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...

Alginic acid-like mucoid exopolysaccharide was isolated from three strains of Pseudomonas aeruginosa obtained from the sputa of patients with cystic fibrosis. Purified mucoid antigens were greater than 99% uronic acid. With a hemagglutination assay, antibody responses to the mucoid exopolysaccharide were documented after immunization of rabbits with either whole mucoid organisms or purified mucoid exopolysaccharide. The mucoid antigen from one strain (no. 2192) was composed predominantly of a single serologic epitope shared among 40 alginate exopolysaccharides from different clinical isolates. The mucoid exopolysaccharide from the other two strains (nos. 1 and 258) had a serotype-specific determinant in addition to the common epitope. Analyses of antibody in sera from normal adults, children, and patients with cystic fibrosis culture-positive and culture-negative for mucoid P. aeruginosa showed a highly significant (P less than 0.001) association between increased hemagglutination titers and positive cultures for mucoid P. aeruginosa.

Eukaryotic cells contain a 700-kDa proteolytic complex (the "proteasome" or multicatalytic endopeptidase complex), whose role in intracellular protein breakdown is unclear. It has been suggested that the proteasome functions in the rapid degradation of oxidant-damaged proteins and in the ATP-dependent proteolytic pathway. To test these possibilities, oxidant-damaged hemoglobin and albumin were produced by treating hemoglobin and albumin with phenylhydrazine, with hydroxyl radicals, or with both hydroxyl and superoxide radicals. After oxidant damage, these proteins were degraded more rapidly in erythrocyte extracts and also by the purified proteasome. However, complete removal of proteasomes from these extracts by immunoprecipitation (or inhibitors of its proteolytic activity) did not reduce the breakdown of oxidant-damaged hemoglobin and decreased degradation of hydroxyl-and superoxide-treated proteins by only 30-40%. Thus, erythrocytes must contain another proteolytic system for degradation of oxidant-damaged proteins. In contrast, immunoprecipitation of proteasomes with polyclonal or monoclonal antibodies prevented the ATP/ubiquitin-dependent degradation of lysozyme and also blocked the ATP-stimulated degradation of ubiquitin-conjugated lysozyme in reticulocyte and skeletal muscle extracts. These data indicate a critical role of the proteasome in the degradation of ubiquitin-conjugated proteins and suggest that the proteasome is associated with or is a component of the larger ubiquitin-conjugate-degrading enzyme complex.

To investigate some aspects of immune function in cystic fibrosis, we measured serum immunoglobulins in 419 patients. Twenty-two per cent of the 154 patients less than 10 years old had hypogammaglobulinemia-G, whereas the older patients had normal or elevated serum immunoglobulins. A single mechanism accounting for the extraordinary prevalence of hypogammaglobulinemia in young patients with cystic fibrosis was not defined in studies of T and B-lymphocyte function in vitro or in studies of IgG metabolism in vivo. Analysis of objective clinical data, including arterial blood gases, chest roentgenograms, and bacteriologic cultures, indicated that the patients with hypogammaglobulinemia had significantly less severe lung disease than did age-matched patients with cystic fibrosis and normal or elevated IgG levels. We conclude that progression of lung disease may be due in part to a hyper-immune response.