Subcellular Localization Dictates Therapeutic Function: Spatially Targeted Delivery of Amuc_1100 by Engineered Lacticaseibacillus paracasei L9 Enhances Intestinal Barrier in Colitis

<b>Background/Objectives</b>: Impaired intestinal barrier function is a hallmark of inflammatory bowel disease (IBD). <i>Akkermansia muciniphila</i> and its outer membrane protein Amuc_1100 can enhance this barrier, but the clinical application of Amuc_1100 is limited by the fastidious growth of its native host. This study aimed to overcome this by utilizing the robust probiotic <i>Lacticaseibacillus paracasei</i> L9 for targeted Amuc_1100 delivery. <b>Methods</b>: We engineered <i>Lc. paracasei</i> L9 to express Amuc_1100 via intracellular (pA-L9), secretory (pUA-L9), and surface-display (pUPA-L9) strategies. Their efficacy was assessed in Lipopolysaccharide (LPS)-induced macrophages and a dextran sulfate sodium (DSS)-induced colitis mouse model, evaluating inflammation, barrier integrity, and mucosal repair. <b>Results</b>: The secretory (pUA-L9) and surface-display (pUPA-L9) strains most effectively suppressed pro-inflammatory cytokines (<i>IL-6</i>, <i>IL-1β</i>, and <i>TNF-α</i>) in macrophages. In mice, both strains alleviated colitis and outperformed native <i>A. muciniphila</i> in improving disease activity. Crucially, they exhibited distinct, specialized functions: pUA-L9 acted as a systemic immunomodulator, reducing pro-inflammatory cytokines (<i>IL-6</i>, <i>IL-1β</i>, and <i>TNF-α</i>), elevating anti-inflammatory mediators (<i>IL-4</i> and <i>IL-10</i>), and promoting goblet cell differentiation; notably, the inhibitory effect of pUA-L9 on <i>IL-6</i> expression was approximately 2-fold greater than that of pUPA-L9. In contrast, pUPA-L9 excelled in local barrier repair, uniquely restoring mucus layer integrity (<i>Muc1</i>, <i>Muc2</i>, and <i>Tff3</i>) and reinforcing tight junctions (<i>ZO-1</i>, <i>Occludin</i>, <i>Claudin1</i>, <i>Claudin3</i>, and <i>Claudin4</i>). In particular, pUPA-L9 increased <i>Muc2</i> expression by approximately 3.6-fold compared with pUA-L9. <b>Conclusions</b>: We demonstrate that the subcellular localization of Amuc_1100 within an engineered probiotic dictates its therapeutic mode of action. The complementary effects of secretory and surface-displayed Amuc_1100 offer a novel, spatially targeted strategy for precision microbiome therapy in IBD.