Targeted deletion of Pf prophages from diverse Pseudomonas aeruginosa isolates impacts quorum sensing and virulence traits

Abstract

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous bacterial virulence phenotypes. Most work on Pf phages has focused on strain Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf strains infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM (PA0718), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, virulence factor production, and virulence. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates, but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages.

Figure 1:. PA0718 (PflM) maintains Pf4 lysogeny.

(A) Schematic of the Pf4 prophage (PA0715-PA0729) integrated into the tRNA-Gly gene PA0729.1 in P. aeruginosa PAO1. The core genome that is conserved amongst Pf strains is indicated. (B) Multiplex PCR was used to measure Pf4 prophage integration and excision from the PAO1 chromosome in the indicated Pf4 single-gene mutants. Excision and integration are differentiated by the size of the PCR product produced. Note that that deleting PA0721 (pfsE) from the Pf4 prophage is lethal to P. aeruginosa (16) explaining why pfsE is not included in the assay. A representative gel is shown. (C) Quantitative PCR (qPCR) was used to measure episomal Pf4 replicative form in PAO1 or ΔPA0718 cells after 18 hours of growth in LB broth. Data are the mean of three replicate experiments, ΔPA0718 values were below the limit of detection for the assay (37 copies per microliter of input material). (D) Supernatants obtained from 18 hour-old cultures of PAO1 or ΔPA0718 were spotted onto lawns of P. aeruginosa ΔPf4. A representative image is shown. (E) PflM and/or XisF4 were expressed from inducible plasmids in P. aeruginosa PAO1. After 18 hours of growth in LB broth, Pf4 integration and excision was measured by multiplex PCR. (F) Filtered supernatants collected from the indicated strains were tittered on lawns of PAO1^ΔPf4 and imaged after 18 hours of growth.

Figure 2:. Targeted deletion of pflM cures diverse P. aeruginosa isolates of their Pf prophage infections.

(A-E) Long-read whole genome sequencing was used to confirm the successful deletion of the indicated Pf prophages. Reads were aligned to 50kb sequences flanking the Pf prophage insertion sites in the parental chromosome. The genomic coordinates for each Pf prophage are shown above each bracket.

Figure 3.

Variation in the operon encoding pflM in Pf prophages.

Figure 4:. Pf phage differentially modulate P. aeruginosa quorum sensing.

(A-E) GFP fluorescence from the transcriptional reporters P_rsaLI-gfp (Las), P_rhlA-gfp (Rhl), or P_pqsA-gfp (PQS) was measured in the indicated strains after 18 hours of growth. GFP fluorescence intensity was normalized to cell growth (OD₆₀₀). Data are the mean ±SEM of seven replicates. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student’s t-test comparing ΔPf strains to the wild-type parent.

Figure 5:. Pf prophage deletion has significant but variable effects on P. aeruginosa biofilm formation.

Crystal violet biofilm assays were performed to measure biofilm formation of the indicated strains after 48h incubation. Data are the mean ±SEM of six replicates. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student’s t-test.

Figure 6:. Pyocyanin production is enhanced in Pf prophage deletion strains.

(A-E) Pyocyanin was CHCl₃-HCl extracted from the supernatants of the indicated cultures after 18h of incubation. Pyocyanin concentration was measured (Abs 520nm). Data are the mean ±SEM of three replicates. **P<0.01, ***P<0.001, ****P<0.0001, Student’s t-test.

Figure 7:. C. elegans actively avoids P. aeruginosa Pf lysogens.

(A) Experimental design: P. aeruginosa and an isogenic ΔPf mutant were spotted onto NMMG plates with wild-type N2 C. elegans at the indicated locations. C. elegans localization to the indicated quadrants was measured hourly. (B) C. elegans association with P. aeruginosa (circles) or isogenic ΔPf mutants (squares) in the indicated strain backgrounds was measured hourly over eight hours (three experiments with N=30 per replicate [90 animals total]). P values were calculated by two-way ANOVA comparing ΔPf strains to the parental strains using the Šidák correction (95% CI threshold), **P<0.01, ***P<0.001, ****P<0.0001.