Structure analysis of Phospholipase C - A virulence factor from Pseudomonas aeruginosa

Antibiotic resistance in Gram-negative bacteria, especially Pseudomonas aeruginosa, presents significant challenges in treating infections, particularly in hospitalized and immunocompromised patients. This pathogen secretes various virulence factors, including phospholipases, which play critical roles in its pathogenicity.This study focused on characterizing two phospholipases, PLCH and PLCN. Both were expressed in *E. coli* and purified using advanced chromatographic techniques. Protein integrity was confirmed through Western blotting, mass spectrometry, and related analyses, which also revealed posttranslational phosphorylation of PLCH.Structural studies identified PLCHR2 as a heterodimer of PLCH and its chaperone PLCR2, with a defined stoichiometric ratio. Biochemical assays demonstrated that both phospholipases could hydrolyze phospholipids in vitro and in vivo, with threonine residues in their active sites proving essential for enzymatic activity. Unlike phospholipases from Gram-positive bacteria, these enzymes did not require cations for activity, although certain ions slightly enhanced their function.Hemolytic activity was observed with PLCHR2 but not PLCN, with pore formation in lipid bilayers visualized using atomic force microscopy. Structural stability studies revealed distinct features in secondary and tertiary structures, with PLCHR2 demonstrating enhanced stability, likely due to the presence of the chaperone. Advanced techniques such as cryo-EM and X-ray crystallography provided further insights into their structural and functional mechanisms.This research represents the first detailed structural study of phospholipases from a Gram-negative bacterium, offering critical insights into their enzymatic mechanisms and potential roles in enhancing pathogenicity. These findings suggest phospholipases as promising therapeutic targets. Future investigations should focus on co-crystallization with inhibitors, cellular assays to determine localization, and exploring their effects on host cells to validate their role in infection.