Bacteriocin Microcin J25's antibacterial infection effects and novel non-microbial regulatory mechanisms: differential regulation of dopaminergic receptors

Abstract

Background: The antibacterial and immunomodulatory activities of bacteriocins make them attractive targets for development as anti-infective drugs. Although the importance of the enteric nervous system (ENS) in the struggle against infections of the intestine has been demonstrated, whether it is involved in bacteriocins anti-infective mechanisms is poorly defined.

Results: Here, we demonstrated that the bacteriocin Microcin J25 (J25) significantly alleviated diarrhea and intestinal inflammation in piglets caused by enterotoxigenic Escherichia coli (ETEC) infection. Mechanistically, macrophage levels were significantly downregulated after J25 treatment, and this was replicated in a mouse model. Omics analysis and validation screening revealed that J25 treatment induced significant changes in the dopaminergic neuron pathway, but little change in microbial structure. The alleviation of inflammation may occur by down-regulating dopamine receptor (DR) D1 and the downstream DAG-PKC pathway, thus inhibiting arachidonic acid decomposition, and the inhibition of macrophages may occur through the up-regulation of DRD5 and the downstream cAMP-PKA pathway, thus inhibiting NF-κB.

Conclusions: Our studies' findings provide insight into the changes and possible roles of the ENS in J25 treatment of ETEC infection, providing a more sophisticated foundational understanding for developing the application potential of J25.

Keywords: Arachidonic acid; Dopaminergic receptors; Enteric nervous system; Macrophage; Microbiota; Microcins.

Fig. 1

The effect of J25 on clinical symptoms, jejunum morphology and immune cells. A Schematic of the experimental design. Red arrows indicate the days on which the samples were collected for analysis. B Fecal fluid content analysis (left) and diarrhea score (right). Scoring standards: 0, normal; 1, loose stool; 2, moderate diarrhea; 3, severe diarrhea. Data are presented as mean ± SEM, n = 12. C Representative images of the jejunum by H&E staining (200×, C1) and histopathological scores (C2) in J25 treated ETEC-challenged piglet experiment. The red arrow indicates inflammatory cell infiltration in the mucosal layer, and the yellow arrow indicates edema status in the submucosa. Data are presented as mean ± SEM, n = 8. D Representative images of NK and macrophage relative proteins immunoreactivity in the jejunum (D1: F4/80, red; DAPI, blue and D2: CD3, red; α2β3, green; DAPI, blue). E Representative images of M1 and M2 subtype macrophage relative proteins immunoreactivity in the jejunum in J25 treated ETEC-challenged piglet experiment. (E1: F4/80, red; CD86, green; DAPI, blue and E2: F4/80, red; Mannose, green; DAPI, blue). F Relative immunolabeling quantification of D and E. Data are presented as mean ± SEM, n = 8. ANOVA followed by Tukey’s multiple comparisons test; different lowercase letters within each group indicate significantly different values (P < 0.05)

Fig. 2

J25 caused little change in the overall microbiota composition. A Alpha diversity comparisons of the microbial communities. Data are presented as mean ± SEM, n = 8. The numbers on the horizontal lines indicate the P-values using a paired Student’s t-test. B PCoA of 16S genes. Using an OTU definition of 97% similarity, and based on Bray_Curtis. C Distance comparisons between groups. NMDS followed by ANOISM was used for each pairwise comparison. D The most important biomarkers identified by random-forest classification in the ETEC and the ETEC + J25 groups, with the biomarker taxa ranking in descending order of importance in terms of model accuracy. Genera that coincided with the association analysis results (Fig. S5) are marked in red. E Relative abundance (%) of marker bacteria, Wilcoxon rank-sum test, BH-FDR corrected

Fig. 3

J25 treatment altered the enteric nervous system responses in ETEC-challenged piglets. A Representative images of β3-tubulin (red), GAFP (green), and DAPI (blue). B Immunolabeling quantification. Measurements were performed using volume density (Vv), which is the quotient from the volume of interest divided by the reference volume, as described in Table S5. n = 8. C–H Enteric neuron marker detection. ALDH1A1: acetaldehyde dehydrogenase 1A1 (marker for dopaminergic neurons); GAD: glutamic acid decarboxylase (marker for GABAergic neurons); TPH: tryptophan hydroxylase (marker for serotonergic neurons); ChAT: choline acetyltransferase (marker for cholinergic neurons); Gls: glutaminase (marker for glutamatergic neurons). n = 3 in C–D and n = 8 in F–H. Data are presented as mean ± SEM. ANOVA followed by Tukey’s multiple comparisons test; different lowercase letters within each group indicate significantly different values (P < 0.05)

Fig. 4

J25 regulates macrophages and inflammation through dopamine and dopamine receptor downstream pathways. A Dopamine receptor mRNA expression levels. Data are presented as mean ± SEM, n = 8. ANOVA followed by Tukey’s multiple comparisons test; different lowercase letters within each group indicate significantly different values (P < 0.05). B Protein levels and data statistics in DRD5 downstream pathways. C Protein levels and data statistics in DRD1 downstream pathways. Data are presented as mean ± SEM, n = 3. ANOVA followed by Tukey’s multiple comparisons test; different lowercase letters within each group indicate significantly different values (P < 0.05)

Fig. 5

Dopaminergic neurons are critical in the anti-infective mechanism of J25. A Schematic of the experimental design. Red arrows indicate the days on which the samples were collected for analysis. B Body weight changes (relative to original weight, set as 100%). Data are presented as mean ± SEM, n = 8. C Representative images of the jejunum by H&E staining (200×) and histopathological scores. Data are presented as mean ± SEM, n = 8. D Protein levels and data statistics in dopaminergic neuron downstream pathways. Data are presented as mean ± SEM, n =3. ANOVA followed by Tukey’s multiple comparisons test; different lowercase letters within each group indicate significantly different values (P < 0.05)

Fig. 6

The inhibition of AA metabolism alleviated macrophage infiltration. A Schematic of the experimental design. Red arrows indicate the days on which the samples were collected for analysis. B Body weight changes (relative to original weight, set as 100%). Data are presented as mean ± SEM, n = 8. C Representative images of the jejunum by H&E staining (200×) and histopathological scores. Data are presented as mean ± SEM, n = 8. D M1 and M2 subtype macrophage relative protein levels and data statistics. Data are presented as mean ± SEM, n = 3. ANOVA followed by Tukey’s multiple comparisons test; different lowercase letters within each group indicate significantly different values (P < 0.05)

Fig. 7

Mechanisms whereby J25 prevents ETEC infection in piglets. The administration of J25 activates cAMP-PKA-mediated NF-κB downregulation through increased DRD5 expression. This dampens EGCs and macrophages, and decrease arachidonic acid (AA) metabolism by lowering DAG-PKC via decreased DRD1, attenuating intestinal inflammation. In addition, downregulated DRD2 by J25 attenuating cAMP inhibition, whereas reinforcing the inhibitory effect of EGCs and macrophages. Reduced AA metabolism further enhanced the inhibitory effect on macrophage infiltration