This study demonstrates that pAKK exacerbates STm infection by promoting M cell differentiation and GP2 receptor expression in mice. While pAKK increased STm colonization in Peyer’s patches (PPs), mesenteric lymph nodes, and spleen, it showed no significant effect on Citrobacter rodentium (Cr) infection. Mechanistically, pAKK enhanced M cell maturation via TLR2-MyD88-NF-κB signaling, independent of RANKL, as shown in in vitro Caco-2 models and ligated intestinal loop assays. Structural analyses revealed pAKK-induced upregulation of M-cell markers (GP2, Spib) and PP expansion.
Functional validation identified Amuc_1100, a pAKK-derived TLR2 ligand, as critical for GP2 upregulation. Disruption of TLR2-MyD88 signaling or RelA (NF-κB) abolished this effect. STm, but not Cr, exploited GP2-mediated M-cell entry, correlating with its intracellular pathogenesis.
These findings highlight context-dependent risks of probiotic use: pAKK’s M-cell activation may inadvertently aid pathogens utilizing this portal. This underscores the need for pathogen-specific safety evaluations when deploying microbiome-based therapies, particularly for infections involving M-cell tropism.
Key findings from the study include:
1. pAKK exacerbates STm infection but has no significant effect on Cr infection. pAKK treatment significantly worsened weight loss, bacterial load in the gut (feces, mesenteric lymph nodes, and spleen), and inflammatory damage in mice infected with STm. However, it had no significant impact on the disease course, bacterial load, or pathological features in mice infected with Cr, indicating that the effects of pAKK on pathogens are pathogen-specific.
2. pAKK promotes the differentiation of intestinal M cells and GP2 expression. pAKK increased the number of Peyer's patches, upregulated the expression of M cell marker genes (such as Ccl20 and Gp2), and enhanced the protein level of the mature M cell receptor GP2, thereby indirectly providing more infection sites for STm to invade the gut.
3. pAKK activates M cell differentiation via the TLR2-MyD88 pathway. In vitro experiments showed that pAKK induces the differentiation of Caco-2 cells into M-like cells through the TLR2-MyD88-dependent canonical NF-κB signaling pathway (not the RANKL pathway). This effect could be blocked by TLR2/MyD88 inhibitors or RelA siRNA. Moreover, the outer membrane protein Amuc_1100 of pAKK alone could activate GP2 expression.
4. The mechanism of pAKK regulation of M cells is independent of the RANKL pathway. The differentiation of M cells induced by pAKK does not rely on the RANKL-noncanonical NF-κB pathway. This finding reveals a novel mechanism by which gut microbes regulate M cell formation through the TLR2-MyD88 axis, which may affect the infection risk of various pathogens that rely on M cell invasion.

This study reveals the dual role of pasteurized pAKK in intestinal infections: while its therapeutic benefits in metabolic disorders are well-established, pAKK may exacerbate pathogen invasion under infectious conditions by promoting microfold M cell differentiation. M cells serve dual roles as initiators of immune surveillance and gateways for infection by enteric pathogens such as Salmonella Typhimurium, Listeria monocytogenes, and certain viruses. Consequently, clinical or dietary applications of pAKK may pose unintended risks in settings where such pathogens are endemic. These findings underscore the necessity of context-specific risk-benefit evaluations when deploying pAKK-based interventions, particularly in populations vulnerable to M cell-tropic pathogens. The work entitled “Pasteurized Akkermansia muciniphila promotes GP2 expression in microfold cells and facilitates Salmonella infection” was published on Protein & Cell (published on March. 04, 2025).
DOI: 10.1093/procel/pwae017