Pf Bacteriophage and Their Impact on Pseudomonas Virulence, Mammalian Immunity, and Chronic Infections

Abstract

Pf bacteriophage are temperate phages that infect the bacterium Pseudomonas aeruginosa, a major cause of chronic lung infections in cystic fibrosis (CF) and other settings. Pf and other temperate phages have evolved complex, mutualistic relationships with their bacterial hosts that impact both bacterial phenotypes and chronic infection. We and others have reported that Pf phages are a virulence factor that promote the pathogenesis of P. aeruginosa infections in animal models and are associated with worse skin and lung infections in humans. Here we review the biology of Pf phage and what is known about its contributions to pathogenesis and clinical disease. First, we review the structure, genetics, and epidemiology of Pf phage. Next, we address the diverse and surprising ways that Pf phages contribute to P. aeruginosa phenotypes including effects on biofilm formation, antibiotic resistance, and motility. Then, we cover data indicating that Pf phages suppress mammalian immunity at sites of bacterial infection. Finally, we discuss recent literature implicating Pf in chronic P. aeruginosa infections in CF and other settings. Together, these reports suggest that Pf bacteriophage have direct effects on P. aeruginosa infections and that temperate phages are an exciting frontier in microbiology, immunology, and human health.

Keywords: Inovirus; Pf phage; Pseudomonas aeruginosa; bacteriophage; cystic fibrosis; immunology; infection; wound.

Figure 1

Structure of the Pf1 virion. (A) Ribbon diagram with Van der Waals surface representation of a portion of the assembled Pf1 virion (PDBID: 1PFI) showing the helical arrangement of CoaB subunits around the P-form ssDNA viral genome. Subunit surface opacity is decreased along the length of the capsid to highlight the alpha-helical secondary structure of CoaB and the unique conformation of the DNA. Amino acids are colored according to charge, with positively-charged residues in blue, negatively-charged residues in red, and neutral residues in gray. (B) Space-filling model of a single CoaB subunit bound to a stretch of cytosines. Arg44 and Lys45 are situated on either side of the DNA backbone and act to stabilize it through electrostatic interactions. (C) Cross-sectional view of five CoaB subunits situated around the packaged viral genome. Amino acids are colored by charge as in (B). Image adapted from reference (69).

Figure 2

Regulation of Pf replication decisions. The Pf4 prophage from P. aeruginosa strain PAO1 is shown. Colored genes correspond to the core Pf genome while gray genes are accessory genes that are variable from strain to strain (see Figure 4 for more details). Pf4r (i.e., the c repressor) maintains lysogeny by repressing the excisionase xisF4. XisF4 promotes transcription of an operon encoding both the replication initiation protein PA0727 and integrase IntF. The transcriptional regulator OxyR suppresses pf4r while the global histone-nucleosome-like transcriptional regulators MvaT and MvaU suppress xisF4. Figure 3 details events in the Pf lifecycle, such as genome replication and assembly of new virions.

Figure 3

The Pf life cycle. Pf infection is initiated when the minor coat protein p3 (PA0724) binds to type IV pili. Pilus retraction draws the phage into the periplasm where p3 binds the secondary receptor TolA. As the phage moves through the inner membrane, the major coat protein CoaB is removed and deposited in the inner membrane. The ssDNA genome is converted into a dsDNA replicative form (RF) and is either integrated into the host chromosome (lysogeny) or used to initiate a chronic infection. The phage initiator protein (IP, PA0727) and host enzymes (DNA polymerase III and UvrD) create additional copies of RF and the ssDNA infective form (IF) via rolling-circle replication. Newly-produced ssDNA molecules are then coated with protein p5 (PA0720) and targeted to the inner membrane where CoaB, which has been inserted into the inner membrane by the hist Sec/YidC machinery, replaces p5 as the virus is extruded through the cell envelope.

Figure 4

Phylogenic analysis of Pf prophages. (A) Forty representative full-length Pf prophage instances were manually collected from the Pseudomonas Genome Database (91). Two DNA sequences encoding Protein III (PA0724) from Inovirus strains M13 and CUS-1 (E. coli) were added to serve as an outgroup for phylogenetic rooting. A multiple sequence alignment of these viral sequences was produced using MAFFT (93) (v7.215, default parameters), then manually trimmed to include only the well-aligned region containing Pf phage core proteins. A maximum-likelihood estimate of the strain phylogeny was produced with FastTree (94) (v2.1.3, default parameters), and visualized with FigTree (v1.4.4). (B) Representative full-length Pf prophage sequences were manually collected from the Pseudomonas Genome Database. Strain Pf4 was used as a reference; open reading frames identified in other Pf strains were color-coded based on genome location relative to Pf4. Gray genes indicate variable moron sequences (see text) outside of the core Pf genome.

Figure 5

Pf phages contribute to biofilm formation. During the early stages of biofilm formation, Pf phages promote bacterial adhesion to surfaces. During later stages, Pf phages serve as structural components in the biofilm matrix where they promote tolerance to desiccation and cationic antimicrobials. Superinfection by Pf phages (see text) promotes differentiation of the biofilm by inducing bacterial lysis in a subpopulation of cells deep within the biofilm.

Figure 6

Pf phages trigger viral pathogen recognition pathways leading to reduced phagocytosis. Phagocytosis is the major way that Pa infections are cleared by the immune system. In the absence of Pf phages, Pa-derived LPS is recognized by TLR4 on the surface of alveolar macrophages (1A), triggering NFkB activation and pro-inflammatory cytokine production (1B). TNF, a major pro-inflammatory effector, is secreted as a result (1C). This drives Pa phagocytosis by alveolar macrophages, leading to bacterial clearance (1D). In the presence of a Pa strain that produces Pf phages, phage particles are internalized in endosomes (2A), triggering TLR3 activation and production of interferons (2B). IFNα/β binds IFNAR on the cell surface (2C) and negatively regulates pro-inflammatory cytokine production, decreasing TNF secretion (2D). This results in decreased phagocytic engulfment of Pa (2E). Together, this supports a model where intracellular Pf phage triggers viral pattern recognition pathways that antagonize bacterial clearance.

Figure 7

Pf phages contribute to the pathogenesis of Pa airway infections in CF. The thick mucus coating the airways in a CF lung facilitates colonization with Pa and other bacterial pathogens. Even in the absence of Pf phages, Pa forms tenacious biofilms which protect bacterial cells from phagosomal clearance and resist penetration by antibiotics (A). However, Pf phages when present organize the polymer-rich biofilm into crystalline, higher order structures (B), This drives sputum viscosity, adhesiveness, and resistance to desiccation. It also enhances the antibiotic tolerance and immune evasion of bacterial colonies within biofilms, thereby contributing to increased bacterial burden. In this way Pf phages enhance the pathogenesis of Pa airway infections in CF.