Mechanistic Insights into the uptake and transport of pyocin S3 via the Ferripyoverdine system

Abstract

Multidrug-resistant Pseudomonas aeruginosa presents a pressing challenge for clinical treatment, driving the search for novel therapeutic strategies. Pyocins, narrow-spectrum proteins produced by P. aeruginosa, offer a promising approach, yet the molecular basis of their receptor dependence remains incompletely understood. The work presented in this thesis advances mechanistic understanding of pyocin S3 (PyoS3), a relatively understudied member of the S-type pyocin family. Using a combination of biochemical assays and genetic analysis, this study establishes that PyoS3 exploits the pyoverdine uptake pathway for cell entry. The type II ferripyoverdine receptor, FpvAII, was identified as the principal uptake route for PyoS3, with knockout and complementation experiments confirming its essential role. Disruption of the pvdE transporter disconnected siderophore production from pyocin susceptibility, abolishing both pyoverdine synthesis and PyoS3 activity. Genetic complementation re-established these functions, thus identifying pvdE as a critical gatekeeper linking pyoverdine biosynthesis to PyoS3 susceptibility. In contrast, deletion of fpvB increased susceptibility and elevated pyoverdine production, suggesting that this secondary receptor modulates siderophore flux rather than serving as a primary entry point. Analytical size-exclusion chromatography provided supporting biochemical evidence of a PyoS3-FpvAII association, although the interaction was weaker and dynamic than the high-affinity mimicry previously described for PyoS2-FpvAI. Additional results suggest that TonB1 may contribute as an energy-transducing partner during the import of pyocin S3, although its binding appears to be conditional and difficult to capture outside a native membrane context. Physiological experiments revealed that iron availability strongly conditions PyoS3 activity, with effective killing requiring extracellular iron concentrations ≤ 1 µM, consistent with upregulation of siderophore receptors under iron limitation. Furthermore, genetic modifiers, including the antibiotic resistance determinant aph(3ˋ)-II, highlight the broader interplay between siderophore regulation, membrane physiology, and bacteriocin susceptibility. Together, these findings establish a mechanistic framework for PyoS3 uptake, emphasize the centrality of siderophore pathways and receptor ecology in shaping susceptibility, and highlight evolutionary trade-offs between iron acquisition and bacteriocin resistance. The results provide a rationale for advancing pyocins as precision antimicrobials, guiding future structural studies and supporting the development of therapeutic strategies based on receptor profiling or rationally designed pyocin cocktails for combating multidrug-resistant P. aeruginosa.