Targeted deletion of Pf prophages from diverse Pseudomonas aeruginosa isolates has differential impacts on 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 virulence phenotypes. Most work on Pf phages has focused on Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf phages 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. The pflM gene shows diversity among different Pf phage isolates; however, all examined pflM alleles encode the DUF5447 domain. We demonstrate that pflM deletion results in prophage excision but not replication, leading to total prophage loss, indicating a role for lysis/lysogeny decisions for the DUF5447 domain. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, pigment production, and virulence against the bacterivorous nematode Caenorhabditis elegans. We find that Pf phages have strain-specific impacts on quorum sensing and biofilm formation, but nearly all suppress pigment production and increase C. elegans avoidance behavior. 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.IMPORTANCEPseudomonas aeruginosa is an opportunistic bacterial pathogen that is frequently infected by filamentous Pf phages (viruses) that integrate into its chromosome, affecting behavior. Although prior work has focused on Pf4 and PAO1, this study investigates diverse Pf in clinical isolates. A simple method targeting the deletion of the Pf lysogeny maintenance gene pflM (PA0718) effectively eliminates Pf prophages from clinical isolates. The research evaluates the impact Pf prophages have on bacterial quorum sensing, biofilm formation, and virulence phenotypes. This work introduces a valuable tool to eliminate Pf prophages from clinical isolates and advances our understanding of P. aeruginosa and filamentous Pf phage interactions.

Keywords: C. elegans; Filamentous Pf bacteriophage; Pseudomonas aeruginosa; biofilms; prophage; pyocyanin; quorum sensing; virulence.

Fig 1

The Pf4 phage gene PA0718 (pflM) maintains lysogeny. (A) The Pf4 prophage is shown. (B) Multiplex PCR was used to measure Pf4 prophage integration and excision from the PAO1 chromosome in Pf4 single-gene mutants, which were generated through allelic exchange. Deletion of the Pf4 repressor (Pf4r) and PA0718 results in prophage excision. Despite our best efforts, we were unable to generate a single-gene mutant of Pf4 gene pfsE, marked with an asterisk. (C) Quantitative PCR (qPCR) was used to measure episomal Pf4 replicative form in cells after 18 hours of growth in lysogeny broth (LB) broth. Data are the mean ± SEM of three replicate experiments. The lower limit of detection for the assay is 37.85 copy numbers per 10,000 cells. **P < 0.01, Student’s t-test. (D) PA0718 and/or XisF4 were expressed from an inducible plasmid in P. aeruginosa PAO1. After 18 hours, Pf4 integration and excision were measured by excision assay. (E) Pf4 virions in filtered supernatants collected from the indicated strains were titered on lawns of P. aeruginosa ∆Pf4. A representative image is shown.

Fig 2

Targeted deletion of pflM cures diverse P. aeruginosa isolates of their Pf prophage infections. A multiplex PCR assay and long-read whole genome sequencing were used to confirm the loss of (A and B) the Pf4 prophage or (C and D) the Pf6 prophage from the PAO1 chromosome. (E-I) Long-read whole genome sequencing was used to confirm the successful deletion of the indicated Pf prophages. Reads were aligned to 50 kb sequences flanking the Pf prophage insertion sites in the parental chromosome. The genomic coordinates for each Pf prophage are shown above each bracket.

Fig 3

Sequence and structural analysis of PflM. (A) Pf prophage annotation was performed with Rapid Annotation using Subsystem Technology (RAST). Inset: Clinker was used to generate alignments of the pflM loci spanning from xisF to PA0719 in the Pf4 reference sequence. DUF5447 and mnt domains in pflM are indicated by orange or green, respectively, in the inset. (B) PflM structures were predicted using AlphaFold2and aligned using ChimeraX. The highlighted region indicates the DUF5447 domain and the Mnt domain is indicated for the PflM sequence from strain DDRC3. (C) A protein sequence logo for PflM was generated with WebLogo3 (v. 2.8.2); dashes represent variable residues. The DUF5447 domain is highlighted.

Fig 4

Pf phage differentially modulates P. aeruginosa quorum sensing. 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. Reporter fluorescence intensity was normalized to cell density (OD₆₀₀) at each time point. 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 ∆Pf strains with the wild-type parent at each time point.

Fig 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 48 hours of 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.

Fig 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 18 hours of incubation. Pyocyanin concentration was measured (Abs 520 nm). Data are the mean ± SEM of three replicates. **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t-test.

Fig 7

C. elegans actively avoids P. aeruginosa Pf lysogens. (A) Experimental design: P. aeruginosa and isogenic ∆Pf mutants were spotted on normal nematode growth medium (NNGM) 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 ∆Pf mutants (squares) in the indicated strain backgrounds was measured hourly over 8 hours (three experiments with N = 30 per replicate [90 animals total]). P values were calculated by two-way ANOVA (analysis of variance) comparing ∆Pf strains with the parental strains using the Šidák correction (95% confidence interval threshold), **P < 0.01, ***P < 0.001, ****P < 0.0001.