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. 2022 Sep 9;13(1):101.
doi: 10.1186/s40104-022-00752-w.

Lactobacillus rhamnosus GR-1 attenuates foodborne Bacillus cereus-induced NLRP3 inflammasome activity in bovine mammary epithelial cells by protecting intercellular tight junctions

Qiang Shan  1 , Ning Liu  1 , Xue Wang  1 , Yaohong Zhu  1 , Jinhua Yin  2   3 , Jiufeng Wang  4
Affiliations

Lactobacillus rhamnosus GR-1 attenuates foodborne Bacillus cereus-induced NLRP3 inflammasome activity in bovine mammary epithelial cells by protecting intercellular tight junctions

Qiang Shan et al. J Anim Sci Biotechnol. .

Abstract

Background: Bacillus cereus is an important pathogen that causes human food poisoning, specifically diarrhea and vomiting. B. cereus can also induce mastitis in dairy cows and has a strong survival ability in milk, as it cannot be inactivated by high-temperature short-time pasteurization. Therefore, B. cereus can enter the market through pasteurized milk and other dairy products, imposing enormous hidden dangers on food safety and human health.

Results: In this study, B. cereus 2101 (BC) was isolated from milk samples of cows with mastitis. BC grew rapidly with strong hemolysis, making it difficult to prevent mastitis and ensure food security. MAC-T cells were treated with BC and/or Lactobacillus rhamnosus GR-1 (LGR-1). Pretreatment with LGR-1 protected the integrity of tight junctions and the expression of zonula occludens-1 (ZO-1) and occludin destroyed by BC. Furthermore, LGR-1 pretreatment reduced the expression of NOD-like receptor family member pyrin domain-containing protein 3 (NLRP3), caspase recruitment and activation domain (ASC), Caspase-1 p20, gasdermin D (GSDMD) p30, inflammatory factors (interleukin (IL)-1β and IL-18), and cell death induced by BC. Moreover, LGR-1 pretreatment reduced NLRP3 inflammasome activity and increased expressions of ZO-1 and occludin induced by lipopolysaccharides (LPS) + ATP stimulation. MAC-T cells were transfected with NLRP3 siRNA or MCC950 and/or treated with BC and/or LGR-1. NLRP3-siRNA transfection and MCC950 attenuated BC-induced NLRP3 inflammasome activity. Expression of inflammatory cytokines and cell death suggested that the inflammatory pathway might play an important role in the induction of the NLRP3 inflammasome by BC and the protection of LGR-1.

Conclusions: These results suggest that LGR-1 might be a probiotic alternative to antibiotics and could be administered to prevent mastitis in dairy cows, thus ensuring food security.

