ADS024, a Bacillus velezensis strain, protects human colonic epithelial cells against C. difficile toxin-mediated apoptosis - PubMed (original) (raw)
ADS024, a Bacillus velezensis strain, protects human colonic epithelial cells against C. difficile toxin-mediated apoptosis
Ying Xie et al. Front Microbiol. 2023.
Abstract
Clostridioides difficile infection (CDI) causes intestinal injury. Toxin A and toxin B cause intestinal injury by inducing colonic epithelial cell apoptosis. ADS024 is a Bacillus velezensis strain in development as a single-strain live biotherapeutic product (SS-LBP) to prevent the recurrence of CDI following the completion of standard antibiotic treatment. We evaluated the protective effects of the sterile filtrate and ethyl acetate extract of conditioned media from ADS024 and DSM7 (control strain) against mucosal epithelial injury in toxin-treated human colonic tissues and apoptosis in toxin-treated human colonic epithelial cells. Ethyl acetate extracts were generated from conditioned culture media from DSM7 and ADS024. Toxin A and toxin B exposure caused epithelial injury in fresh human colonic explants. The sterile filtrate of ADS024, but not DSM7, prevented toxin B-mediated epithelial injury in fresh human colonic explants. Both sterile filtrate and ethyl acetate extract of ADS024 prevented toxin-mediated apoptosis in human colonic epithelial cells. The anti-apoptotic effects of ADS024 filtrate and ethyl acetate extract were dependent on the inhibition of caspase 3 cleavage. The sterile filtrate, but not ethyl acetate extract, of ADS024 partially degraded toxin B. ADS024 inhibits toxin B-mediated apoptosis in human colonic epithelial cells and colonic explants.
Keywords: C. difficile; apoptosis; biologic; infection; single-strain live biotherapeutic product; therapy.
Copyright © 2023 Xie, Chupina Estrada, Nelson, Feng, Pothoulakis, Chesnel and Koon.
Conflict of interest statement
Adiso Therapeutics sponsored this study and a part of HK and LC salaries. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Figures
FIGURE 1
ADS024 sterile filtrate and ethyl acetate extract possess anti-apoptotic effects in human colonic epithelial cells. (A–C) Apoptosis assays of human colonic epithelial NCM460 cells. The Annexin V luminescent signal above 100% indicated the occurrence of apoptosis. (A) Serum-starved NCM460 cells were treated with PBS or 0.1% v/v of ADS024 and DSM7 sterile filtrate (FS) from 1 to 0.0001-fold. (B) Serum-starved NCM460 cells were treated with PBS or 1% v/v of ADS024 and DSM7 isopropanol extract (IPA) from 1 to 0.0001-fold. (C) Serum-starved NCM460 cells were treated with PBS or 1% v/v of ADS024 and DSM7 ethyl acetate extract (EA) from 1- to 0.0001-fold. (A–C) After adding sterile filtrates (FS), IPA extracts, and EA extracts, the cells were added with Promega RealTime-Glo Annexin V apoptosis assay reagents in a 1:1,000 ratio. Thirty minutes later, the cells were added with PBS, toxin A (0.1 μg/ml), or toxin B (0.1 μg/ml). After 24 h, the luminescent (apoptosis) signals were read by a 96-plate reader. Toxins increased the apoptosis signal. ADS024 FS at 0.0001X reduced toxin B-induced apoptosis. EA extracts of ADS024 and DSM7 at 1X reduced toxin A- and B-induced apoptosis. The results were pooled from 3–4 independent experiments. One-way ANOVA tests were used.
FIGURE 2
ADS024 sterile filtrate reduced toxin B-mediated epithelial injury in fresh human colonic explants. Fresh human colonic explants were placed in serum-free RPMI1640 media with PBS or 0.1% v/v of ADS024 and DSM7 sterile filtrate (FS) at 1X. Thirty minutes later, toxin A and toxin B (0.1 μg/ml) were added and further incubated for 24 h. (A) H&E-stained images at 100X magnification. Black lines indicated the integrity of the top lining of colonic mucosa in PBS control and toxin B- and ADS024 filtrate-treated groups. Arrows indicated toxin-mediated disruption of mucosal integrity and thickness. (B) Histology scores for epithelial injury (0–3). ADS024 sterile filtrate 1X reduced toxin-B mediated epithelial injury. Results were pooled from samples from 10 patients. One-way ANOVA tests were used.
FIGURE 3
ADS024 isopropanol extract did not affect toxin-mediated epithelial injury in fresh human colonic explants. Fresh human colonic explants were placed in serum-free RPMI1640 media with PBS or 0.1% v/v of IPA extracts of ADS024 and DSM7 at 1X. Thirty minutes later, toxin A and toxin B (0.1 μg/ml) were added and further incubated for 24 h. (A) H&E-stained images at 100X magnification. Black lines indicated the integrity of the top lining of colonic mucosa in PBS control groups. Arrows indicated toxin-mediated disruption of mucosal integrity and thickness. (B) Histology scores for epithelial injury (0–3). ADS024 and DSM7 IPA extracts did not affect toxin-mediated epithelial injury. Results were pooled from samples from 10 patients. One-way ANOVA tests were used.
FIGURE 4
ADS024 ethyl acetate extract reduced toxin-mediated epithelial injury in fresh human colonic explants. Fresh human colonic explants were placed in serum-free RPMI1640 media with PBS or 0.1% v/v of ADS024 and DSM7 EA extracts at 1X. Thirty minutes later, toxin A and toxin B (0.1 μg/ml) were added and further incubated for 24 h. (A) H&E-stained images at 100X magnification. Black lines indicated the integrity of the top lining of colonic mucosa in PBS control groups and toxin- and EA extract-treated groups. Arrows indicated toxin-mediated disruption of mucosal integrity and thickness. (B) Histology scores for epithelial injury (0–3). EA extracts of ADS024 and DSM7 reduced toxin-mediated epithelial injury. Results were pooled from samples from 10 patients. One-way ANOVA tests were used.
