scholarly journals The Pore-Forming Hemolysin BL Enterotoxin from Bacillus cereus: Subunit Interactions in Cell-Free Systems

Toxins ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 807
Author(s):  
Franziska Ramm ◽  
Marlitt Stech ◽  
Anne Zemella ◽  
Hendrik Frentzel ◽  
Stefan Kubick
Keyword(s):  
Bacillus Cereus ◽  
Chinese Hamster ◽  
Iodide Uptake ◽  
Cytotoxic Effects ◽  
Free System ◽  
Multiple Protein ◽  
Uptake Assay ◽  
Membrane Bound ◽  
Hemolysin Bl ◽  
Cell Free Protein Synthesis

The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by Bacillus cereus. Previous studies have described the formation of the Hbl complex and aimed to identify the toxin’s mode of action. In this study, we analyzed the assembly of Hbl out of its three individual subunits L1, L2 and B in a soluble as well as a putative membrane bound composition using a Chinese hamster ovary (CHO) cell-free system. Subunits were either coexpressed or synthesized individually in separate cell-free reactions and mixed together afterwards. Hemolytic activity of cell-free synthesized subunits was demonstrated on 5% sheep blood agar and identified both synthesis procedures, coexpression as well as individual synthesis of each subunit, as functional for the synthesis of an active Hbl complex. Hbl’s ability to perforate cell membranes was evaluated using a propidium iodide uptake assay. These data suggested that coexpressed Hbl subunits augmented cytotoxic activity with increasing concentrations. Further, a pre-pore-complex of L1-L2 showed cytotoxic effects suggesting the possibility of an interaction between the cell membrane and the pre-pore-complex. Overall, this study shows that cell-free protein synthesis is a fast and efficient way to study the assembly of multiple protein subunits in soluble as well as vesicular fractions.

Comprehensive bioinformatics analysis of cell‐free protein synthesis: identification of multiple protein properties that correlate with successful expression

2009 ◽  
Vol 24 (4) ◽  
pp. 1095-1104 ◽  
Author(s):  
Atsushi Kurotani ◽  
Tetsuo Takagi ◽  
Mitsutoshi Toyama ◽  
Mikako Shirouzu ◽  
Shigeyuki Yokoyama ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Bioinformatics Analysis ◽  
Free Protein ◽  
Multiple Protein ◽  
Protein Properties ◽  
Cell Free Protein Synthesis ◽  
Successful Expression

Growth, Germination and Toxigenic Activity of Bacillus cereus in Milk Products

1988 ◽  
Vol 51 (9) ◽  
pp. 707-710 ◽  
Author(s):  
H. C. WONG ◽  
Y. L. CHEN ◽  
C. L. F. CHEN
Keyword(s):  
Bacillus Cereus ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fermented Milk ◽  
Vegetative Cells ◽  
Ovary Cells ◽  
Milk Products ◽  
Pasteurized Milk ◽  
Sterilized Milk ◽  
Reconstituted Milk

Growth, germination and toxigenic activity of Bacillus cereus in pasteurized milk, fruit-flavored reconstituted milk and fermented milk at 30°C were studied. Vegetative cells of B. cereus rapidly increased to 108 CFU/ml in 8 h and 106 CFU/ml in 12 h in pasteurized milk and fruit-flavored reconstituted milk respectively. In fermented milk, vegetative cells of B. cereus were killed or inactivated in 40 min; while about 31% of spores survived the 7 d test period. Spore germination of B. cereus was inhibited by fruit-flavored reconstituted milk and fermented milk. Chinese hamster ovary cells were transformed and/or lysed by pasteurized milk and sterilized milk with B. cereus cultured for 8 h and also by laboratory-prepared fermented milk with B. cereus introduced at the beginning of fermentation an incubated for 12 or 72 h.

scholarly journals Novel Effective Bacillus cereus Group Species “Bacillus clarus” Is Represented by Antibiotic-Producing Strain ATCC 21929 Isolated from Soil

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Marysabel Méndez Acevedo ◽  
Laura M. Carroll ◽  
Manjari Mukherjee ◽  
Emma Mills ◽  
Lingzi Xiaoli ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Hela Cells ◽  
Species Complex ◽  
Taxonomic Classification ◽  
Phenotypic Characterization ◽  
Pathogenic Potential ◽  
Cytotoxic Effects ◽  
Content Type ◽  
Group Species

