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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.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.
摘要：
目的：铜绿假单胞菌是一种机会致病菌，可引起住院和慢性病患者的严重感染。在感染期间,铜绿假单胞菌经历适应性变化以逃避宿主防御和治疗干预，增加死亡率和发病率。脂质结构改变是铜绿假单胞菌分离物在CF慢性肺部感染期间经历的一种这样的改变。研究这种脂质的遗传驱动因素A结构变异对于理解感染期间的铜绿假单胞菌适应至关重要。这里,我们描述了两种具有酰基链位点特异性的脂质A双加氧酶，每个都有不同的进化起源。Further,我们表明，这些酶的功能丧失发生在CF临床分离株，提示潜在的病态适应性表型。研究这些细菌适应提供了对CF气道对在慢性感染期间持续存在的铜绿假单胞菌表型的选择压力的见解。了解这些适应性变化可能最终为临床医生在慢性感染期间更好地控制细菌种群。
