Characterization of Pseudomonas aeruginosa Bacteriophage L5 Which Requires Type IV Pili for Infection

Abstract

Pseudomonas aeruginosa is a common opportunistic human pathogen. With the emergence of multidrug-resistant (MDR) clinical infection of P. aeruginosa, phage therapy has received renewed attention in treating P. aeruginosa infections. Moreover, a detailed understanding of the host receptor of lytic phage is crucial for selecting proper phages for therapy. Here, we describe the characterization of the P. aeruginosa bacteriophage L5 with a double-stranded DNA genome of 42,925 bp. The genomic characteristics indicate that L5 is a lytic bacteriophage belonging to the subfamily Autographivirinae. In addition, the phage receptors for L5 were also identified as type IV pili, because the mutation of pilZ, which is involved in pili synthesis, resists phage infection, while the complementation of pilZ restored its phage sensitivity. This research reveals that L5 is a potential phage therapy candidate for the treatment of P. aeruginosa infection.

Keywords: Autographivirinae; Pseudomonas aeruginosa; phage (bacteriophage); phage receptors; phage-host interaction.

Figure 1

The morphological characteristics of Pseudomonas aeruginosa phage L5. (A) Plaque morphology of L5 on P. aeruginosa strain PAO1r; (B) Transmission electron micrograph image of L5. The scale bar represents 20 nm; (C) The multiplicity of infection (MOI) of L5; (D) The one-step growth curve of L5 on P. aeruginosa strain PAO1r. Error bars indicate standard deviation.

Figure 2

Genome organization of P. aeruginosa phage L5. Arrows represent predicted CDSs, with the direction of the arrow indicating the transcription direction. The different colors indicate different functional modules of gene products. The L5 genome can be divided into six functional modules.

Figure 3

(A) Phylogenetic analysis of P. aeruginosa phage L5 and related phages. The terminase large subunits of selected phages were compared using the ClustalW program, and the phylogenetic tree was generated using the neighbor-joining method with 1,000 bootstrap replicates. (B) Comparison of the genome sequence of phage PAXYB1(top) with L5 (bottom). Predicted ORFs and the direction of transcription are indicated by block arrows. Conserved regions are shaded in gray. The color intensity corresponds to the sequence identity level (83 to 100%). Genomic comparisons were performed using BLASTn. Similarities with E values lower than 0.00001 are plotted. The figure was produced using Easyfig 2.2.5 (Sullivan et al., 2011).

Figure 4

(A) Twitching motility zones by the wild-type sensitive strain PAO1r, the L5-resistant mutant strain PAO1rRL5, the knockout strain PAO1rΔpilZ, and the complementation strain PAO1rΔpilZ::pilZ. (B) Phage Adsorption Assay of P. aeruginosa phage L5 for the strain PAO1r and the strain PAO1rRL5. The phage adsorption percent was calculated as described in the MATERIALS and METHODS.

Figure 5

Phage Plaquing Assay of P. aeruginosa phage L5. The strain PAO1r and the strain PAO1rΔpilZ::pilZ are susceptible to L5, while the strain PAO1rRL5 and strain PAO1rΔpilZ are resistant to infection.