Keywords: Bacillus cereus; Intercellular tight junctions; Lactobacillus rhamnosus GR-1; NLRP3 inflammasome.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Lactobacillus rhamnosus GR-1 (LGR-1) alleviated Bacillus cereus 2101 (BC)-induced cell death. MAC-T cells were treated with BC (MOI = 0.5, 5 or 50) and/or LGR-1 (MOI = 1, 10, 100, or 1000) for 3 h. (A) The blood plate shows that the strain was hemolytic. Transmission electron microscopy (TEM) showed that the strain was rod-shaped. The scale bar is shown in the lower left corner. The color was purple after staining with the Gram Staining Kit. (B) Virulence factors of BC. M. Marker; 1. HBLA; 2. HBLB; 3. HBLD; 4. NHEA; 5. NHEB; 6. NHEC; 7. InhA; 8. CytK; 9. HlyA; 10. HlyIII; 11. EntFM. (C) Growth curves of BC. (D) Cell death induced by BC. (E) Cell death induced by LGR-1 and/or BC. One-way ANOVA was used for comparison between groups. The data are mean ± SEM of three independent experiments. *** P < 0.001
Fig. 2
Fig. 2
LGR-1 protects BC damaged intercellular tight junctions. MAC-T cells were treated with BC (MOI = 5) or LPS (500 ng/mL) + ATP (5 mmol/L) and/or LGR-1 (MOI = 100). (A) The ultrastructure of LGR-1 and/or BC treated cells was visualized by TEM. The scale bar is shown in the lower right corner. (B) Expression of ZO-1 in cells measured by immunofluorescence analysis; the scale bar is shown in the lower right corner. (C) and (F) Protein levels of ZO-1 and occludin in MAC-T. (D) and (G) Relative protein level of ZO-1. (E) and (H) Relative protein level of occludin. The data for the CONTROL group were used to normalize the data of each treatment group. White arrows indicate the nuclear membrane and black arrowheads indicate junctional complexes. Comparisons among groups were calculated using one-way ANOVA. Data are means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001
Fig. 3
Fig. 3
LGR-1 attenuated BC-induced NLRP3 inflammasome activity. MAC-T cells were treated with BC (MOI = 5) and/or LGR-1 (MOI = 100) for 3 h. (A) and (B) Protein levels of NLRP3, ASC, Caspase-1 p20, GSDMD p30. (C) IL-1β and IL-18 mRNA level. Expression of NLRP3, ASC, GSDMD, and Caspase-1 in cells measured by immunofluorescence analysis; scale bar shown in the lower right corner. (D) NLRP3. (E) ASC. (F) GSDMD. (G) Caspase-1. The data for the CONTROL group were used to normalize the data of each treatment group. Comparisons among groups were calculated using one-way ANOVA. Data are means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001
Fig. 4
Fig. 4
BC-induced NLRP3 inflammasome activity requires K+ efflux. MAC-T cells were treated with BC (MOI = 5) or LPS (500 ng/mL) + ATP (5 mmol/L) in the absence (-) or presence ( +) of 50 mmol/L KCl ( +) or at increasing concentrations of KCl (5, 25, 50 and 75 mmol/L). (A) IL-1β mRNA level. (B) IL-18 mRNA level. (C) Cell death in MAC-T. The data for the CONTROL group were used to normalize the data of each treatment group. Comparisons among groups were calculated using one-way ANOVA. Data are means ± SEM of three independent experiments. *P < 0.05 and ***P < 0.001
Fig. 5
Fig. 5
LGR-1 attenuated LPS + ATP-induced NLRP3 inflammasome activity. MAC-T cells were treated with LPS (500 ng/mL) + ATP (5 mmol/L) and/or LGR-1 (MOI = 100). (A) Protein levels of NLRP3 and Caspase-1 p20. (B) Protein levels of GSDMD p30. (C) IL-1β mRNA level. (D) IL-18 mRNA level. (E) Relative protein level of NLRP3. (F) Relative protein level of Caspase-1 p20. (G) Relative protein level of GSDMD p30. The data for the CONTROL group were used to normalize the data of each treatment group. Comparisons among groups were calculated using one-way ANOVA. Data are means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001
Fig. 6
Fig. 6
BC induced NLRP3 inflammasome activity. MAC-T cells were treated with BC (MOI = 5) and/or MCC950 (100 nmol/L). (A) Protein levels of NLRP3, Caspase-1 p20, GSDMD p30. (B) IL-1β mRNA level. (C) IL-18 mRNA level. (D) Relative protein level of NLRP3. (E) Relative protein level of Caspase-1 p20. (F) Relative protein level of GSDMD p30. The data for the CONTROL group were used to normalize the data of each treatment group. Comparisons among groups were calculated using one-way ANOVA. Data are means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001
Fig. 7
Fig. 7
NLRP3 knockdown attenuated inflammatory responses. MAC-T cells were transfected with NLRP3 siRNA for 5 h and/or treated with BC (MOI = 5) and/or LGR-1 (MOI = 100) for another 3 h. (A) NLRP3 mRNA level. (B) Protein level of NLRP3. (C) Relative protein level of NLRP3. (D) Protein level of Caspase-1 p20. (E) IL-1β mRNA level. (F) IL-18 mRNA level. (G) Cell death in MAC-T. (H) Relative protein level of Caspase1 p20. In A and C, data for the si-CONTROL group were used to normalize data of each treatment group. Comparisons among groups were calculated using the t-test. Data are means ± SEM of three independent experiments. In E–H, the data for the CONTROL group were used to normalize the data of each treatment group. Comparisons among groups were calculated using one-way ANOVA. Data are means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001
Fig. 8
Fig. 8
Schematic of the proposed model. LGR-1 can reduce BC-induced K+ efflux, inhibit the NLRP3 inflammasome activity, decrease the expressions of IL-1β and IL-18, reduce the production of active N-terminal fragment of GSDMD, and reduce both pyroptosis and the inflammatory response by protecting intercellular tight junctions. The solid black arrows indicate demonstrated effects, the dashed arrows indicate potential effects, red lines indicate negative or protective effects, and solid red arrows represent changes in expression
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