Nonmucoid Pseudomonas aeruginosa expresses alginate in the lungs of patients with cystic fibrosis and in a mouse model

Abstract

Background: In patients with cystic fibrosis (CF), lung infection with mucoid Pseudomonas aeruginosa strains overexpressing the exopolysaccaride alginate is preceded by colonization with nonmucoid strains. We investigated the kinetics, impact of environmental signals, and genetics of P. aeruginosa alginate expression in a mouse model and in patients with CF.

Methods: Using indirect immunofluorescence, microarray technology and real-time reverse-transcription polymerase chain reaction, we assessed alginate gene expression during aerobic and anaerobic growth of the nonmucoid strain PAO1 in vitro, in a mouse lung-infection model and in sputum specimens from patients with CF infected with nonmucoid or mucoid P. aeruginosa strains.

Results: Anaerobic conditions increased the transcription of alginate genes in vitro and in murine lungs within 24 h. Alginate production by PAO1 in murine lungs and by nonmucoid P. aeruginosa strains in patients with CF was reversible after in vitro culture under aerobic conditions. A subpopulation of P. aeruginosa clones revealing stable alginate production was detected in murine lungs 2 weeks after infection.

Conclusions: Anaerobiosis and lung infection rapidly induce alginate production by gene regulation in nonmucoid P. aeruginosa. This trait may contribute to early persistence, leading to chronic P. aeruginosa infection once stable mucoid strains are generated.

Figure 1

Production of nonmucoid Pseudomonas aeruginosa alginate grown anaerobically in vitro that does not change its phenotype. On Pseudomonas isolation agar, P. aeruginosa strain PAO1 revealed a nonmucoid phenotype when cultivated aerobically (A) or anaerobically (C), whereas its isogenic mutant PDO300 showed only a mucoid phenotype (B). When indirect immunofluorescence was used, alginate production of PAO1 was detectable under anaerobic growth conditions (F) but not under aerobic growth conditions (D), whereas PDO300 was alginate-positive under aerobic growth conditions (E). Bars in D–F, 1 μm.

Figure 2

Increase in Pseudomonas aeruginosa alginate gene expression under anaerobic growth conditions. Differential gene expression by strain PAO1, grown for 1–4 days under anaerobic growth conditions (−O₂) or aerobically for 2 h (+O₂), was assessed by use of the microarray technique. Values are given as means ± SDs. Dotted lines indicate cutoff levels for differential transcription that is increased or decreased. napC and arcC genes represent positive controls for anaerobiosis. gyrA is a constitutively transcribed P. aeruginosa gene.

Figure 3

Pseudomonas aeruginosa algD transcription in murine lungs and in sputum specimens from patients with cystic fibrosis (CF), as measured by real-time reverse-transcription polymerase chain reaction. Total RNA from lung homogenates of mice infected with P. aeruginosa strain PAO1 in agar beads or from sputum specimens from patients with CF was isolated. algD expression was quantified in relation to the gyrA expression in each specimen. Data are presented as the fold change vs. the referent strain, PAO1. In vitro data showed increased algD expression after 8 h of anaerobic growth (−O₂) vs. aerobic growth (+O₂). Infected mice showed low algD expression 1 h after infection, whereas a significant increase was detected 24 h after infection. Similarly, algD expression was detected in sputum specimens from patients with CF whose respiratory cultures grew only nonmucoid (NM) P. aeruginosa and in sputum specimens from patients with CF whose respiratory cultures grew only mucoid (M) P. aeruginosa colonies. Control experiments were performed by growing M and NM strains in vitro. In vitro, M strains expressed high levels of algD transcripts, whereas only low expression was measured for NM strains.

Figure 4

Subpopulation of Pseudomonas aeruginosa selected in the lungs of agar bead–infected C57BL/6 mice. C57Bl/6 mice were infected with 10⁶–10⁷ cfu of P. aeruginosa strain PAO1/lung, and mortality was determined. Other groups of mice were killed at the indicated time points, for the determination of the no. of colony forming units per lung and the percentage of infected mice. Shown is the growth curve of strain PAO1 in murine lungs. Dots represent individual measurements of the no. of colony forming units per lung, and horizontal lines represent median values. Mortality (mean ± SD) and infection rates (mean ± SD) of PAO1-infected mice and no. of colony forming units per lung (median) are also shown. Values were selected from 2–6 different experiments. n, no. of pooled mice analyzed for each condition.

Figure 5

Expression of alginate by Pseudomonas aeruginosa within agar beads in murine lungs. Mice were infected with PAO1 embedded in agar beads, and lungs were investigated histologically and for alginate by indirect immunofluorescence. A, Agar bead (arrows), deposited in the bronchial lumen (L) 1 h after intratracheal injection. B, No alginate detectable by indirect immunofluorescence in an agar bead (arrows). C, 4′,6′-Diamidino-2-phenylindole dihydrochloride (DAPI) staining of the same tissue section as that in panel B, 1 h after infection. D, Bacterial macrocolonies in an agar bead (arrows) 1 day after infection. E, Expression of alginate by bacterial macrocolonies 1 day after infection. F, DAPI staining of the same tissue section as that in panel E. Bars in A–F, 100 μm.

Figure 6

Pseudomonas aeruginosa alginate gene expression in mouse lungs and in vitro under aerobic growth conditions. P. aeruginosa strain PAO1 expressed alginate as detected by indirect immunofluorescence in lung homogenates of C57Bl/6 mice, infected for 1 (A), 7 (B), and 14 (C) days. Bacterial colonies analyzed after 1 (D) or 7 (E) days of infection lost this phenotypic trait when they were subcultured in vitro under aerobic conditions; after 14 days of infection (F), colonies remained positive for alginate gene expression after aerobic subculture. Bars in A–F, 1 μm.

Figure 7

Pseudomonas aeruginosa alginate gene expression in sputum specimens from patients with cystic fibrosis (CF) and its phenotype on agar plates. P. aeruginosa cultured from sputum specimens from 2 different patients with CF showed a mucoid (A) or a nonmucoid (D) phenotype when they were plated on Pseudomonas isolation agar for 24 h. However, P. aeruginosa bacterial cells in the sputum from the patient with CF whose respiratory cultures grew only mucoid (B) or only nonmucoid P. aeruginosa (E) expressed alginate when they were stained by indirect immunofluorescence. C and F, 4′,6′-Diamidino-2-phenylindole dihydrochloride staining of the same sputum as that in panels B and E. Bar in B, C, E, and F, 10 μm.