Live and pasteurized Akkermansia muciniphila decrease susceptibility to Salmonella Typhimurium infection in mice

Abstract

Introduction: The gut microbiome is vital for providing resistance against colonized pathogenicbacteria. Recently, specific commensal species have become recognized as important mediators of host defense against microbial infection by a variety of mechanisms.

Objectives: To examine the contribution of live and pasteurized A. muciniphila to defend against the intestinal pathogen Salmonella Typhimurium in a streptomycin-treated mouse model of infection.

Methods: C57B6J mice were pretreated with phosphate-buffered saline (PBS), live Akkermansia muciniphila (AKK), and pasteurized A. muciniphila (pAKK) for two weeks, then mice were infected by S. Typhimurium SL 1344. 16S rRNA-based gut microbiota analysis was performed before and after infection. Bacterial counts in feces and tissues, histopathological analysis, gut barrier-related gene expression, and antimicrobial peptides were examined. Co-housing was performed to examine the role of microbiota in the change of susceptibility of mice to infection.

Results: AKK and pAKK markedly decreased Salmonella fecal and systemic burdens and reduced inflammation during infection. Notably, further characterization of AKK and pAKK protective mechanisms revealed different candidate protective pathways. AKK promoted gutbarrier gene expression and the secretion of antimicrobial peptides, and co-housing studies suggested that AKK-associated microbial community played a role in attenuating infection. Moreover, pAKK had a positive effect on NLRP3 in infected mice. We verified that pretreatment of pAKK could promote the expression of NLRP3, and enhance the antimicrobial activity of macrophage, likely through increasing the production of reactive oxygen (ROS), nitric oxide (NO), and inflammatory cytokines.

Conclusion: Our study demonstrates that live or pasteurized A. muciniphila can be effective preventive measures for alleviating S. Typhimurium-induced disease, highlighting the potential of developing Akkermansia-based probiotics or postbiotics for the prevention of Salmonellosis.

Keywords: Akkermansia muciniphila; Gut barriers; Gut microbiota; Inflammasomes; RegIII lectins; Salmonella Typhimurium.

Graphical abstract

Fig. 1

AKK and pAKK treatment impacted tight junction protein expression, SCFA production, and gut microbial community composition in mice before infection. C57BL/6 mice were orally gavaged with PBS (CK). Live A. muciniphila (AKK) or pasteurized A. muciniphila (pAKK) for 14 days and euthanized on day 14. (A) Weight change. (B) Representatives image of colon (left) and colon length (right). (C) Representative H&E stained cecal sections of mice. Images were taken at × 10 magnification. The mRNA expression of (D) Cldn3 (Claudin3), (E) Ocln  (Occludin), (F) Tjp1 (ZO-1), (G) Muc2 (Mucin2), (H) Reg3b, and (I) Reg3g. (J) Quantification of SCFAs derived from fecal contents. (K-R) Microbial communities. (K) Shannon index and (L) PCoA analysis were shown. Each dot represented an individual in each group. (M) Relative abundance of bacterial phyla in three representative animals per group. (N) Boxplots displaying the relative abundance fractions of Bacteroidetes and Firmicutes; (O) The ratio of Firmicutes to Bacteroidetes; (P) The difference in the bacterial family among three groups. A-J: n = 5, K-P: n = 6. Statistical significance was determined by ANOVA or Kruskal-Wallis test for multiple comparisons, and post-hoc Tukey test or Mann-Whitney test for comparisons between two groups. *^,#p < 0.05, **^,##p < 0.01, and ***^,###p < 0.001.

Fig. 2

AKK and pAKK treatment alleviated S. Typhimurium infection in mice. (A) Experimental design. After the 14-day intervention, C57BL/6 mice were orally gavaged with streptomycin, then challenged with 10⁷ CFU/100 μL of S. Typhimurium SL1344. Here are two parts of the experiment. One part of the mice was sacrificed on day 23 for monitoring the trend of weight change and fecal shedding of pathogens, and the other part of the mice was sacrificed on day 19 for the determination of organ and tissue bacterial burdens and histopathology analysis. (B) Weight loss and (C) S. Typhimurium shedding in feces. S. Typhimurium burdens in tissues or intestinal contents, including (D) liver, (E) spleen, (F) kidney, (G) MLN, (H) SI, (I) cecal content, (J) colon, and (K) colon content. (L) Representative image of colon (left) and colon length (right). (M) Representative H&E stained cecal sections of mice (left) and associated histopathological scores after infection (right). Images were taken at × 10 magnification. (N) AB/PAS stained cecal sections of mice (left) and the number of goblet cells per crypt (right). Images were taken at × 10 magnification. B-C: n = 6, D-N: n = 7 ∼ 10. Statistical significance was determined by ANOVA or Kruskal-Wallis test for multiple comparisons, and post-hoc Tukey test or Mann-Whitney test for comparisons between two groups. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 3

