c-di-GMP levels control infection of P. aeruginosa by bacteriophage Clew-1.

A) Efficiency of plating experiment in which 3µL of a 10x dilution series of bacteriophage Ocp-2 or Clew-1 were spotted on wild-type PAO1F, or PAO1F ΔfliF2. The adjacent graph shows the compiled results from 11 experiments. B) Maximum likelihood phylogenetic tree of Clew-1 relative to other Bruynogheviruses (including the type phage, LUZ24) and phage Bjorn as an outgroup. Branch lengths are measured in number of substitutions per site in the terminase large subunit. C) transmission electron micrograph of the Clew-1 phage. D) Efficiency of plating experiment as in (A) assaying the effect of expressing the phosphodiesterase PA2133 from a plasmid. E) Efficiency of plating experiment assaying the effect of deleting wspF on Clew-1 resistance. (* p<0.05, **** p<0.0001 by Student’s T-test (A, E) or 1-way ANOVA with Šídák’s multiple comparisons test (D))

Bacteriophage Clew-1 uses Psl as a receptor to infect P. aeruginosa.

A) TnSeq experiment in which a pool of mariner transposon mutants of strain PAO1F were infected with phage Clew-1 for 2h. The number of insertions in the output pool were plotted against the ratio of the output and input pool. B) Similar TnSeq analysis as in A) but using PAO1F ΔfliF2. C) Efficiency of plating analysis on ΔfliF2 ΔpslC and ΔfliF2 ΔpslD, Psl biosynthesis mutants, either harboring an empty vector or a complementing plasmid (n=6). Clew-1 values were compared by 1-way ANOVA with Šídák’s multiple comparisons test (** p<0.01, n.s. .. not significant).

The ΔfliF2 mutation changes the fraction of cells that phage Clew-1 binds to.

A) Efficiency of center of infection analysis. The indicated strain was infected for 5 minutes at an MOI of 0.01, the bacteria were pelleted, washed 3x with PBS, then diluted and mixed with an excess of the ΔfliF2 mutant strain, top agar and plated on an LB agar plate. The number of plaques was used to calculate the number of phage that attached and productively infected the initial strain. B) Phage Clew-1 was labeled with DyLight594 fluorophores, bound to the indicated wild-type or mutant bacteria (15 minutes in LB), washed and fixed with paraformaldehyde. Phage attached to bacteria were imaged by fluorescence microscopy and attachment was quantified over 5 biological replicates, shown in C). Attachment was compared by 1-way ANOVA with Šídák’s multiple comparisons test. * p< 0.05, *** p<0.001, **** p<0.0001.

Phage Clew-1 binds to Psl.

A) Sterile filtered mid-log culture supernatants of PAO1F ΔfliF2 or PAO1F ΔfliF2 ΔpslC were incubated with phage Clew-1, as well as magnetic protein-A beads and, where indicated, a rabbit, anti-Psl antiserum. Beads were collected, washed 3x, and phage in the input and output samples were quantified by qPCR (7 independent replicates.) B) Phage Clew-1 was incubated for 1h in SM buffer with affinity purified, biotinylated Psl (biotin-Psl) and magnetic protein A beads, or magnetic streptavidin beads (SA), where indicated. Beads were collected and washed 3x, and phage in the input and output samples were quantified by qPCR (3 independent replicates). Statistical significance was determined by ANOVA with Sidák post-hoc test (**** p<0.0001).

Phage Clew-1 can infect P. aeruginosa in biofilms.

A) Biofilms of wild type P. aeruginosa PAO1F were established overnight in 5mL culture tubes (1mL culture), the tubes were washed with PBS and 1.2mL LB, LB with 10^10 pfu phage Clew-1, or LB-with 10^10 pfu phage Ocp-2 were added back to each tube (1 was fixed with EtOH to represent the 1-day old biofilm). The following day all biofilms were washed with PBS and stained with crystal violet. B) PAO1F biofilms were established overnight in a 96-well plate (150µL of culture, 6 technical replicates/condition), washed and incubated overnight with 200µL of LB or LB with 10^9 pfu bacteriophage Clew-1 or Ocp-2. The biofilms were then washed, fixed, and stained with crystal violet, which was then solubilized with 30% acetic acid and quantified spectrophotometrically at 590 nm. The day 1 controls were set to 100% (5 biological replicates). C) Growth of phage Clew-1 or Ocp-2 was assayed by establishing a static biofilm in 5 mL culture tubes overnight (1mL culture volume). The biofilms were washed with PBS, then 1mL of LB with 10^5 pfu/mL of phage Clew-1 or Ocp-2 were added back. Biofilms were incubated at 37°C for 2h 15 minutes, the culture supernatants were filter sterilized and input and output phage concentrations were tittered (6 biological replicates). Statistical significance was determined by ANOVA with Šídák’s multiple comparisons test test (** p<0.01, *** p<0.001).

Phage Clew-1 reduces the bacterial burden in a mouse cornea model of infection.

A) Mice corneas were scratched and infected with 5*10^4 cfu strain PAO1F/pP25-GFPo, which produces GFP constitutively. Infected corneas were treated with 2*10^9 pfu phage Clew-1 or a PBS control at 3h and 24h post infection. B) At 48h post infection, the corneas were imaged by confocal microscopy to estimate the opacity (driven largely by the infiltration of neutrophils) and C) GFP fluorescence (produced by infecting P. aeruginosa). D) Eyes were also homogenized and plated for CFU to determine the total bacterial burden at the end of the experiment. Significance was determined by Mann-Whitney test (** p<0.01, n.s. not significant).