Characterization of Pseudomonas aeruginosa lipid A structural variants in cystic fibrosis

Abstract

Pseudomonas aeruginosa is the most common Gram-negative bacteria to cause chronic lung infection in people with cystic fibrosis (pwCF), leading to structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous genetic and phenotypic changes, adapting to the airway environment while establishing chronic infection. The work presented in this thesis characterizes one specific P. aeruginosa adaptation that occurs during CF lung infection: lipid A structural modification. Lipid A is the membrane anchor of lipopolysaccharide (LPS) (i.e., endotoxin), which comprises approximately 75% of the outer membrane of Gram-negative bacteria and is a potent agonist of toll-like receptor (TLR)-4, an innate immune receptor. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4 and the subsequent immune response. We hypothesize that lipid A structural alteration is beneficial for P. aeruginosa survival and pathogenesis by manipulating the host immune response during lung infection. Using a cohort of CF-derived P. aeruginosa isolates, we identify lipid A structural variation in isolates from 20% of pwCF. These lipid A structural alterations are driven by non-synonymous mutations in three lipid A genes: lpxO1, lpxO2, and pagL. We then characterize two P. aeruginosa lipid A enzymes encoded by lpxO1 and lpxO2 that can be mutated during chronic lung infection in CF. These two lipid A enzymes have distinct functions, mediating lipid A 2-hydroxylation in a site-specific manner. We also characterize P. aeruginosa isolates obtained from another inflammatory lung disease, diffuse panbronchiolitis, which result in the synthesis of structurally-distinct lipid A structures, suggesting that lipid A structural variation is not CF-specific. Lastly, we evaluate the impact of P. aeruginosa lipid A structure on host immune recognition and response. In vivo P. aeruginosa lacks PagL-mediated lipid A deacylation, which subsequently induces a stronger cytokine response. P. aeruginosa lipid A that lacks LpxO2-mediated 2-hydroxylation has reduced inflammatory potential, whereas LpxO1-mediated 2-hydroxylation has no measurable impact. This demonstrates distinct roles for each of the lipid A 2-hydroyxlation enzymes during in vivo P. aeruginosa infection. Taken together, P. aeruginosa lipid A structure plays an important role in pathogenesis during lung infection.