Background: In the CF lung, Pa biofilms form as dense aggregates of ~10-1,000 cells. While co-colonization of Pa with other organisms has been linked to poorer health outcomes, little is known about the dynamics of these multi-kingdom communities. Specifically, there is a significant gap in understanding of the interactions between Pa and the emerging CF pathogen Aspergillus fumigatus (Af). One of the biggest challenges in studying these interactions is the lack of a robust animal model, therefore we have utilized a synthetic CF sputum media (SCFM2). SCFM2 mimics the physical and nutritional conditions of human sputum, including supporting the formation of aggregates of similar sizes to those observed in the CF lung. In a novel application of this model, we have adapted SCFM2 to support the growth of human lung epithelial cells (H411) and Af, providing a multi-kingdom biofilm model that more closely recapitulates chronic infection of the CF lung. Here, we use this system to identify physiological pathways and spatial organization that are critical for Pa colonization with Af in the CF lung.
Methods: High resolution confocal laser scanning microscopy (CLSM) was used to visualize the development of the biofilms over time, including Pa aggregate position, growth, and motility in relation to Af and H411 cells. Proteomic analysis of cell lysates and supernatants by Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer was utilized to identify critical pathways, specific to the culture of Pa and Af in the presence of epithelial cells in synthetic sputum.
Results: We have now developed an adapted SCFM2 model to include human epithelial cells and active growth of the emerging CF pathogen Af, to identify physiological and physical phenotypes specific to multispecies interactions in an environment similar to the CF lung.We found that during co-culture with Af, Pa aggregates were able to closely associate with hyphal regions of Af, suggesting a co-existance strategy in which Af remained metabolically active. Mass spectrometry data provided a robust profile of both secretory and intracellular proteins for Pa, Af, and H411 cells. High abundance proteins included a a quorum sensing (QS) regulated lysyl endopeptidase and the acyl homoserine lactone acylase QuiP, which has a specificity for long chain AHLs. These data suggest that QS is involved in the mediation of interactions between Pa, Af and human cells in SCFM2. We hypothesize that is heavily influenced by the intra aggregate signaling which has previously been shown to predominate in this model.
Conclusions: At present, the underlying mechanisms of aggregate formation and interactions in the presence of other microbes are not well defined. Here we present a model to study such interactions at high resolution, with the ability to apply proteomic approaches to identify pathways that support them. Understanding the mechanisms that contribute to Pa persistence is urgently important, as multi-drug resistant Pa infections increase in frequency within the CF clinic. However, studying Pa aggregates in isolation may not provide the full picture – highlighting the need to study more complex microbial communities, specifically with emerging pathogens.
Acknowledgements: This work was supported by the Cystic Fibrosis Foundation (DARCH19G0) awarded to S.E.D and a student traineeship award awarded to A.D.G
