Effect of Pseudomonas Aeruginosa Exoproteins on Nasal Mucosal Barrier in Chronic Rhinosinusitis

Abstract

Chronic rhinosinusitis (CRS) is characterized by a multitude of symptoms caused by chronic inflammation of the sinus mucosa. The condition is linked to recurrent infections and compromised mucosal barriers. It often occurs in association with asthma and affects the majority of people with cystic fibrosis (CF). There is a significant negative socio-economic impact associated with CRS in Western societies. Recalcitrant CRS is associated with Pseudomonas aeruginosa infections; however, little is known about the exact pathophysiology of CRS and the role of biofilms and P. aeruginosa infections. As a result, understanding the mechanisms through which these microorganisms and their exoproteins affect the mucosa and relate to chronic inflammation and relapsing infections is a continuous and intense effort worldwide. Additionally, proteomic analysis of the biofilm exoproteins may contribute to understanding the mechanism of the mucosal barrier disruption and involvement of specific proteins. In part one of this thesis, our research focused on the pathogenicity of Pseudomonas aeruginosa clinical isolates isolated from patients suffering from CRS and grown in both planktonic and biofilm forms. Our study examined the effect of exoproteins secreted by those isolates on in vitro models of the nasal epithelial barrier. According to our data, planktonic exoproteins of P. aeruginosa significantly disrupted the membrane structure and caused mucosal barrier dysfunction when 80μg/ml planktonic exoproteins were applied on primary human nasal epithelial cell cultures (HNECs) grown at Air Liquid Interface (ALI) for 2 hours. The Trans Epithelial Electrical Resistance (TEER) was decreased significantly (p<0.5) which supports barrier disruption. Significantly increased permeability of FITC dextrans (p<0.5) and discontinuous immunolocalization of tight junction proteins were observed when planktonoic exoproteins were applied to HNEC-ALI cultures. Additionally, a significant association was found between the severity of barrier disruption induced by exoproteins harvested from planktonic bacteria, their effect on inducing pro-inflammatory cytokine (IL-6) secretion and the presence of asthma in corresponding patients. This mucosal barrier damage is thought to be a crucial factor in CRS development. In the second part of the thesis, we focused on the growth of the P. aeruginosa clinical isolates in anaerobic conditions in planktonic and biofilm form and the effect of the exoproteins on mucosal barrier disruption. Several studies have shown a significant association between the presence of anaerobic bacteria and CRS and studies also revealed that P. aeruginosa could grow within the thick mucus secretions in airway lumen and survive in a low oxygen environment. So P. aeruginosa exoproteins were harvested from both planktonic and biofilm forms grown in anaerobic conditions and applied to HNEC-ALI cultures. Significant mucosal barrier disruption along with an induction of inflammation were observed when both planktonic and biofilm exoproteins from isolates grown in anaerobic conditions were used. In aerobic conditions however, exoproteins from planktonic but not biofilm forms could disrupt the mucosal barrier structure and induce inflammation. Proteomic analysis of the biofilm exoproteins from aerobic and anaerobic growth conditions indicated unique protein signatures of P. aeruginosa exoproteins in both conditions. An upregulation of biofilm exoproteins involved in Quorum sensing and denitrification was identified in anaerobic conditions. Our third study involved investigating the susceptibility of P. aeruginosa to different antibiotics in its planktonic and biofilm forms grown in aerobic and anaerobic conditions. Additionally, we examined the antibiotic susceptibility between isolates from three different countries according to the presence of CF. Results showed that P. aeruginosa isolates were more resistant to antibiotics (Ciprofloxacin, Gentamicin and Levofloxacin) in anaerobic than aerobic conditions in both planktonic and biofilm forms. The MIC values for Gentamicin and the MBEC values for Levofloxacin and Gentamicin were significantly higher for isolates grown in anaerobic conditions compared to the same isolates grown in aerobic conditions. In a country-wise analysis, some variability was seen with MBEC values between countries. Namely, MBEC values were significantly higher for Gentamicin in Australian isolates grown in anaerobic conditions compared to the same isolates grown in aerobic conditions, however, those differences were not seen for isolates from the US and The Netherlands. In contrast, Levofloxacin showed significantly higher MBEC values in anaerobic conditions among all three countries. Our study also demonstrated that the metabolic activity in anaerobically cultured biofilms was significantly higher compared to aerobic biofilms. In contrast, eDNA concentrations and biofilm biomass were higher in aerobic conditions compared to anaerobic conditions even though the latter was seen for the non-CF isolates only. Our study paves the way for future in vivo experiments to investigate the host-microbe interaction during chronic infection. A future novel therapeutic approach to CRS treatment could potentially target specific microbial exoproteins identified in this study.