ABSTRACT Gram-positive, spore-forming members of the Bacillus cereus group species complex are widespread in natural environments and display various degrees of pathogenicity. Recently, B. cereus group strain Bacillus mycoides Flugge ATCC 21929 was found to represent a novel lineage within the species complex, sharing a relatively low degree of genomic similarity with all B. cereus group genomes (average nucleotide identity [ANI] < 88). ATCC 21929 has been previously associated with the production of a patented antibiotic, antibiotic 60-6 (i.e., cerexin A); however, the virulence potential and growth characteristics of this lineage have never been assessed. Here, we provide an extensive genomic and phenotypic characterization of ATCC 21929, and we assess its pathogenic potential in vitro. ATCC 21929 most closely resembles Bacillus paramycoides NH24A2T (ANI and in silico DNA-DNA hybridization values of 86.70 and 34.10%, respectively). Phenotypically, ATCC 21929 does not possess cytochrome c oxidase activity and is able to grow at a range of temperatures between 15 and 43°C and a range of pH between 6 and 9. At 32°C, ATCC 21929 shows weak production of diarrheal enterotoxin hemolysin BL (Hbl) but no production of nonhemolytic enterotoxin (Nhe); at 37°C, neither Hbl nor Nhe is produced. Additionally, at 37°C, ATCC 21929 does not exhibit cytotoxic effects toward HeLa cells. With regard to fatty acid composition, ATCC 21929 has iso-C17:0 present in highest abundance. Based on the characterization provided here, ATCC 21929T (= PS00077AT = PS00077BT = PSU-0922T = BHPT) represents a novel effective B. cereus group species, which we propose as effective species “Bacillus clarus.” IMPORTANCE The B. cereus group comprises numerous closely related lineages with various degrees of pathogenic potential and industrial relevance. Species-level taxonomic classification of B. cereus group strains is important for risk evaluation and communication but remains challenging. Biochemical and phenotypic assays are often used to assign B. cereus group strains to species but are insufficient for accurate taxonomic classification on a genomic scale. Here, we show that antibiotic-producing ATCC 21929 represents a novel lineage within the B. cereus group that, by all metrics used to delineate prokaryotic species, exemplifies a novel effective species. Furthermore, we show that ATCC 21929 is incapable of producing enterotoxins Hbl and Nhe or exhibiting cytotoxic effects on HeLa cells at human body temperature in vitro. These results provide greater insight into the genomic and phenotypic diversity of the B. cereus group and may be leveraged to inform future public health and food safety efforts.

scholarly journals Exposure to Bacillus cereus in Water Buffalo Mozzarella Cheese

Foods ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1899
Author(s):  
Angela Michela Immacolata Montone ◽  
Federico Capuano ◽  
Andrea Mancusi ◽  
Orlandina Di Maro ◽  
Maria Francesca Peruzy ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Dairy Products ◽  
Water Buffalo ◽  
Diarrheal Disease ◽  
Food Poisoning ◽  
Mozzarella Cheese ◽  
Food Borne ◽  
Genes Encoding ◽  
Spoilage Bacterium ◽  
Hemolysin Bl

Bacillus cereus is a spoilage bacterium and is recognized as an agent of food poisoning. Two food-borne illnesses are caused by B. cereus: a diarrheal disease, associated with cytotoxin K, hemolysin BL, non-hemolytic enterotoxin and enterotoxin FM, and an emetic syndrome, associated with the cereulide toxin. Owing to the heat resistance of B. cereus and its ability to grow in milk, this organism should be considered potentially hazardous in dairy products. The present study assessed the risk of B. cereus poisoning due to the consumption of water buffalo mozzarella cheese. A total of 340 samples were analyzed to determine B. cereus counts (ISO 7932:2005); isolates underwent molecular characterization to detect the presence of genes encoding toxins. Eighty-nine (26.1%) samples harbored B. cereus strains, with values ranging from 2.2 × 102 to 2.6 × 106 CFU/g. Isolates showed eight different molecular profiles, and some displayed virulence characteristics. Bacterial counts and the toxin profiles of isolates were evaluated both separately and jointly to assess the risk of enteritis due to B. cereus following the consumption of buffalo mozzarella cheese. In conclusion, the results of the present study showed that the risk of poisoning by B. cereus following the consumption of this cheese was moderate.

Increasing membrane-bound MCSF does not enhance OPGL-driven osteoclastogenesis from marrow cells

2001 ◽  
Vol 280 (1) ◽  
pp. E103-E111 ◽  
Author(s):  
X. Fan ◽  
D. Fan ◽  
H. Gewant ◽  
C. L. Royce ◽  
M. S. Nanes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chinese Hamster ◽  
Osteoclast Formation ◽  
Local Bone ◽  
Cell Layers ◽  
Membrane Bound ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Osteoprotegerin Ligand ◽  
Fixed Cell

Macrophage colony-stimulating factor (MCSF) and osteoprotegerin ligand (OPGL), both produced by osteoblasts/stromal cells, are essential factors for osteoclastogenesis. Whether local MCSF levels regulate the amount of osteoclast formation is unclear. Two culture systems, ST-2 and Chinese hamster ovary-membrane-bound MCSF (CHO-mMCSF)-Tet-OFF cells, were used to study the role of mMCSF in osteoclast formation. Cells from bone marrow (BMM) or spleen were cultured with soluble OPGL on glutaraldehyde-fixed cell layers; osteoclasts formed after 7 days. Osteoclast number was proportional to the amount of soluble OPGL added. In contrast, varying mMCSF levels in the ST-2 or CHO-mMCSF-Tet-OFF cell layers, respectively by variable plating or by addition of doxycycline, did not affect BMM osteoclastogenesis: 20–450 U of mMCSF per well generated similar osteoclast numbers. In contrast, spleen cells were resistant to mMCSF: osteoclastogenesis required ≥250 U per well and further increased as mMCSF rose higher. Our results demonstrate that osteoclast formation in the local bone environment is dominated by OPGL. Increasing mMCSF above basal levels does not further enhance osteoclast formation from BMMs, indicating that mMCSF does not play a dominant regulatory role in the bone marrow.

