PIT3: A phage-derived protein with anti-virulence activity against the Type III Secretion System in Pseudomonas aeruginosa

Samiya Siddiqui

Samiya Siddiqui

KU Leuven

Laboratory of Gene Technology

I am a second-year PhD student at the Laboratory of Gene Technology (LoGT), KU Leuven, Belgium, supported by the HEC Overseas Scholarship for PhD, Pakistan. My research focuses on bacteriophage-encoded proteins and their impact on bacterial virulence and the phenotypic characteristics of Pseudomonas aeruginosa. This includes the use of DNA and membrane dyes visualized by fluorescence microscopy, alongside recombinant protein expression, purification, and analyses of protein-protein and protein-DNA interactions. Currently, I am working on Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) to determine the DNA-binding specificity of YuAgp37, a phage-encoded protein we have designated PIT3 (Phage-encoded Inhibitor of the Type III Secretion System). This work aims to lay the groundwork for exploring phage proteins as anti-virulence agents in the fight against MDR Pseudomonas aeruginosa.

Authors: Siddiqui S1, Hendrix H1, Lavigne R1

Affiliations: (1). Laboratory of Gene Technology, KU Leuven Kasteelpark Arenberg (21), (3001) Heverlee (Belgium)

Pseudomonas aeruginosa is a Gram-negative, nosocomial, opportunistic pathogen capable of causing a variety of infections, particularly in immunocompromised individuals. Over the past decades, P. aeruginosa has rapidly developed multidrug resistance (MDR) against a wide range of antibiotics, posing a significant challenge to modern medicine. As a result, alternative therapeutic strategies targeting bacterial virulence rather than viability can be a promising approach to combat infections. One such strategy is the use of anti-virulence agents, including phage-derived proteins, to attenuate bacterial pathogenicity by disarming the pathogen without exerting selective pressure for resistance. This project investigates the anti-virulence potential of phage-derived proteins, focusing on YuAgp37, designated as PIT3 (Phage-encoded Inhibitor of Type III Secretion System)—identified in a screen for inhibitors of the Type III Secretion System (T3SS). The study was conducted in two phases. First, candidate phage proteins were screened for their ability to reduce T3SS activity, with PIT3 demonstrating a consistent reduction in ExoS activity. Second, PIT3 was heterologously expressed in E. coli and purified for downstream analyses. The purified protein was then used to assess molecular interactions at both the DNA and protein levels. Electrophoretic mobility shift assays indicated concentration-dependent DNA binding, while pull-down assays were conducted to identify potential protein-protein interactions. These findings support a potential regulatory role for PIT3 and lay the groundwork for further exploration of phage proteins as anti-virulence agents in the fight against multidrug-resistant P. aeruginosa.