The <i>Chlamydia pneumoniae</i> inclusion membrane protein Cpn0308 interacts with host protein ACBD3

<i>Chlamydia pneumoniae</i> is an obligate intracellular bacterium of eukaryotic cells characterized by a unique biphasic life cycle; its biosynthesis and replication must occur within a cytoplasmic vacuole or inclusion. Certain inclusion membrane proteins have been demonstrated to mediate the interactions between intra-inclusion chlamydial organisms and the host cell. It has been demonstrated previously that the <i>C. pneumoniae</i>-encoded Cpn0308 localizes to the inclusion membrane; however, its function remains unknown. In the current study, a yeast two-hybrid assay was conducted to screen Cpn0308 as a bait against a HeLa cell cDNA library, revealing its binding to the host protein acyl-coenzyme A binding domain-containing 3 (ACBD3). The interaction between Cpn0308 and ACBD3 was confirmed through co-immunoprecipitation and GST (Glutathione S-transferase) pull-down assays. The two proteins were also co-localized in HeLa cells co-expressing Cpn0308 and ACBD3, as well as in <i>C. pneumoniae</i>-infected cells, as observed under confocal fluorescence microscopy. Given that ACBD3 plays a crucial role in maintaining host cell lipid homeostasis and its Golgi dynamic domain is responsible for interacting with Cpn0308, we hypothesize that the Cpn0308-ACBD3 interaction may facilitate <i>C. pneumoniae</i>'s acquisition of host lipids, thereby benefiting chlamydial survival. This study lays a foundation for further elucidating the mechanisms of Cpn0308-mediated <i>C. pneumoniae</i> pathogenesis.IMPORTANCEThe biosynthesis and replication of <i>Chlamydia pneumoniae</i> (<i>Cpn</i>) must occur within the cytoplasmic vacuoles or inclusions of host cells. Inclusion bodies play a crucial role in mediating the interactions between <i>Cpn</i> and host cells. Cpn0308 is localized to the inclusion membrane; however, its function is unknown. In this study, Cpn0308 was found to bind to host protein acyl-coenzyme A binding domain-containing 3 (ACBD3) through some standard approaches. Co-localization of the two proteins was observed in both original HeLa cells and Cpn-infected HeLa cells. ACBD3 plays a significant role in maintaining lipid homeostasis in host cells; we speculate that the Cpn0308-ACBD3 interaction may facilitate the acquisition of host lipids by <i>C. pneumoniae</i>, thereby enhancing chlamydial survival.