Pseudomonas aeruginosa Lipid A Structural Variants Induce Altered Immune Responses.

Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection that is not seen in any other disease state. Lipid A, the membrane anchor of LPS (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent Toll-like receptor 4 (TLR4) agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4 and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of the P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human BAL fluid. This structure triggers increased proinflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CF transmembrane conductance regulator function. It is interesting that there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lacks PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.

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

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.IMPORTANCEPseudomonas 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.

Characterization of Pseudomonas aeruginosa from subjects with diffuse panbronchiolitis.

Diffuse panbronchiolitis (DPB) is a rare, idiopathic inflammatory disease primarily diagnosed in East Asian populations. DPB is characterized by diffuse pulmonary lesions, inflammation of the respiratory bronchioles, and bacterial infections of the airway. Historically, sputum cultures reveal Pseudomonas aeruginosa in 22% of DPB patients, increasing to 60% after 4 years from disease onset. Although DPB patients have a known susceptibility to respiratory P. aeruginosa infections, as is observed in other chronic lung diseases such as cystic fibrosis (CF), the characterization of DPB P. aeruginosa strains is limited. In this study, we characterized 24 strains obtained from a cohort of DPB patients for traits previously associated with virulence, including growth, motility, antibiotic susceptibility, lipopolysaccharide structure, and genomic diversity. Our cohort of DPB P. aeruginosa strains exhibits considerable genomic variability when compared with isolates from people with cystic fibrosis chronically colonized with P. aeruginosa and acute P. aeruginosa infection isolates. Similar to CF, DPB P. aeruginosa strains produce a diverse array of modified lipid A structures. Antibiotic susceptibility testing revealed increased resistance to erythromycin, a representative agent of the macrolide antibiotics used to manage DPB patients. Differences in the O-antigen type among P. aeruginosa strains collected from these different backgrounds were also observed. Ultimately, the characterization of DPB P. aeruginosa strains highlights several unique qualities of P. aeruginosa strains collected from chronically diseased airways, underscoring the challenges in treating DPB, CF, and other obstructive respiratory disease patients with P. aeruginosa infections. IMPORTANCE: Diffuse panbronchiolitis (DPB), a chronic lung disease characterized by persistent P. aeruginosa infection, serves as an informative comparator to more common chronic lung diseases, such as cystic fibrosis (CF). This study aimed to better address the interplay between P. aeruginosa and chronically compromised airway environments through the examination of DPB P. aeruginosa strains, as existing literature regarding DPB is limited to case reports, case series, and clinical treatment guidelines. The evaluation of these features in the context of DPB, in tandem with prevailing knowledge of P. aeruginosa strains collected from more common chronic lung diseases (e.g., CF), can aid in the development of more effective strategies to combat respiratory P. aeruginosa infections in patients with chronic lung diseases.