Divergent Pseudomonas aeruginosa LpxO enzymes perform site-specific lipid A 2-hydroxylation

ABSTRACT Pseudomonas aeruginosa can survive in a myriad of environments, partially due to modifications of its lipid A, the membrane anchor of lipopolysaccharide. We previously demonstrated that divergent late acyltransferase paralogs, HtrB1 and HtrB2, add acyloxyacyl laurate to lipid A 2- and 2′-acyl chains, respectively. The genome of P. aeruginosa also has genes which encode two dioxygenase enzymes, LpxO1 and LpxO2, that individually hydroxylate a specific secondary laurate. LpxO1 acts on the 2′-acyloxyacyl laurate (added by HtrB2), whereas LpxO2 acts on the 2-acyloxyacyl laurate (added by HtrB1) in a site-specific manner. Furthermore, while both enzyme pairs are evolutionarily linked, phylogenomic analysis suggests the LpxO1/HtrB2 enzyme pair as being of ancestral origin, present throughout the Pseudomonas lineage, whereas the LpxO2/HtrB1 enzyme pair likely arose via horizontal gene transfer and has been retained in P. aeruginosa over time. Using a murine pulmonary infection model, we showed that both LpxO1 and LpxO2 enzymes are functional in vivo, as direct analysis of in vivo lipid A structure from bronchoalveolar lavage fluid revealed 2-hydroxylated lipid A. Gene expression analysis reveals increased lpxO2 but unchanged lpxO1 expression in vivo, suggesting differential regulation of these enzymes during infection. We also demonstrate that loss-of-function mutations arise in lpxO1 and lpxO2 during chronic lung infection in people with cystic fibrosis (CF), indicating a potential role for pathogenesis and airway adaptation. Collectively, our study characterizes lipid A 2-hydroxylation during P. aeruginosa airway infection that is regulated by two distinct lipid A dioxygenase enzymes. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infection in hospitalized and chronically ill individuals. During infection, P. aeruginosa undergoes adaptive changes to evade host defenses and therapeutic interventions, increasing mortality and morbidity. Lipid A structural alteration is one such change that P. aeruginosa isolates undergo during chronic lung infection in CF. Investigating genetic drivers of this lipid A structural variation is crucial in understanding P. aeruginosa adaptation during infection. Here, we describe two lipid A dioxygenases with acyl-chain site specificity, each with different evolutionary origins. Further, we show that loss of function in these enzymes occurs in CF clinical isolates, suggesting a potential pathoadaptive phenotype. Studying these bacterial adaptations provides insight into selection pressures of the CF airway on P. aeruginosa phenotypes that persist during chronic infection. Understanding these adaptive changes may ultimately provide clinicians better control over bacterial populations during chronic infection. Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infection in hospitalized and chronically ill individuals. During infection, P. aeruginosa undergoes adaptive changes to evade host defenses and therapeutic interventions, increasing mortality and morbidity. Lipid A structural alteration is one such change that P. aeruginosa isolates undergo during chronic lung infection in CF. Investigating genetic drivers of this lipid A structural variation is crucial in understanding P. aeruginosa adaptation during infection. Here, we describe two lipid A dioxygenases with acyl-chain site specificity, each with different evolutionary origins. Further, we show that loss of function in these enzymes occurs in CF clinical isolates, suggesting a potential pathoadaptive phenotype. Studying these bacterial adaptations provides insight into selection pressures of the CF airway on P. aeruginosa phenotypes that persist during chronic infection. Understanding these adaptive changes may ultimately provide clinicians better control over bacterial populations during chronic infection.