scholarly journals Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1082 ◽  
Author(s):  
Gallardo-Vara ◽  
Ruiz-Llorente ◽  
Casado-Vela ◽  
Ruiz-Rodríguez ◽  
López-Andrés ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Membrane Glycoproteins ◽  
Ovary Cells ◽  
Auxiliary Component ◽  
Extracellular Region ◽  
Galectin 3 ◽  
Human Proteins ◽  
Membrane Bound ◽  
New Research

Endoglin is a 180-kDa glycoprotein receptor primarily expressed by the vascular endothelium and involved in cardiovascular disease and cancer. Heterozygous mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia type 1, a vascular disease that presents with nasal and gastrointestinal bleeding, skin and mucosa telangiectases, and arteriovenous malformations in internal organs. A circulating form of endoglin (alias soluble endoglin, sEng), proteolytically released from the membrane-bound protein, has been observed in several inflammation-related pathological conditions and appears to contribute to endothelial dysfunction and cancer development through unknown mechanisms. Membrane-bound endoglin is an auxiliary component of the TGF-β receptor complex and the extracellular region of endoglin has been shown to interact with types I and II TGF-β receptors, as well as with BMP9 and BMP10 ligands, both members of the TGF-β family. To search for novel protein interactors, we screened a microarray containing over 9000 unique human proteins using recombinant sEng as bait. We find that sEng binds with high affinity, at least, to 22 new proteins. Among these, we validated the interaction of endoglin with galectin-3, a secreted member of the lectin family with capacity to bind membrane glycoproteins, and with tripartite motif-containing protein 21 (TRIM21), an E3 ubiquitin-protein ligase. Using human endothelial cells and Chinese hamster ovary cells, we showed that endoglin co-immunoprecipitates and co-localizes with galectin-3 or TRIM21. These results open new research avenues on endoglin function and regulation.

A nonradioactive iodide uptake assay for sodium iodide symporter function

2010 ◽  
Vol 396 (1) ◽  
pp. 91-95 ◽  
Author(s):  
Fanny Waltz ◽  
Lucie Pillette ◽  
Yves Ambroise
Keyword(s):  
Sodium Iodide ◽  
Sodium Iodide Symporter ◽  
Iodide Uptake ◽  
Uptake Assay

Regulation of Rho protein binding to membranes by rhoGDI: inhibition of releasing activity by physiological ionic conditions

1999 ◽  
Vol 77 (1) ◽  
pp. 59-69 ◽  
Author(s):  
Diane Bilodeau ◽  
Sylvie Lamy ◽  
Richard R Desrosiers ◽  
Denis Gingras ◽  
Richard Béliveau
Keyword(s):  
Ionic Strength ◽  
Brush Border ◽  
Rat Kidney ◽  
Regulatory Protein ◽  
Rho Proteins ◽  
Glutathione S Transferase ◽  
Free System ◽  
Brush Border Membranes ◽  
A Cell ◽  
Membrane Bound

The Rho GDP dissociation inhibitor (GDI) is an ubiquitously expressed regulatory protein involved in the cycling of Rho proteins between membrane-bound and soluble forms. Here, we characterized the Rho solubilization activity of a glutathione S-transferase (GST) - GDI fusion protein in a cell-free system derived from rat kidney. Addition of GST-GDI to kidney brush border membranes resulted in the specific release of Cdc42 and RhoA from the membranes, while RhoB and Ras were not extracted. The release of Cdc42 and RhoA by GST-GDI was dose dependent and saturable with about 50% of both RhoA and Cdc42 extracted. The unextracted Rho proteins were tightly bound to membranes and could not be solubilized by repeated GST-GDI treatment. These results demonstrated that kidney brush border membranes contained two populations of RhoA and Cdc42. Furthermore, the GST-GDI solubilizing activity on membrane-bound Cdc42 and RhoA was abolished at physiological conditions of salt and temperature in all tissues examined. When using bead-immobilized GST-GDI, KCl did not reduced the binding of Rho proteins. However, washing brush border membranes with KCl prior treatment by GST-GDI inhibited the extraction of Rho proteins. Taken together, these results suggest that the binding of GDI to membrane-bound Cdc42 and RhoA occurs easily under physiological ionic strength conditions, but a complementary factor is required to extract these proteins from membranes. These observations suggest that the shuttling activity of GDI upon Rho proteins could be normally downregulated under physiological conditions.Key words: rhoGDI, rho proteins, ionic strength, kidney.

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