Elucidating the molecular targets of bacterial nanosyringes

Abstract

Contractile injection systems (CIS) are bacterial nanomachines that deliver toxic effectors into target cells. The Photorhabdus virulence cassette (PVC) is one such system, and while its structural organisation has been studied, the mechanisms of host recognition remain poorly understood. This thesis focuses on the tail fibre protein, Pvc13, with the aim of elucidating its structural and functional role in PVC–host interactions.

Biochemical characterisation demonstrated that Pvc13 can be expressed and purified in sufficient yield, assembling into fibres of approximately 55 nm in length. However, the protein was unstable in isolation, displaying a tendency towards aggregation and proteolysis. Attempts to resolve its structure by cryo-electron microscopy were hindered by sample heterogeneity and filament breakage, preventing high-resolution reconstruction.

Bioinformatic analyses provided deeper insight into Pvc13 organisation and evolutionary context. Sequence comparisons revealed strong conservation with Afp13 but no detectable sequence homology to bacteriophage tail fibres or R-type pyocins, suggesting a distinct evolutionary lineage. AlphaFold modelling predicted a trimeric architecture with a distal tip cavity featuring a negatively charged funnel and hydrophobic core, consistent with a charge-guided capture mechanism. Additional predictions from MODA and OPM PPM3.0 indicated that the C-terminal knob may mediate lateral interactions with membranes.

Experimental assays with purified Pvc13 showed tentative but inconsistent evidence of membrane association. In contrast, assays with intact PVCs demonstrated clearer lipid interactions, though without specificity for a single lipid species, consistent with a weak, charge-based tethering mechanism. These findings align with recent functional studies of engineered PVCs and suggest a cooperative two-step binding model: initial transient tethering via the knob, followed by stronger distal tip engagement to trigger contraction.

This thesis therefore provides a framework for understanding Pvc13 function, integrating biochemical, computational, and functional data into a mechanistic hypothesis that informs both fundamental CIS biology and future translational applications.

Date of Award	Sept 2025
Original language	English
Awarding Institution	Aston University
Supervisor	Alice Rothnie (Supervisor) & Alan Goddard (Supervisor)