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-cell-density (HCD) replication in the lung, allowing this bacterium to induce virulence controlled by the quorum-sensing 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.

FIG. 1.

Constitutive expression of P. aeruginosa biofilm/alginate biosynthetic genes results in high production of alginate at all growth phases by the clinical isolate pool from the CF patient relative to the production by strain PAO1. Although the growth of the pooled isolates from the CF patient and the growth of PAO1 are comparable, the pooled isolates from the CF patient showed excessive alginate production at all growth phases.

FIG. 2.

Average fold changes for pairwise comparisons of glp genes, plcH, and mexY, which were not detected due to the statistical stringency (P ≤ 0.05) imposed on the GeneChip data for glp genes (Table 4) and plcH and mexY (see Table S1 in the supplemental material). (A to E) Pairwise comparisons of three data sets for PAO1 grown in 1× M9 medium containing 0.4% PC (indicated by the numbers 1 to 3 on the x axis) to three data sets for PAO1 grown in 1× M9 medium containing 20 mM citrate (indicated by the letters A to C on the x axis). (F and G) Pairwise comparisons of two data sets for clinical in vivo samples (indicated by the numbers 1 and 2 on the x axis) to three data sets for the clinical isolate pool grown in 1× M9 medium containing 20 mM citrate (indicated by the letters A to C on the x axis). The glycerol uptake facilitator gene glpF (A), the glycerol metabolism regulator gene glpR (B), the glycerol kinase gene glpK (C), the glycerol-3-phosphate transporter gene glpT (D), and the glycerol-3-phosphate dehydrogenase gene glpD (E) all demonstrated induction when PAO1 was grown on PC compared to when PAO1 was grown on citrate, yielding average changes of 4.4-, 2.4-, 7.0-, 3.5-, and 4.0-fold, respectively. The hemolytic phospholipase C precursor gene plcH (F) and the RND multidrug efflux transporter gene mexY (G) were induced more on average in vivo than in the clinical isolate pool grown in 1× M9 containing 20 mM citrate, with average changes of 2.8- and 2.2-fold, respectively. Real-time RT-PCR confirmation data for glpK and glpD are shown in Table 4, and confirmation data for plcH and mexY are shown in Table 3.

FIG. 3.

(A) Lung surfactant, made of 90% lipids and 10% proteins, coats the trachea, bronchioles, and alveoli of the lung. (B) Of the 90% lipids, 80% consists of PC, which can be cleaved by P. aeruginosa lipases and phospholipase C into three constituents, including fatty acids (FA), glycerol, and phosphorylcholine. (C) Glycerol and phosphorylcholine constituents are further metabolized by known Glp and Bet enzymes of P. aeruginosa (42, 49). The regulators GlpR and BetR control the expression of these enzymes. GlpT and GlpF of the cytoplasmic membrane facilitate glycerol-3-phosphate and glycerol transport, respectively. Based on the E. coli model (8), predicted steps in the Fad pathway of P. aeruginosa, which has not been characterized, are shown. In addition, the regulation of the fad genes in P. aeruginosa is an enigma. FadL, an outer membrane protein, is involved in fatty acid transport. FAD, flavin adenine dinucleotide; FADH₂, reduced flavin adenine dinucleotide; CoA, coenzyme A.

FIG. 4.

fadBA5 operon is involved in PC degradation. (A) P. aeruginosa fusion strain PAO1-PfadBA5-lacZ was grown in LB, PC, citrate, and palmitate (C_16:0), and the β-galactosidase (β-Gal) assay was performed (20). The results indicate the reason for no apparent difference in the fold change in the fadBA5 operon expression shown in Table 4 for growth on PC relative to growth on citrate. The lack of fadBA5 induction when P. aeruginosa strain PAO1 was grown on 1× M9 medium plus PC compared to growth on citrate seems to be due to the high expression of this operon when bacteria were grown in 1× M9 medium plus citrate compared to the expression during growth on PC. (B and C) ΔfadBA5 mutant showed a defect in growth on palmitate compared to wild-type strain PAO1 (WT) (B), and this mutant had a defect in PC degradation compared to wild-type strain PAO1 (C), although the defect was not as dramatic as the defect with palmitate, probably due to growth on other carbon sources (glycerol and phosphorylcholine of PC). Both panel B and panel C show that the ΔfadBA5 mutant had a lower growth rate and overall lower final cell density, which were due to a partial defect in the ability to degrade fatty acid as one of the components of PC as a nutrient. Data for construction of the mutant and fusion are not shown.