β-lactam antibiotic treatments and the employment of undefined persistence mechanisms, along with the lack of suitable animal models to understand the etiology of infection make its eradication complicated and development of innovative in vitro alternatives urgent. The human airway epithelium is the first line of defense against inhaled pathogens, acting as a physical, chemical, and immunological barrier. Modeling human-derived lung epithelial barrier in vitro could represent a valid tool for the characterization of host-pathogen interaction and shed light on Moraxella catarrhalis persistence systems. To that purpose highly physiological human-derived in vitro models has been assembled. Co-cultures of epithelial and stromal cells on porous transwell membrane allowed to obtain fully-differentiate human epithelial tissues displaying physiological features of in vivo lung tissue, such as the formation of a pseudostratified architecture composed of different cell types, and a functional epithelial barrier with an active mucociliary clearance system. In this context, we aimed to monitor Mcat infection progression to investigate pathogen-associated invasive and persistence strategies in the host environment. At the early stages of infection, Mcat targeted preferentially epithelial ciliated cells and showed the ability to adapt to the new environment by modulating the expression of specific virulence factors. Interestingly, when the infection occurred in concomitance with NTH, the expression of genes involved in iron uptake and β-lactam antibiotic resistance were differently regulated respect to single-infected models suggesting the presence of mechanisms of transcriptional regulation that were active between the two pathogens. After the initial adaptation phase Mcat was observed to actively invade the host epithelium by modulating the eukaryotic cytoskeleton for internalization inside cells. Interestingly, fluorescence and electron microscopy analysis revealed Mcat ability to subvert host clearance mechanisms and to replicate inside the cell cytosol giving rise to massive intracellular colonies that subsequently expanded by invading neighboring cells. Importantly, these dense biofilm-like structures appeared to form inside the epithelial layer and subsequently emerged to originate large surface-associated bacterial communities suggesting a colonization mechanism that relies on both intracellular and extracellular bacterial lifestyle. This aggressive invasion phenotype caused significant remodeling of the pseudostratified host tissue, especially at the level of luminal cells with activation of stimulatory mechanisms for tissue regeneration. In conclusion, in-house assembled in vitro systems made it possible to identify potential invasive and persistence strategies used by Mcat in complex, self-sustaining airway infection context, creating the basis for the identification of bacterial antigens that could be used as candidates for new potential vaccines interventions.

Downloads

Actions (login required)

Modifica documento