Cystic fibrosis sputum supports growth and cues key aspects of Pseudomonas aeruginosa physiology

Abstract

The opportunistic human pathogen Pseudomonas aeruginosa causes persistent airway infections in patients with cystic fibrosis (CF). To establish these chronic infections, P. aeruginosa must grow and proliferate within the highly viscous sputum in the lungs of CF patients. In this study, we used Affymetrix GeneChip microarrays to investigate the physiology of P. aeruginosa grown using CF sputum as the sole source of carbon and energy. Our results indicate that CF sputum readily supports high-density P. aeruginosa growth. Furthermore, multiple signals, which reduce swimming motility and prematurely activate the Pseudomonas quinolone signal cell-to-cell signaling cascade in P. aeruginosa, are present in CF sputum. P. aeruginosa factors critical for lysis of the common CF lung inhabitant Staphylococcus aureus were also induced in CF sputum and increased the competitiveness of P. aeruginosa during polymicrobial growth in CF sputum.

FIG. 1.

Growth of P. aeruginosa in MOPS medium containing 20 mM glucose (•), 6.3 mM glucose (○), or 10% CF sputum (▪) as the sole source of carbon and energy. Bacteria were grown with shaking (250 rpm) at 37°C and sampled during exponential growth (10⁸ CFU of P. aeruginosa/ml) for Affymetrix GeneChip analysis. Representative growth curves are shown.

FIG. 2.

Proposed pathway for aromatic amino acid and quinolone/quinoline biosynthesis in P. aeruginosa. Dual arrowheads indicate multiple steps. Genes encoding proteins critical for quinolone/quinoline production are shown in boldface type.

FIG. 3.

Increased production of PQS during growth in CF sputum. TLC was used to monitor PQS production by P. aeruginosa grown with CF sputum, glucose, or glucose containing a CF sputum ethyl acetate extract (see Materials and Methods). Growth in glucose containing an ethyl acetate extract of CF sputum medium was used to test if CF sputum medium contained sufficient P. aeruginosa quorum sensing signals to induce PQS production. Synthetic PQS (150 ng) was included as a reference. Sampling occurred during late exponential phase (approximately 6 × 10⁸ CFU of P. aeruginosa/ml), and similar results were obtained for mid-exponential phase bacteria. Spot densitometry of triplicate experiments revealed that CF sputum-grown P. aeruginosa produced 4.9 ± 0.56 times more PQS than glucose-grown bacteria.

FIG. 4.

Induction of pqsA and increased production of PQS during growth with amino acids. (A) Semiquantitative RT-PCR was used to examine pqsA transcript levels during growth with glucose, succinate, Casamino Acids, tryptic soy broth, or CF sputum. Bacteria were grown to identical densities, and the constitutively expressed clpX gene was used as the control. (B) P. aeruginosa was grown in MOPS succinate (as the control); MOPS succinate with 1 mM serine (Ser); MOPS succinate with 1 mM tryptophan (Trp); and MOPS succinate with Trp, phenylalanine (Phe), and tyrosine (Tyr) (0.33 μM each). PQS was extracted and quantitated as outlined in Materials and Methods. Data are expressed as increases (n-fold) in PQS production during growth with amino acids compared to growth in succinate alone (PQS produced in MOPS succinate with amino acids/PQS produced in MOPS succinate). Bacteria were sampled at identical densities in late exponential phase.

FIG. 5.

Induction of PQS-controlled genes during growth in CF sputum. Semiquantitative RT-PCR using RNA harvested from P. aeruginosa grown with glucose or CF sputum as the sole source of carbon and energy. Genes encoding proteins important for quinoline production (pqsA) and production of the PQS-controlled virulence factors pyocyanin (phzE2) and hydrogen cyanide (hcnA) were tested. For visualization, 5 μl of the resulting PCR was separated by agarose gel electrophoresis and stained with ethidium bromide. The constitutively expressed clpX gene was used as the control.

FIG. 6.

Lysis of S. aureus by P. aeruginosa during planktonic growth in brain heart infusion (A) and MOPS-CF sputum (B) media. P. aeruginosa and S. aureus were inoculated to equal densities in test tubes and grown with shaking (250 rpm) at 37°C. Bacterial numbers were determined by differential plating (see Materials and Methods). For all time points, standard deviations were less than 10% of the mean. P. aeruginosa and S. aureus have similar growth rates and maximum cell densities in these media (data not shown).