Evaluation of host-pathogen interactions for the design of novel therapies against Pseudomonas aeruginosa

Pseudomonas aeruginosa is a highly antimicrobial-resistant pathogen that causes difficult-to-treat acute and chronic infections. Hence, new antibiotic therapies against P. aeruginosa are urgently needed. Understanding host-bacterial interactions is critical for the design of innovative treatments against infections. This project evaluated the interplay between P. aeruginosa and the human host from different perspectives: (a) the effect of the versatility and daptability of P. aeruginosa within the host by the in vitro characterisation of immune responses of epithelial cells stimulated by P. aeruginosa sequential isolates from two Cystic Fibrosis (CF) patients (b) the elucidation of host receptors for P. aeruginosa; and (c) the evaluation of TLR4 ligands as adjuvants for vaccines against P. aeruginosa. The immune responses against CF sequential isolates were independent on their status as “early” or “late” isolates, but isolate-dependent, confirming a divergent evolution of the isolates within the host. One of the main achievements was the identification and validation of the role of the human protein disulfide isomerases (PDI) A1 and PDIA3 in P. aeruginosa attachment to epithelial cells using a novel unbiased 2D proteomic approach. The pre-treatment of human bronchial epithelial cells (16HBE14o-) with the PDI inhibitor, LOC14, decreased P. aeruginosa attachment to these cell lines by 2.5 fold (p= 0.0188), P. aeruginosa attachment to HEK293T cells overexpressing PDIA1 and PDIA3 was 6.01 and 6.52 fold higher than the control (empty plasmid), respectively (p=0.0344), and bacterial attachment to CRISPR cell lines A549 pdia3-/- was 1.71 fold lower than to wild-type A549 cells (p=0.0344). This discovery might lead to the design of novel antimicrobial therapies or the use of currently available drugs that target human PDIs. Finally, rOprF (recombinant outer membrane protein F) in combination with FP18 adjuvant was validated as a suitable adjuvant-antigen system for vaccines against P. aeruginosa. FP18 reduced bacterial counts in the lungs and dissemination of bacteria to spleens, it enhanced the production of antibodies and IFNγ stimulation, relative to immunisation with antigen alone in a mice model of acute P. aeruginosa pneumonia. The results might lead to novel host-directed therapies against the challenging pathogen, P. aeruginosa.