P. aeruginosa Isolates From CF Patients With Intermittent, but Not With Chronic Infection Show Inhibition of Antiviral Responses
It is well-known that P. aeruginosa adapts to the lung microenvironment in CF patients by downregulating several virulence factors over time including secreted proteinases when establishing a chronic infection. Therefore, we made use of four longitudinal P. aeruginosa pairings from four different CF patients (CF1 to CF4). All pairings are clonally related as was shown by RFLP before (6) and were isolated from the same patients with at least 8 years time span (Figure 4A). Whereas, the early isolates of each patient showed considerable protease activity, the late-stage isolates had significantly lower protease activity (Figure 4B). Analyzing the effect of the early isolates on the antiviral response to RSV, all isolates were able to suppress the induction of MX1 or OAS1. In line with their reduced protease activity, 3 out of 4 of later isolates displayed a lower or completely missing capacity to suppress the antiviral response (Figure 4C). An exception was isolate CF1.2, but as shown in Figure 4B this isolate also showed only a minor reduction in protease activity. Of note, expression of MX1 or OAS1 correlated significantly with the protease activity measured (Figure 4D). Many of the secreted proteases are under the regulation of the transcription factor LasR. Since LasR is known to become mutated in progressed stages of P. aeruginosa infection in CF patients (26), we sequenced LasR of all isolates used (CF1-4). We identified mutations in the late-stage isolates and in P. aeruginosa Boston (Figure 4E). Those mutated strains were the strains that had no inhibitory effect on MX1 and OAS induction (Figure 4C). Of note, no mutation of LasR was identified in CF1.2, the only strain that was still able to inhibit the antiviral response. Additionally, a mutation in LasR was detected in the early strain CF2.1 that was the one with low protease activity even at early time of infection (Figure 4B). The mutations were either non-synonymous mutations (Boston, CF2.1), deletions resulting in a frameshift and nonsense peptide sequence (CF2.2, CF4.2) or a synonymous mutation resulting in a seldom-used codon/t-RNA combination for P. aeruginosa (CF3.2) (Figure 4E). In line with this, PA7, a group 3 P. aeruginosa strain (also not able to suppress antiviral response, Figure 1E), has only 93% identical nucleotides compared to the reference strain PAO1 which results in three amino acid exchanges (K70 >R; S142 >N; N182 >S).
Figure 4  Late P. aeruginosa CF isolates display low protease activity and decreased ability to inhibit antiviral response. (A) Characteristics of longitudinal isolates of P. aeruginosa from CF patients. (B) Protease activity of P. aeruginosa CF isolates was assessed using skim milk agar. (C) BEAS2B cells were pretreated with conditioned medium of P. aeruginosa from (A) or control medium and subsequently infected with RSV. Induction of MX1 and OAS1 mRNA were analyzed by qRT-PCR after 14 h. (D) Spearman correlation of protease activity and induction of MX1 or OAS1 mRNA measured in (B,C). (E) LasR genetic mutations and resulting changes in protein translation found in CF isolates described in (A). All experiments n = 4, ANOVA and Bonferroni post-test was used for statistical analysis. Significant differences were considered at *p < 0.05, **p < 0.01, and ***p < 0.001 as compared to the control condition. n.s., not significant.