Genotypic and phenotypic analyses of a Pseudomonas aeruginosa chronic bronchiectasis isolate reveal differences from cystic fibrosis and laboratory strains.

BACKGROUND: Pseudomonas aeruginosa is an environmentally ubiquitous Gram-negative bacterium and important opportunistic human pathogen, causing severe chronic respiratory infections in patients with underlying conditions such as cystic fibrosis (CF) or bronchiectasis. In order to identify mechanisms responsible for adaptation during bronchiectasis infections, a bronchiectasis isolate, PAHM4, was phenotypically and genotypically characterized. RESULTS: This strain displays phenotypes that have been associated with chronic respiratory infections in CF including alginate over-production, rough lipopolysaccharide, quorum-sensing deficiency, loss of motility, decreased protease secretion, and hypermutation. Hypermutation is a key adaptation of this bacterium during the course of chronic respiratory infections and analysis indicates that PAHM4 encodes a mutated mutS gene responsible for a ~1,000-fold increase in mutation rate compared to wild-type laboratory strain P. aeruginosa PAO1. Antibiotic resistance profiles and sequence data indicate that this strain acquired numerous mutations associated with increased resistance levels to ß-lactams, aminoglycosides, and fluoroquinolones when compared to PAO1. Sequencing of PAHM4 revealed a 6.38 Mbp genome, 5.9 % of which were unrecognized in previously reported P. aeruginosa genome sequences. Transcriptome analysis suggests a general down-regulation of virulence factors, while metabolism of amino acids and lipids is up-regulated when compared to PAO1 and metabolic modeling identified further potential differences between PAO1 and PAHM4. CONCLUSIONS: This work provides insights into the potential differential adaptation of this bacterium to the lung of patients with bronchiectasis compared to other clinical settings such as cystic fibrosis, findings that should aid the development of disease-appropriate treatment strategies for P. aeruginosa infections.

Overexpression of MexCD-OprJ reduces Pseudomonas aeruginosa virulence by increasing its susceptibility to complement-mediated killing.

We evaluated the resistance to complement-mediated killing of a collection of isogenic Pseudomonas aeruginosa strains expressing different antimicrobial resistance phenotypes. Only the nfxB mutant demonstrated increased susceptibility to complement compared with that for the wild-type strain. This increment was due to the overexpression of MexCD-OprJ, which led to increased C3 opsonization and a reduced ability to infect the lungs of mice. Our results show that the acquisition of antibiotic resistance may alter the interplay of P. aeruginosa with the host immune system.

Surfactant protein A blocks recognition of Pseudomonas aeruginosa by CKAP4/P63 on airway epithelial cells.

We used isogenic mutant strains that were deficient or over-expressed capsule to study the function of the alginate exopolysaccharide in the interaction of Pseudomonas aeruginosa with the human airway epithelial cells (AEC) in the presence or absence of surfactant protein A (SP-A). SP-A prevented the invasion of AEC by alginate-producing P. aeruginosa strains because of a direct effect on the AEC. Monoclonal antibodies to CKAP4/P63, the principal SP-A-binding receptor on AEC, or inhibition of its expression using specific siRNA reduced the invasion of both highly encapsulated and poorly encapsulated strains, but not the invasion of the acapsular mutant. Treatment of AEC with SP-A, monoclonal antibodies to CKAP4/P63, or CKAP4/P63-specific siRNA decreased the binding of purified alginate exopolysaccharide to AEC. Alginate binding to AEC reduced SP-A release by these cells. Because the alginate exopolysaccharide is surface-exposed, levels of SP-A may be crucial to modulate the interaction of P. aeruginosa with AEC.

Lysine trimethylation of EF-Tu mimics platelet-activating factor to initiate Pseudomonas aeruginosa pneumonia.

UNLABELLED: Pseudomonas aeruginosa is a ubiquitous microorganism and the most common Gram-negative bacterium associated with nosocomial pneumonia, which is a leading cause of mortality among critically ill patients. Although many virulence factors have been identified in this pathogen, little is known about the bacterial components required to initiate infection in the host. Here, we identified a unique trimethyl lysine posttranslational modification of elongation factor Tu as a previously unrecognized bacterial ligand involved in early host colonization by P. aeruginosa. This modification is carried out by a novel methyltransferase, here named elongation factor Tu-modifying enzyme, resulting in a motif that is a structural mimic of the phosphorylcholine present in platelet-activating factor. This novel motif mediates bacterial attachment to airway respiratory cells through platelet-activating factor receptor and is a major virulence factor, expression of which is a prerequisite to pneumonia in a murine model of respiratory infection. IMPORTANCE: Phosphorylcholine is an interesting molecule from the microbiological and immunological point of view. It is a crucial epitope for the virulence of many important human pathogens, modulates the host immune response, and is involved in a wide number of processes ranging from allergy to inflammation. Our current work identifies a novel bacterial surface epitope structurally and functionally similar to phosphorylcholine. This novel epitope is crucial for initial colonization of the respiratory tract by Pseudomonas aeruginosa and for development of pneumonia. This opens up new targets for the development of novel drugs to prevent P. aeruginosa pneumonia, which is particularly important given the frequent emergence of multidrug-resistant strains.