Development of a PapG-AcmA surface-associated antigen on Lactobacillus reuteri as a vaccine strategy against uropathogenic Escherichia coli

Urinary tract infections (UTIs) are highly prevalent across diverse populations, with uropathogenic Escherichia coli (UPEC) recognized as the leading cause, responsible for more than 85% of cystitis and acute pyelonephritis cases. Because bacterial attachment to the uroepithelium is the critical initial step in colonization and infection, blocking this adhesion represents one of the most effective preventive strategies. The PapG adhesin, located at the tip of P-fimbriae in P-type UPEC, is a key mediator of this attachment and therefore a promising vaccine target. In this study, a chimeric antigen was designed by fusing the 198-amino-acid receptor-binding domain of PapG (GenBank accession No. YP_543821.1) to the 85-amino-acid cell-wall anchoring domain of AcmA (accession No. NC_011751.1) via a pentameric EAAAK flexible linker, yielding a 314-amino-acid construct. The gene was subcloned into the pET21a expression vector and expressed in E. coli Origami (DE3) under 1 mM IPTG induction. The purified recombinant protein was verified by SDS-PAGE and Western blot analysis, and its surface association on Lactobacillus reuteri was confirmed by ELISA. Seventy BALB/c mice were allocated into five groups (14 mice per group) and received a triple-dose vaccination regimen at two-week intervals: alum-adjuvanted + PapG, PapG + formulated with Freund's adjuvant, PapG-AcmA-associated L. reuteri, PBS-treated L. reuteri, and PBS alone as a negative control. Antigen-specific antibody responses and cytokine secretion profiles were subsequently evaluated. The findings indicate that adjuvanted PapG formulations elicit robust systemic Th1 and Th2 immune responses. Meanwhile, oral administration of L. reuteri surface-associated PapG-AcmA provides partial mucosal protection against UPEC colonization, likely driven by localized immunomodulation, and may serve as a promising mucosal vaccine platform.