AKK and pAKK modulated gut microbial community composition in the streptomycin-treated S. Typhimurium infection mice model. (A) Shannon index and (B) PCoA analysis were shown. Each dot represented an individual in each group. (C) Differences in microbial taxa before and after treatment of streptomycin. (D) Relative abundance of bacterial phyla in three representative animals per group. (E) Relative abundance of Bacteroidetes and Firmicutes. (F) Shannon index and (G) PCoA analysis were shown. Each dot represented an individual in each group. (H) Relative abundance of bacterial phyla in three representative animals per group. (I) Quantification of fecal propionic acid. A-H: n = 6, I: n = 5. Statistical significance was determined by ANOVA or Kruskal-Wallis test for multiple comparisons, and post-hoc Tukey test or Mann-Whitney test for comparisons between two groups. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 4

Influence of AKK and pAKK on the expression of tight junction proteins and RegIII lectins in Salmonella-infected mice. The mRNA expression of (A) Cldn3, (B) Ocln, (C) Tjp1, and (D) Muc2. (E) Western blots for tight junction proteins. (F) Quantification of bands in E. (G) Immunofluorescence image of tight junction proteins. The blue color represents colonic cells stained with DAPI, the red color represents Cldn3 and Ocln stained with Alexa Fluor  647, and the green color represents Tjp1 and Muc2 stained with Alexa Fluor  488. Images were taken at × 10 magnification. (H) The mRNA expression of Lcn2. (I) Lipocalin-2 levels determined by ELISA. The mRNA expression of (J) Reg3b and (K) Reg3g. (L) Western blots of RegIII proteins. (M) Quantification of bands in L. n = 3 ∼ 5. Statistical significance was determined by ANOVA for multiple comparisons and post-hoc Tukey test for comparisons between two groups. *p < 0.05, **p < 0.01, and ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Effect of AKK and pAKK on inflammasome expression and production of caspase-1 and IL-1β in infected mice. The mRNA expression of (A) TLR4, (B) NLRP3, (C) Caspase-1, and (D) IL-1β. (E) Western blots of inflammasome-related proteins. (F) Quantification of bands in E. n = 3 ∼ 5. Statistical significance was determined by ANOVA for multiple comparisons and post-hoc Tukey test for comparisons between two groups. *p < 0.05, **p < 0 0.01, and ***p < 0.001.

Fig. 6

Effect of AKK and pAKK on antimicrobial activity of macrophage and its NO and ROS production. The numbers of intracellular Salmonella in macrophages pretreated with (A) pAKK at different MOI for 24 h, (B) AKK at different MOI for 24 h, (C) pAKK or AKK at the same MOI for 24 h, and (D) pAKK or AKK at an MOI of 10 for 6 h or 24 h. (E) The antimicrobial kinetics of macrophages. (F) The fluorescence intensity of GFP-S. Typhimurium SL1314. (G) Representative image of GFP-S. Typhimurium. (H-N) Macrophages were pretreated with pAKK or AKK for 24 h, then infected with S. Typhimurium SL1314 for 3 h. After that, (H) ROS, (I) NO, (K) IL-1β, and (L) IL-6 were measured. (J) Western blotting (up) and band quantification (down) of iNOS. (M) Western blotting (left) and band quantification (right) of NLRP3. (N) Immunofluorescence image of NLRP3. (O) The number of intracellular Salmonella in macrophages pretreated with MCC950 for 24 h before pAKK treatment. Statistical significance was determined by ANOVA for multiple comparisons and post-hoc Tukey test for comparisons between two groups.*p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 7

The protective effect of AKK was transferable through gut microbiota to co-housed naïve mice. (A) Experimental design. Naïve mice (co-AKK) were co-housed with AKK-treated mice (AKK) for 14 days, then all mice were orally gavaged with streptomycin before challenging with 10⁷ CFU/100 μL of S. Typhimurium SL1344. (B) Weight loss. (C) S. Typhimurium shedding in feces throughout the infection. S. Typhimurium burdens in tissues, including (D) liver, (E) kidney, (F) MLN, (g) spleen, (H) SI, (I) colon, n = 5. (J) Representative H&E stained cecal sections of mice (left) and histopathological scores (right). Images were taken at × 10 magnification. The cytokines production of (K) D-lactate in serum, (L) Lipocalin-2, (M) IL-6, (N) TNF-α, and (O) IL-1β in cecum, n = 3. Statistical significance was determined by ANOVA for multiple comparisons and post-hoc Tukey test for comparisons between two groups. *p < 0.05.