Dataset Information

Site-specific activity of the acyltransferases HtrB1 and HtrB2 in Pseudomonas aeruginosa lipid A biosynthesis.

ABSTRACT: Pseudomonas aeruginosa (PA) is an opportunistic Gram-negative pathogen associated with nosocomial infections, acute infections and chronic lung infections in patients with cystic fibrosis. The ability of PA to cause infection can be attributed to its ability to adapt to a multitude of environments. Modification of the lipid A portion of lipopolysaccharide (LPS) is a vital mechanism Gram-negative pathogens use to remodel the outer membrane in response to environmental stimuli. Lipid A, the endotoxic moiety of LPS, is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria making it a critical factor for bacterial adaptation. One way PA modifies its lipid A is through the addition of laurate and 2-hydroxylaurate. This secondary or late acylation is carried out by the acyltransferase, HtrB (LpxL). Analysis of the PA genome revealed the presence of two htrB homologs, PA0011 (htrB1) and PA3242 (htrB2). In this study, we were able to show that each gene identified is responsible for site-specific modification of lipid A. Additionally, deletions of either gene altered resistance to specific classes of antibiotics, cationic antimicrobial peptides and increased membrane permeability suggesting a role for these enzymes in maintaining optimal membrane organization and integrity.

SUBMITTER: Hittle LE

PROVIDER: S-EPMC4626592 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Site-specific activity of the acyltransferases HtrB1 and HtrB2 in Pseudomonas aeruginosa lipid A biosynthesis.

Hittle Lauren E LE Powell Daniel A DA Jones Jace W JW Tofigh Majid M Goodlett David R DR Moskowitz Samuel M SM Ernst Robert K RK

Pathogens and disease 20150729 8

Pseudomonas aeruginosa (PA) is an opportunistic Gram-negative pathogen associated with nosocomial infections, acute infections and chronic lung infections in patients with cystic fibrosis. The ability of PA to cause infection can be attributed to its ability to adapt to a multitude of environments. Modification of the lipid A portion of lipopolysaccharide (LPS) is a vital mechanism Gram-negative pathogens use to remodel the outer membrane in response to environmental stimuli. Lipid A, the endoto ...[more]

PMID: 26223882

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Project description: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.

Project description:Pseudomonas aeruginosa strain PA14 is a multi-host pathogen that infects plants, nematodes, insects, and vertebrates. Many PA14 factors are required for virulence in more than one of these hosts. Noting that plants have a fundamentally different cellular architecture from animals, we sought to identify PA14 factors that are specifically required for plant pathogenesis. We show that synthesis by PA14 of the disaccharide trehalose is required for pathogenesis in Arabidopsis, but not in nematodes, insects, or mice. In-frame deletion of two closely-linked predicted trehalose biosynthetic operons, treYZ and treS, decreased growth in Arabidopsis leaves about 50 fold. Exogenously co-inoculated trehalose, ammonium, or nitrate, but not glucose, sulfate, or phosphate suppressed the phenotype of the double ?treYZ?treS mutant. Exogenous trehalose or ammonium nitrate does not suppress the growth defect of the double ?treYZ?treS mutant by suppressing the plant defense response. Trehalose also does not function intracellularly in P. aeruginosa to ameliorate a variety of stresses, but most likely functions extracellularly, because wild-type PA14 rescued the in vivo growth defect of the ?treYZ?treS in trans. Surprisingly, the growth defect of the double ?treYZ?treS double mutant was suppressed by various Arabidopsis cell wall mutants that affect xyloglucan synthesis, including an xxt1xxt2 double mutant that completely lacks xyloglucan, even though xyloglucan mutants are not more susceptible to pathogens and respond like wild-type plants to immune elicitors. An explanation of our data is that trehalose functions to promote the acquisition of nitrogen-containing nutrients in a process that involves the xyloglucan component of the plant cell wall, thereby allowing P. aeruginosa to replicate in the intercellular spaces in a leaf. This work shows how P. aeruginosa, a multi-host opportunistic pathogen, has repurposed a highly conserved "house-keeping" anabolic pathway (trehalose biosynthesis) as a potent virulence factor that allows it to replicate in the intercellular environment of a leaf.

Dataset Information

Site-specific activity of the acyltransferases HtrB1 and HtrB2 in Pseudomonas aeruginosa lipid A biosynthesis.

Publications

Site-specific activity of the acyltransferases HtrB1 and HtrB2 in Pseudomonas aeruginosa lipid A biosynthesis.

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