A Filamentous Bacteriophage Protein Inhibits Type IV Pili To Prevent Superinfection of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in a variety of settings. Many P. aeruginosa isolates are infected by filamentous Pf bacteriophage integrated into the bacterial chromosome as a prophage. Pf virions can be produced without lysing P. aeruginosa. However, cell lysis can occur during superinfection, which occurs when Pf virions successfully infect a host lysogenized by a Pf prophage. Temperate phages typically encode superinfection exclusion mechanisms to prevent host lysis by virions of the same or similar species. In this study, we sought to elucidate the superinfection exclusion mechanism of Pf phage. Initially, we observed that P. aeruginosa that survive Pf superinfection are transiently resistant to Pf-induced plaquing and are deficient in twitching motility, which is mediated by type IV pili (T4P). Pf utilize T4P as a cell surface receptor, suggesting that T4P are suppressed in bacteria that survive superinfection. We tested the hypothesis that a Pf-encoded protein suppresses T4P to mediate superinfection exclusion by expressing Pf proteins in P. aeruginosa and measuring plaquing and twitching motility. We found that the Pf protein PA0721, which we termed Pf superinfection exclusion (PfsE), promoted resistance to Pf infection and suppressed twitching motility by binding the T4P protein PilC. Because T4P play key roles in biofilm formation and virulence, the ability of Pf phage to modulate T4P via PfsE has implications in the ability of P. aeruginosa to persist at sites of infection. IMPORTANCE Pf bacteriophage (phage) are filamentous viruses that infect Pseudomonas aeruginosa and enhance its virulence potential. Pf virions can lyse and kill P. aeruginosa through superinfection, which occurs when an already infected cell is infected by the same or similar phage. Here, we show that a small, highly conserved Pf phage protein (PA0721, PfsE) provides resistance to superinfection by phages that use the type IV pilus as a cell surface receptor. PfsE does this by inhibiting assembly of the type IV pilus via an interaction with PilC. As the type IV pilus plays important roles in virulence, the ability of Pf phage to modulate its assembly has implications for P. aeruginosa pathogenesis.

Keywords: Pf4; PilC; Pseudomonas aeruginosa; filamentous bacteriophage; superinfection exclusion; twitching motility; type IV pili.

FIG 1

Pf4 superinfection transiently suppresses twitching motility and promotes resistance to Pf4-induced plaquing. Twitch assays were performed by stabbing the indicated strain through the agar on a petri dish to the plastic surface below. After 24 h, the agar was removed and bacteria on the plastic dish were stained with Coomassie (upper panels A, C, E, and G). To measure sensitivity of P. aeruginosa to Pf4 superinfection, lawns of the indicated strains were spotted with 10⁶ PFU of Pf4 in 3 μL (lower panels B, D, F, and H). Strains tested include (A and B) PAO1 superinfected with Pf4, (C and D) uninfected PAO1, (E and F) the twitch-deficient type IV pili mutant ΔpilA, and (G and H) Pf4 superinfected PAO1 that was subcultured in phage-free broth and replated. Scale bar 5 mm.

FIG 2

PfsE suppresses twitching motility and protects P. aeruginosa from Pf4 superinfection. (A) The Pf4 prophage is composed of core genes that are essential for the phage to complete its life cycle (Pf4r to PA0728) and flanking moron regions (gray) that add “more on” to the core genome (54). (B) Genes PA0717–PA0728 in the core Pf4 genome were placed under the control of an arabinose-inducible promoter and expressed individually in P. aeruginosa PAO1^ΔPf4. Twitching was measured in the indicated strains after 24 h; arrows indicate strains with reduced twitching motility. Bar 5 mm. (C) 10⁶ PFU of Pf4 were spotted onto lawns of PAO1^ΔPf4 expressing the indicated phage protein. (D) Growth curves in liquid culture for PAO1^ΔPf4 bacteria carrying the indicated expression vector. Cultures were grown in LB supplemented with 0.1% arabinose. Results are the mean ± SEM of three experiments.

FIG 3

Type IV pili are not apparent on cells expressing PfsE. Transmission electron microscopy of the indicated strains of P. aeruginosa was performed. Midlogarithmic cells were washed, fixed, placed on a grid, and negatively stained with uranyl acetate. Arrows indicate potential pili on PAO1 cells. White triangles indicate flagella.

FIG 4

Pf4 superinfection and expression of PfsE promotes resistance to type IV pili (T4P)-dependent bacteriophages. Representative images of P. aeruginosa PAO1 lawns spotted with 10⁶ PFU of Pf4, JBD26 (both T4P-dependent phages), or CMS1 (a T4P-independent phage). See also Fig. S1.

FIG 5

Pf4 virions not encoding pfsE are more virulent against P. aeruginosa. Wild-type and mutant Pf4 virions were induced by expressing both the Pf4 excisionase (xisF4) and integrase (intF4) in trans in wild-type, ΔintF4, or ΔintF4/pfsE backgrounds. Note that deletion of pfsE alone from the Pf4 prophage was not possible. Wild-type, ΔintF4, and ΔintF4/pfsE Pf4 virion titers were measured by qPCR and normalized to 6.95 × 10⁷ virions per mL. Virions were then spotted as a 10× dilution series on lawns of ΔPf4 to enumerate PFU. Results are mean ± SEM of three experiments, unpaired Student's t test, ****, P < 0.0001.

FIG 6

Conserved aromatic residues in PfsE are essential for the inhibition of type IV pili and resistance against Pf4 superinfection. (A) WebLogo (55) was used to construct a protein sequence logo for 312 PfsE sequences from Pf prophages infecting P. aeruginosa strains in the Pseudomonas genome database (24). Note that aromatic residues in the YXWGW motif (residues 16–20) are conserved (arrows). (B) There is 66% sequence identity (20/30) between two of the most highly diverged PfsE homologues found in Pf prophages residing in the P. aeruginosa PAO1 or E80 chromosomes. (C) Twitch motility was measured in PAO1^ΔPf4 expressing wild-type or modified PfsE. Results are mean ± SEM of 16 experiments, unpaired Student's t test, **, P < 0.01, ****, P < 0.0001. (D) Lawns of PAO1^ΔPf4 carrying the indicated expression vectors were induced with 0.01% arabinose and spotted with 3 μL of 10⁹ PFU/mL stocks of Pf4, JBD26 (pili-dependent phages), or CMS1 (a pili-independent phage). (E) AlphaFold (52) was used to predict the structure of PfsE. (F) A bacterial adenylate cyclase two-hybrid (BACTH) assay (25) was used to detect interactions between PfsE and the indicated T4P proteins. Interactions between bait and prey proteins are detected by β-galactosidase activity, as indicated by the production of blue pigment. Representative colonies are shown. (G) A 6×-His tag was added to the N- or C-terminus of PfsE, expressed in PAO1^ΔPf4, and twitching was measured. Scale bar 5 mm.

FIG 7

Divergent PfsE sequences inhibit twitching motility in various P. aeruginosa strains. (A) PfsE was expressed in trans in the indicated strains of P. aeruginosa and twitching motility measured after 24 h. Representative images are shown. (B) Twitching motility was assessed in PAO1^ΔPf4 carrying either the empty vector pHERD20T or p_BAD-pfsE-E80 by standard twitch assay. Representative images are shown.