Investigation of the role of recombinant phage tail proteins in adhesions to bacteria

Aim: Proteus mirabilis and Proteus vulgaris are significant opportunistic pathogens implicated in serious clinical conditions such as catheter-associated urinary-tract infections (CAUTI), pyelonephritis, sepsis and stone formation. Their surface structures, biofilm-forming ability and rising antibiotic resistance complicate both diagnosis and treatment. The principal aim of this dissertation is the recombinant production and comprehensive evaluation of the diagnostic potential of the receptor-binding proteins (RBPs) Gp35 and Gp106, derived from the P. mirabilis phage RP6 and the P. vulgaris phage RP7, respectively. The study characterised these proteins in terms of specificity, sensitivity, binding spectrum, performance in biological matrices, phage-adsorption blocking capacity and absence of antibacterial or cytotoxic effects. Materials and Methods: Gene sequences encoding Gp35 and Gp106 proteins were amplified by PCR with specific primers and cloned into the pET-30a(+) expression vector. After verification of cloning, the recombinant proteins were expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Proteus-specific binding of the purified RBPs was analysed by crystal-violet staining and ELISA. Competitive ELISA (C-ELISA) with varying bacterial loads determined detection limits, while binding spectra were assessed using 49 clinical isolates. Binding performance in urine and other biological matrices was examined, and competitive adsorption assays evaluated whether the recombinant proteins bind the same receptors as their cognate phages. Antibacterial activities of recombinant proteins were investigated with spot-test analysis, and cytotoxicity was assessed on human fibroblast (BJ) cells using the WST-1 assay. Results: Gp35 and Gp106 bound exclusively to Proteus isolates and produced no signal with heterologous bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus). In C-ELISA sensitivity tests, both proteins achieved target-specific binding at ≈10³ CFU mL⁻¹. Binding-spectrum analysis showed attachment of Gp35 to 82.5 % and Gp106 to 90 % of Proteus isolates, indicating a broader host range for Gp106. In several isolates, the RBPs bound despite the inability of their corresponding phages to infect, suggesting that RBP-based diagnostics may offer wider coverage than phage assays. In biological samples, the RBPs retained specificity and bound successfully to target bacteria in urine. Competitive adsorption assays revealed that Gp35 and Gp106 inhibited RP6 and RP7 phage attachment to host cells by 65–80 %, demonstrating that both proteins occupy the same specific receptors and can block phage infection. Neither RBP exhibited antibacterial activity, confirming their sole function in target recognition and binding, and neither displayed cytotoxicity toward human fibroblast (BJ) cells. Conclusion: This thesis presents the first systematic characterisation of Proteus-specific recombinant RBPs. Gp35 and Gp106 combine high specificity, broad spectrum, compatibility with clinical samples and confirmed target-receptor recognition, making them strong candidates for diagnostic applications. Their lack of antibacterial or cytotoxic effects permits safe use in assays that require viable cells. Compared with other RBP-based diagnostic systems reported in the literature, this study lays an important foundation for rapid, sensitive and specific detection of Proteus infections. Beyond diagnostics, these RBPs also hold promise as biological tools in targeted-therapy strategies—such as conjugation to nanoparticles for directed delivery of antimicrobial agents.