Type III secretion phenotypes of Pseudomonas aeruginosa strains change during infection of individuals with cystic fibrosis

Abstract

Pseudomonas aeruginosa is a frequent cause of respiratory exacerbations in individuals with cystic fibrosis. An important virulence determinant of this pathogen is its type III protein secretion system. In this study, the type III secretion properties of 435 P. aeruginosa respiratory isolates from 56 chronically infected individuals with cystic fibrosis were investigated. Although it had been previously reported that 75 to 90% of P. aeruginosa isolates from patients with hospital-acquired pneumonia secreted type III proteins, only 12% of isolates from cystic fibrosis patients did so, with nearly all of these isolates secreting ExoS and ExoT but not ExoU. Despite the low overall prevalence of type III protein-secreting isolates, at least one secreting isolate was cultured from one-third of cystic fibrosis patients. Interestingly, the fraction of cystic fibrosis patient isolates capable of secreting type III proteins decreased with duration of infection. Although 90% of isolates from the environment, the presumed reservoir for the majority of P. aeruginosa strains that infect patients with cystic fibrosis, secreted type III proteins, only 49% of isolates from newly infected children, 18% of isolates from chronically infected children, and 4% of isolates from chronically infected adults with cystic fibrosis secreted these proteins. Within individual patients, isolates of clonal origin differed in their secretion phenotypes, indicating that as strains persisted in cystic fibrosis patient airways, their type III protein secretion properties changed. Together, these findings indicate that following infection of cystic fibrosis patient airways, P. aeruginosa strains gradually change from a type III protein secretion-positive phenotype to a secretion-negative phenotype.

FIG. 1.

Immunoblot analysis showing the type III protein secretion phenotypes of representative P. aeruginosa isolates from CF patients. Individual isolates were grown under conditions that induced type III protein secretion. Immunoblot analysis was then performed on culture supernatants using a mixture of antisera against type III proteins. The migrations of the effector proteins ExoU, ExoT, and ExoS are shown to the left of the panel. P. aeruginosa isolate designations are listed above the membrane. The samples shown are five individual colonies from a single respiratory sample from patient N001 and five individual colonies from a single respiratory sample from patient N016. PA103 is a control strain that secretes ExoU and ExoT. Strain 388 is a control strain that secretes ExoT and ExoS. CF patient isolates differed in their type III secretion phenotypes.

FIG. 2.

Percentage of isolates from CF patients and the environment that secreted type III proteins. “Environment” refers to isolates obtained from vegetables, rivers, soil, lakes, and well water. “Newly infected children” refers to isolates from children with CF who grew P. aeruginosa from a respiratory culture for the first time in 2 years. “Chronically infected children” refers to isolates from children with CF who had at least one respiratory culture within the preceding year that grew P. aeruginosa. “Chronically infected adults” refers to isolates from adults with CF, all of whom had P. aeruginosa grow from multiple prior respiratory cultures. “Duration of Infection” refers to the approximate mean duration of infection associated with each of these groups. Error bars represent 95% CIs. The prevalence of TTS⁺ isolates decreased with duration of infection of the CF patient airways.

FIG. 3.

Type III protein secretion phenotypes and clonality of P. aeruginosa isolates from representative patients with CF. RAPD PCR genotyping was used to assess the clonality of isolates obtained over time from individual patients with CF, and immunoblot analysis was used to determine type III secretion phenotype. The date on which the culture was obtained is indicated on the left. Individual isolates are represented by a circle or a square. All isolates with similar or identical RAPD PCR genotyping patterns from a single patient are represented by the same geometric shape. TTS⁺ isolates are shown in black, whereas TTS⁻ isolates are shown in white. Isolates that were chosen for further analysis by pulsed-field gel electrophoresis are indicated by arrows with letters. (A) Representative patients who were infected with both TTS⁺ and TTS⁻ isolates that originated from a single clonal strain. (B) Patients who were infected by two unrelated strains, one of which consisted of both TTS⁺ and TTS⁻ isolates. (C) Patients who were infected with a TTS⁺ strain and an unrelated TTS⁻ strain.

FIG. 4.

Pulsed-field gel electrophoresis analysis of P. aeruginosa isolates from representative CF patients. Isolates from three patients, designated CFN1, CFN17, and CFN26, were chosen for characterization. The examined isolates correspond to those marked by arrows and letters in Fig. 3. Using the criteria of Tenover et al. (54), it was determined that isolates A and B of patient CFN1 are closely related; isolates A, B, D, and E of patient CFN17 are closely related or identical, whereas isolate C is distinct; and isolates A, B, C, and D of patient CFN26 are all closely related to one another.