Deciphering Rab32-associated antibacterial responses: Proteomics reveals the heterogeneity of macrophage defense against bacterial pathogen infection

• Rab32, a Ras superfamily GTPase, exhibits broad-spectrum antimicrobial activity in macrophages against various bacterial pathogens. • Proteomic analysis reveals Rab32 deficiency alters host cell response and susceptibility to bacterial infections. • Temporal proteomic profiling reveals distinct host response patterns, highlighting the complexity of host-pathogen interactions and Rab32’s regulatory role. Rab32, a member of the Ras superfamily of small GTPases, has been extensively reported to participate in the infection processes of various bacteria. However, the molecular mechanisms involved are highly complex and diverse. This study focuses on elucidating the role of Rab32 in the invasion of macrophages by different bacterial pathogens and their subsequent intracellular replication within these cells, to thoroughly investigate its function in antibacterial defense. Initially, we employed CRISPR/Cas9 technology to generate a Rab32-knockout Raw264.7 macrophage cell line. Then, we infected Rab32 knockout and wild-type Raw264.7 cells with a panel of bacterial pathogens, including Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes , and Pseudomonas aeruginosa . Finally, we collected samples at various time points post-infection for proteomic analysis, aiming to delineate the proteomic landscape of the antibacterial response in the absence of Rab32. Our results indicated that Rab32 deletion increased intracellular proliferation rates of five bacterial species within macrophages by 5- to 8-fold. Subsequently, using proteomic analysis, this study elucidated the dynamic infection profiles of these five bacterial species in wild-type and Rab32-deficient macrophages. This study also identified the Rab32-associated signaling pathways triggered by each bacterial infection. Finally, through bioinformatics analysis, this study identified a panel of 11 proteins that potentially collaborate with Rab32 in defense against bacterial incursions, including Alox5, Selenbp1, Serpinb6b, Eno3, Serpinb1a, Gmds, Srgap3, Kctd12, Cd36, Fahd2, and Evl. Our study revealed that infection with five bacterial species in Rab32-deficient macrophages induces a unique Rab32-associated antibacterial response, highlighting the heterogeneity of signaling pathways modulated by Rab32 in response to distinct bacterial infections. Nonetheless, this study identified 11 proteins that may collaborate with Rab32 to combat bacterial pathogen infections. Elucidating the functional interplay between these proteins and Rab32 will provide robust support for the development of clinical antimicrobial agents and the formulation of therapeutic strategies.