In Vivo Evidence of Pseudomonas aeruginosa Nutrient Acquisition and Pathogenesis in the Lungs of Cystic Fibrosis Patients

Abstract

One of the hallmarks of Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients is very-high-celldensity (HCD) replication in the lung, allowing this bacterium to induce virulence controlled by the quorumsensing systems. However, the nutrient sources sustaining HCD replication in this chronic infection are largely unknown. Here, we performed microarray studies of P. aeruginosa directly isolated from the lungs of CF patients to demonstrate its metabolic capability and virulence in vivo. In vivo microarray data, confirmed by real-time reverse transcription-PCR, indicated that the P. aeruginosa population expressed several genes for virulence, drug resistance, and utilization of multiple nutrient sources (lung surfactant lipids and amino acids) contributing to HCD replication. The most abundant lung surfactant lipid molecule, phosphatidylcholine (PC), induces key genes of P. aeruginosa pertinent to PC degradation in vitro as well as in vivo within the lungs of CF patients. The results support recent research indicating that P. aeruginosa exists in the lungs of CF patients as a diverse population with full virulence potential. The data also indicate that there is deregulation of several pathways, suggesting that there is in vivo evolution by deregulation of a large portion of the transcriptome during chronic infection in CF patients. To our knowledge, this is the first in vivo transcriptome analysis of P. aeruginosa in a natural infection in CF patients, and the results indicate several important aspects of P. aeruginosa pathogenesis, drug resistance, nutrient utilization, and general metabolism within the lungs of CF patients.Pseudomonas aeruginosa is the major cause of morbidity and mortality in lung diseases, including cystic fibrosis (CF) (6, 11, 32) and nosocomial pneumonia (3,40). Over 93% of CF patients between 18 and 24 years old have been reported to have P. aeruginosa infections (11). In addition, nosocomial pneumonia is the second most common of all nosocomial infections, and P. aeruginosa was the most frequently isolated microbe involved from 1992 to 1997 (37). The pathogenesis of this organism has been intensively studied with respect to virulence and virulence expression (5,26,31,36,39), biofilm production (10, 43), and quorum sensing (15,27,28,30). Several virulence factors that P. aeruginosa expresses (e.g., exotoxin A, exoenzyme S, cytotoxin, proteases, lipases, phospholipases, alginate, and hydrogen cyanide) all contribute to severe lung damage. High-cell-density (HCD) replication is necessary for many of these events to occur, and Ͼ10 9 bacteria/ml of sputum have been found in the lungs of CF patients (43, 46). The ability of P. aeruginosa to obtain nutrients in the lung for HCD replication and maintenance is the quintessential factor leading to quorum-sensing-induced virulence expression, which is a hallmark of chronic lung infections in CF patients. However, the nutrient requirements of P. aeruginosa in vivo are unknown. A recent in vitro study by Palmer et al. (25), in which P. aerugi...

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