Major membrane protein of Mycobacterium avium subp. paratuberculosis activates immune and autophagic pathways in bovine monocyte-derived macrophages

Authors: KIM, JONG HYUK - University Of Florida; LEE, DONGHEE - University Of Florida; HALL, KEVIN - University Of Florida; JO, HYUNJI - University Of Florida; Bannantine, John; DAVIS, WILLIAM - Washington State University; DE SOUZA, CLEVERSON - University Of Florida

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/17/2025
Publication Date: 2/21/2025
Citation: Kim, J., Lee, D., Hall, K., Jo, H., Bannantine, J.P., Davis, W.C., De Souza, C. 2025. Major membrane protein of Mycobacterium avium subp. paratuberculosis activates immune and autophagic pathways in bovine monocyte-derived macrophages. Veterinary Immunology and Immunopathology. https://doi.org/10.1016/j.vetimm.2025.110901.
DOI: https://doi.org/10.1016/j.vetimm.2025.110901

Interpretive Summary: In this study, we assessed the global gene expression levels of bovine white blood cells (called macrophages) before and after exposure to a strong Mycobacterium antigen that my lab has previously implicated in virulence. This particular Mycobacterium is a pathogen that causes Johne's disease in cattle and sheep. The reason for conducting this experiment is to understand how the pathogen uses macrophages to advance the disease. Normally, macrophages control bacterial infections, but not so with Mycobacterium. We discovered that certain macrophage pathways designed to kill the bacteria are crippled and non-functional when the cell encounters this antigen. These new results offer insights into macrophage-based immunity. Also, clues to development of novel prophylactic strategies against this disease have been revealed.

Technical Abstract: Mycobacterium avium subspecies paratuberculosis (Map), the etiological agent of Johne’s disease in ruminants, poses challenges to veterinary health and food safety. Despite an immune response that partially controls early infection, Map persists in macrophages through mechanisms not well understood. Here, we explored how the Map major membrane protein (MMP) modulates immune pathways in bovine monocyte-derived macrophages (MoMFs). Using high-resolution transcriptomics, we identified that MMP stimulation rapidly activates genes linked to pro-inflammatory cytokine signaling, antigen processing, and presentation via MHC I and II pathways. Gene Ontology and KEGG pathway enrichment analyses highlighted upregulation of TNF, IL-17, and NF-'B signaling cascades, suggesting an immune signaling that may foster cytotoxic T cell development. Phosphorylation assays confirmed that MMP triggers MAPK activation within minutes, implicating both p38 and JNK1/2 in early macrophage responses. Machine learning approaches revealed subtle yet significant MMP-specific gene signatures including ATG5 and ATG12, implicated in autophagosome assembly. These findings point to a dynamic interplay between antibacterial autophagy and immunostimulatory pathways elicited by MMP in bovine macrophages. Importantly, our results suggest the relevance of MMP as a potential vaccine target, as it not only elicits immune-activating signals but also engages host defenses critical to restricting Map survival. Overall, this work provides an ex vivo framework for delineating the molecular underpinnings of Map infection, offering new insights into macrophage-based immunity and informing development of novel therapeutic and prophylactic strategies against paratuberculosis. Our data open avenues for translational studies, illuminating the interplay between MMP, macrophages, and protective host immunity.