LAUSR

The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen Pseudomonas aeruginosa. The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen’s virulence. Using cryo–electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics. Editor’s summary Viruses that infect bacteria are ubiquitous even among bacterial pathogens. Pseudomonas aeruginosa is a widespread opportunistic pathogenic bacterium that has multiple potential virulence factors, including a retractable type 4 pilus that propels the cell. Thongchol et al. have found that one Pseudomonas phage called PP7 infects its host cell by means of a virus protein called Mat that attached to the bacterium’s pilus. The pilus retracts and drags the phage to the bacterial cell surface. At the point of virus entry into the cell, the pilus is bent and snaps off, thus disabling and reducing the bacterium’s ability to infect its own host. —Caroline Ash INTRODUCTION Retractile pili in bacteria play an important role in numerous biological processes, such as DNA and protein transfer, motility, adhesion, surface sensing, biofilm formation, and pathogenesis. Single-stranded RNA (ssRNA) bacteriophages (phages) are small viruses that specifically target these retractile pili. With a small positive-strand RNA genome of approximately 4000 nucleotides, ssRNA phages typically encode four proteins: maturation protein (Mat), coat protein (Coat), RNA-dependent RNA replicase (Rep), and single-gene lysis protein (Sgl). The Mat is crucial for phage maturity and pilus recognition. However, how ssRNA phages use the Mat-pilus interaction to enter the host cell has remained mysterious for the more than six decades since the discovery of the first ssRNA phage. RATIONALE The ssRNA phage PP7 infects Pseudomonas aeruginosa O1 (PAO1) through the type IV pilus (T4P), a prominent virulence factor associated with motility. Unveiling the mechanisms under which PP7 exploits T4P for cellular entry will shed light on fundamental aspects of phage-bacterium interactions and phage biology and may open an avenue for antimicrobial strategies. RESULTS Using fluorescence microscopy, we observed the detachment of PAO1 T4P during PP7 infection. Intriguingly, T4P detachment induced by ultraviolet (UV)–inactivated PP7 (UV-PP7), whose viral RNA cannot enter the cell, mirrors that of the wild-type (WT) PP7. This indicates that T4P detachment occurs at the cell envelope during PP7 entry, independently of PP7 replication inside the cell. Alongside T4P detachment, both WT PP7 and UV-PP7 treatments impede T4P dynamics. This combined effect drastically reduces cell twitching motility. PP7 mature virions, resolved by means of single-particle cryo–electron microscopy, feature two Mat proteins forming a heterotypic dimer. One Mat is exposed for T4P interaction, whereas the other is entirely internalized within the capsid. This mature PP7 virion structure diverges substantially from the single-Mat structure found in canonical Escherichia coli phages, such as MS2 and Qβ, challenging our understanding of the structure of mature ssRNA phages. To delve into the phage entry mechanism, we determined the structures of T4P and the PP7/T4P complex, which revealed that PP7 binds to T4P by interacting with a single pilin subunit through the Pilus-Interacting Region (PIR) of the Mat. Through examination of various mutants of the host retraction ATPases and the pilus, we discovered that pilus detachment is influenced by the pilus retraction force and speed, as well as the affinity between phage Mat and its bound pilin. Further mechanical considerations led to the pilus bending hypothesis, which was substantiated by our Langevin dynamics simulation of the Mat bound to T4P during the phage entry process. CONCLUSION We observed that T4P can be detached or removed by an ssRNA phage and revealed the molecular mechanism for T4P detachment. We propose that similar mechanisms are widespread among ssRNA phages and their respective retractile pilus systems. Recent bioinformatic studies of environmental samples have identified thousands of ssRNA phage genomes, which await mechanistic analysis. This work could serve as a benchmark for investigating other phage and virus systems of different organisms. PP7 infection impairs P. aeruginosa motility. (A) Fluorescence images showing that T4P are detached upon PP7 infection. (B) Structures of PP7, T4P, and the PP7/T4P complex are resolved with single-particle cryo–electron microscopy. (C) PP7 hijacks T4P retraction for infection and detaches T4P. (D) Cell twitching motility is impaired owing to pilus detachment and nonfunctional T4P (the schematic diagram is not drawn to scale).