FIGURE 5
Protease inhibitors abolished the anti-apoptotic effect of ADS024 sterile filtrate. (A–C) Toxin digestion assays. Sterile filtrates, IPA extracts, and EA extracts of ADS024 and DSM7 from 1X to 10– 6X were added to serum-free RPMI1640 containing 50 ng/500 μl toxin A or toxin B. (C) ADS024 filtrates were further processed by MWCO columns. The centrifuged eluates were diluted to 0.0001X and added to serum-free RPMI1640 containing 50 ng/500 μl toxin B. (A–C) The mixtures were incubated at 37°C for 1 h and then stopped by adding a PIC and EDTA mixture (#PI78429, Thermo Scientific) in 1:100 dilution ratio. The toxin levels in the mixtures were measured by ELISA. ADS024 FS at 1–0.0001X partially degraded toxin B. MWCO at 50kDa abolished this effect. (D) Apoptosis assay. Serum-starved NCM460 cells were pretreated with ADS024 sterile filtrate at 0.0001X with or without protease inhibitor cocktail at 1X. Reagents of Promega RealTime-Glo Annexin V apoptosis assay in a 1:1,000 ratio were added simultaneously. Thirty minutes later, toxin A or toxin B (0.1 μg/ml) was added to start the apoptosis process. After 24 h, the luminescent (apoptosis) signals were read by a 96-plate reader. The Annexin V luminescent signal above 100% indicated the occurrence of apoptosis. The ADS024 sterile filtrate-mediated inhibition of apoptosis in toxin B-treated cells was reversed by pretreatment of PIC at 1X. Results were pooled from 3–4 independent experiments. One-way ANOVA tests were used.
FIGURE 6
ADS024 inhibited caspase three cleavage in toxin-treated human colonic epithelial cells. (A,B) Apoptosis array. Serum-starved primary human colonic epithelial cells were pretreated with ADS024 ethyl acetate extract at 1X or sterile filtrate at 0.0001X dilution for 30 min, followed by toxin B (0.1 μg/ml). A total of 24 h later, the cells were collected for Proteome Profiler Human Apoptosis Array (ARY009, R&D Systems). (A) Bio-Rad ChemiDoc Imaging system captured the images. The rectangles highlighted the inactive procaspase three and active cleaved caspase three. The images are representative of three independent experiments. (B) Bio-Rad Image Lab Software performed quantitation of cleaved caspase 3/procaspase 3 signal. Toxin B increased cleaved caspase 3/pro-caspase 3 ratio, which was reduced by ADS024 EA extract 1X and sterile filtrate 0.0001X dilution. (C) Cleaved caspase 3 ELISA. Serum-starved primary human colonic epithelial cells were pretreated with ethyl acetate extracts of ADS024 and DSM7 at 1X or sterile filtrates of ADS024 and DSM7 at 0.0001X dilution for 30 min, followed by the addition of toxin B (0.1 μg/ml). The cells were lysed by RIPA buffer with 1X PIC and 1X EDTA. The cleaved caspase three levels in cell lysates were measured by ELISA. (D) Apoptosis assays. Serum-starved NCM460 cells were pretreated with ethyl acetate extracts of ADS024 and DSM7 at 1X and sterile filtrates of ADS024 and DSM7 at 0.0001X dilution with or without adding 10 μM procaspase activating compound 1 or PAC1 (#10009317, Cayman Chemical). PAC1 was used to activate caspase three cleavage. Reagents of Promega RealTime-Glo Annexin V apoptosis assay in a 1:1,000 ratio were added simultaneously. Thirty minutes later, toxin A or toxin B (0.1 μg/ml) was added to start the apoptosis process. After 24 h, the luminescent (apoptosis) signals were read by a 96-plate reader. The Annexin V luminescent signal above 100% indicated the occurrence of apoptosis. The ADS024 EA extract-mediated inhibition of toxin A- and B-dependent apoptosis was reversed by PAC1 pretreatment. The ADS024 sterile filtrate-mediated inhibition of apoptosis in toxin B-treated cells was reversed by PAC1 pretreatment. Results were pooled from 3–4 independent experiments. One-way ANOVA tests were used.
FIGURE 7
ADS024 inhibited caspase three cleavage in toxin-treated human colonic explants. (A) Immunofluorescence staining of cleaved caspase 3 (red), pan-cytokeratin (green), and nuclei (blue) in fresh human colonic explants treated with or without toxin A, toxin B, PBS, ADS024 filtrate, control EA, ADS024 EA, or DSM7 EA. The gray bars in the lower-left corner indicate 200 microns. Apoptosis (red, cleaved caspase) occurred in colonic epithelial cells (green, pan-cytokeratin) as red and green signals either overlapped or in proximity. (B) Quantitative analysis of images representing cleaved caspase three (red) and epithelial cells (green) ratio after normalized to blue nuclear signal. The high red/green ratio represented increased apoptosis. Toxin B-mediated cleaved caspase three was reduced by ADS024 filtrate treatment. Toxin A- and toxin B-mediated caspase three cleavages were reduced by ADS024 and DSM7 EA extract treatment. Results were representative of samples from 10 patients. One-way ANOVA tests were used. (C) A comparison of ADS024 and DSM7 properties.
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