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. 2016 Feb 3:7:69.
doi: 10.3389/fmicb.2016.00069. eCollection 2016.

Cytotoxic Potential of Bacillus cereus Strains ATCC 11778 and 14579 Against Human Lung Epithelial Cells Under Microaerobic Growth Conditions

Kathleen Kilcullen  1 , Allison Teunis  1 , Taissia G Popova  1 , Serguei G Popov  1
Affiliations

Cytotoxic Potential of Bacillus cereus Strains ATCC 11778 and 14579 Against Human Lung Epithelial Cells Under Microaerobic Growth Conditions

Kathleen Kilcullen et al. Front Microbiol. .

Abstract

Bacillus cereus, a food poisoning bacterium closely related to Bacillus anthracis, secretes a multitude of virulence factors including enterotoxins, hemolysins, and phospholipases. However, the majority of the in vitro experiments evaluating the cytotoxic potential of B. cereus were carried out in the conditions of aeration, and the impact of the oxygen limitation in conditions encountered by the microbe in natural environment such as gastrointestinal tract remains poorly understood. This research reports comparative analysis of ATCC strains 11778 (BC1) and 14579 (BC2) in aerobic and microaerobic (static) cultures with regard to their toxicity for human lung epithelial cells. We showed that BC1 increased its toxicity upon oxygen limitation while BC2 was highly cytotoxic in both growth conditions. The combined effect of the pore-forming, cholesterol-dependent hemolysin, cereolysin O (CLO), and metabolic product(s) such as succinate produced in microaerobic conditions provided substantial contribution to the toxicity of BC1 but not BC2 which relied mainly on other toxins. This mechanism is shared between CB1 and B. anthracis. It involves the permeabilization of the cell membrane which facilitates transport of toxic bacterial metabolites into the cell. The toxicity of BC1 was potentiated in the presence of bovine serum albumin which appeared to serve as reservoir for bacteria-derived nitric oxide participating in the downstream production of reactive oxidizing species with the properties of peroxynitrite. In agreement with this the BC1 cultures demonstrated the increased oxidation of the indicator dye Amplex Red catalyzed by peroxidase as well as the increased toxicity in the presence of externally added ascorbic acid.

Keywords: Bacillus cereus; cereolysin O; culture filtrates; cytotoxicity; lung epithelial cells.

