Screening the receptors for Mycoplasma penetrans P35 lipoprotein and characterization of its functional binding domains

<i>Mycoplasma penetrans</i>, a prokaryotic microorganism initially isolated from the urine of a patient infected with <i>human immunodeficiency virus</i> (HIV), possesses a distinctive elongated flask-like shape and a tip-like structure. This unique morphology has been shown to facilitate its ability to invade cells both <i>in vitro</i> and <i>in vivo</i>. The adhesion of <i>M. penetrans</i> to host cells relies on lipid-associated membrane proteins (LAMPs), especially P35 lipoprotein, which is exposed on the mycoplasmal surface. In this study, modified Virus Overlay Protein Binding Assay (VOPBA) was employed to identify P35-interacting proteins from membrane protein extracts of SV40-immortalized human uroepithelial (SV-HUC-1) cells. Through recombinant protein binding assays, siRNA-mediated knockdown, ELISA, Far-Western blot, and inhibition experiments, the binding mechanisms and functional domains were further elucidated. Results demonstrated that the P35 lipoprotein interacts with γ-actin (ACTG1). Recombinant P35 specifically bound to both recombinant and endogenous ACTG1 on the host cell membrane. ACTG1 partially inhibited the adhesion of P35 and <i>M. penetrans</i> to host cells. In SV-HUC-1 cells transfected with ACTG1-siRNA, adhesion of P35 and <i>M. penetrans</i> was significantly reduced. Further studies identified the functional domains responsible for binding between P35 and ACTG1 at amino acid residues 35-42 and 179-186. These findings suggest that ACTG1 on the host cell membrane may act as a receptor for the P35 lipoprotein, facilitating the adhesion of <i>M. penetrans</i> to host cells. The identified critical binding regions of P35 represent potential targets for therapeutic interventions against <i>M. penetrans</i> infections.