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Figures

FIGURE 1
FIGURE 1
The growth (dashed lines) and toxicity (solid lines) of BC1 (A) and BC2 (B) under microaerobic and aerobic conditions (filled and open circles, correspondingly). Overnight cultures were grown in aerated LB medium and used to inoculate CSFM (1:100). For static microaerobic cultures the inoculated medium was dispensed into 24-well plate (2 mL per well) and incubated at 37°C and 5% CO2 without shaking. For aerobic cultures, 20 mL of inoculated CSFM in a 50 mL Falcon tube were shaken at 200 rpm and 37°C. The cultures were harvested every hour post-inoculation and their OD measured at 600 nm. Bacteria were pelleted and the Sups were used to expose HSAECs for 2 h at 37°C. Cell viability was assessed relative to untreated HSAECs using resazurin as described in the Section “Materials and Methods.”
FIGURE 2
FIGURE 2
BC1 and BC2 cultures accumulate high levels of toxicity when grown in microaerobic and aerobic conditions for 20 h (A). The toxicity of microaerobic Sup can be partially abrogated by cholesterol (B). (A) BC1 and BC2 were cultured in CSFM as described in the Figure 1 legend. The Sups were collected, serially diluted in CSFM, and used to treat HSAECs for 20 min. The viability was assessed with resazurin relative to unexposed cells. (B) Where indicated, cholesterol was added to microaerobic Sups at 10 μg mL-1 for 1 h. For each strain the data for two dilutions in the dynamic range of the assay are shown.
FIGURE 3
FIGURE 3
The activity of Sups results in cell membrane damage of the exposed HSAECs. Sups from microaerobic cultures of BC1 (A) and BC2 (B) in DMEM supplemented with 1 g L-1 of BSA were serially diluted with fresh medium and incubated with 10 μg mL-1 of cholesterol (ch) for 1 h at room temperature. Then 100 μl of Sups at the indicated dilutions were added to the monolayers of HSAECs and incubated for 20 min. The cell membrane permeability was assessed with the CytoTox-ONE Homogeneous Membrane Integrity kit (Promega) based on the release of LDH. Toxicity toward HSAECs was analyzed using resazurin. The viability and permeability corresponding assays were calculated as fluorescence relative to the untreated control or completely lysed cells, respectively.
FIGURE 4
FIGURE 4
The cultivation of B. cereus in medium supplemented with BSA enhances the cytotoxicity of Sups. DMEM was supplemented with or without 1 g L-1 of BSA and Sups were generated from 20-h microaerobic cultures. Dilutions were prepared with the medium used for growing cultures and their toxicity to HSAECs assessed in triplicates using resazurin.
FIGURE 5
FIGURE 5
CLO is inactivated in Sups concentrated in the absence of BSA. (A) Sups from BC1 microaerobic cultures grown in DMEM with or without 1 g L-1 of BSA were filtered using Amicon centrifugal filters with a 3 kDa cut-off pore size. The retentates were diluted with corresponding medium to the original volume and assayed for CLO activity in triplicates after incubation with or without 10 μg mL-1 cholesterol for 1 h at room temperature. (B) Retentates from filtration were run on SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA in PBST and reacted with rabbit polyclonal anti-streptolysin O antibody followed by an anti-rabbit IgG, HRP-linked antibody. Blots were developed using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific).
FIGURE 6
FIGURE 6
Succinic acid (SA) increases the toxicity of microaerobic Sups grown in medium containing BSA. Strain BC1 was inoculated into DMEM medium with 1 g L-1 of BSA and grown under microaerobic conditions for 20 h. Sups were diluted in DMEM titrated using HCl to the pH of Sups (5.4). After 20-min HSAECs exposure, Sups were removed and the cells were further incubated for 2 h at 37°C, 5% CO2 in DMEM supplemented with 0, 2, or 5 mM SA. After incubation, medium was removed and cell viability was assessed using resazurin. Controls included the same concentration of SA in the medium titrated to pH 5.4.
FIGURE 7
FIGURE 7
B. cereus produces reactive oxidizing species under microaerobic conditions. BC1 and BC2 microaerobic cultures were grown in DMEM with 1 g L-1 of BSA, 0.1 mM of AR reagent, and 0.2 U L-1 of HRP using 24-well plates (1 mL per well). Sups were collected every hour and their absorbance was read in triplicates, via spectrophotometer at 571 nm. The reaction mixture including all components except the bacterial inoculum was used as a control.
FIGURE 8
FIGURE 8
Ascorbic acid (AA) potentiates the toxicity of Sups in BSA-dependent manner. (A,B) Strains BC1 and BC2 were inoculated into DMEM medium with 1 g L-1 of BSA and grown under microaerobic conditions. Dilutions were made with medium titrated using HCl to the pH of grown cultures. Then, 100 mM stock of ascorbic acid in water was prepared and added to Sups at a concentration of 0, 0.5, 1, and 2 mM and incubated for 2 h at room temperature. The corresponding pH values were 5.42, 5.34, 5.28, and 5.08 in the case of BC1 and 5.35, 5.29, 5.11, and 4.96 in the case of BC2. HSAECs were exposed to the Sups for 20 min and viability was determined. (C) Strain BC1 was grown with and without BSA, and the effect of added AA was assessed as in (A) and (B). Controls represent viability of mock-treated HSAECs.
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References

    1. Andreeva Z. I., Nesterenko V. F., Yurkov I. S., Budarina Z. I., Sineva E. V., Solonin A. S. (2006). Purification and cytotoxic properties of Bacillus cereus hemolysin II. Protein Expr. Purif. 47 186–193. 10.1016/j.pep.2005.10.030 - DOI - PubMed
    1. Asano S. I., Nukumizu Y., Bando H., Iizuka T., Yamamoto T. (1997). Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis. Appl. Environ. Microbiol. 63 1054–1057. - PMC - PubMed
    1. Baida G. E., Kuzmin N. P. (1995). Cloning and primary structure of a new hemolysin gene from Bacillus cereus. Biochim. Biophys. Acta 1264 151–154. 10.1016/0167-4781(95)00150-F - DOI - PubMed
    1. Balavoine G. G. A., Geletii Y. V. (1999). Peroxynitrite scavenging by different antioxidants. Part I: convenient Assay. Nitric Oxide 3 40–54. 10.1006/niox.1999.0206 - DOI - PubMed
    1. Bendich A., Machlin L. J., Scandurra O., Burton G. W., Wayner D. D. M. (1986). The antioxidant role of vitamin C. Adv. Free Radic. Biol. Med. 2 419–444. 10.1016/S8755-9668(86)80021-7 - DOI

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