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Home / Equine Research Bank / Microbiol Spectr / Efficient and Scalable Process to Produce Novel and Highly B...
Microbiology spectrum2022; 10(4); e0235622; doi: 10.1128/spectrum.02356-22

Efficient and Scalable Process to Produce Novel and Highly Bioactive Purified Cytosolic Crystals from Bacillus thuringiensis.

Abstract: Bacillus thuringiensis (Bt) is a Gram-positive soil bacterium that is widely and safely applied in the environment as an insecticide for combatting insect pests that damage crops or are disease vectors. Dominant active ingredients made by Bt are insect-killing crystal (Cry) proteins released as crystalline inclusions upon bacterial sporulation. Some Bt Cry proteins, e.g., Cry5B (formally Cry5Ba1), target nematodes (roundworms) and show exceptional promise as anthelmintics (cures for parasitic nematode diseases). We have recently described inactivated bacteria with cytosolic crystal(s) (IBaCC) in which bioactive Bt Cry crystals (containing Cry5B) are fully contained within the cytosol of dead bacterial ghosts. Here, we demonstrate that these IBaCC-trapped Cry5B crystals can be liberated and purified away from cellular constituents, yielding purified cytosolic crystals (PCC). Cry5B PCC contains ~95% Cry5B protein out of the total protein content. Cry5B PCC is highly bioactive against parasitic nematode larvae and adults . Cry5B PCC is also highly active against experimental human hookworm and infections in rodents. The process was scaled up to the 100-liter scale to produce PCC for a pilot study to treat two foals infected with the ascarid spp. Single-dose Cry5B PCC brought the fecal egg counts of both foals to zero. These studies describe the process for the scalable production of purified Bt crystals and define a new and attractive pharmaceutical ingredient form of Bt Cry proteins. Bacillus thuringiensis crystal proteins are widely and safely used as insecticides. Recent studies have shown they also can cure gastrointestinal parasitic worm (nematode) infections when ingested. However, reproducible, scalable, and practical techniques for purifying these proteins have been lacking. Here, we address this severe limitation and present scalable and practical methods for large-scale purification of potently bioactive B. thuringiensis crystals and crystal proteins. The resultant product, called purified cytosolic crystals (PCC), is highly compatible with ingestible drug delivery and formulation. Furthermore, there are growing applications in agriculture and insect control where access to large quantities of purified crystal proteins is desirable and where these methods will find great utility.
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Publication Date: 2022-08-10 PubMed ID: 35946940PubMed Central: PMC9430767DOI: 10.1128/spectrum.02356-22Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • N.I.H.
  • Extramural
  • Research Support
  • U.S. Gov't
  • Non-P.H.S.

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research article explores the efficient production of purified cytosolic crystals from the bacterium Bacillus thuringiensis, demonstrating its effectiveness against parasitic worms and potential as a pharmaceutical ingredient.

Research Objective and Methodology

  • The objective of this research was to create a scalable process to produce purified crystals from the bacterium Bacillus thuringiensis (Bt), which are highly bioactive against parasitic nematodes (roundworms).
  • This process included the liberation and purification of Cry5B crystals from the cytosol of inactive bacterial ghosts. Cry proteins from Bt are known for their insecticidal properties.
  • The researchers focused on Cry5B proteins, which specifically target nematodes and can be used as anthelmintics or cures for nematode parasites.

Findings and Implications

  • The researchers have successfully demonstrated that Cry5B cytosolic crystals (named as purified cytosolic crystals – PCC), which are ~95% Cry5B protein, can be efficiently extracted and purified.
  • Tests against parasitic nematode larvae and adults found the Cry5B PCC to be highly bioactive, with promising implications for the treatment of human hookworm infections and other parasitic diseases.
  • When the process was scaled up, Cry5B PCC succeeded in reducing to zero the fecal egg counts of two foals infected with ascarids, a type of parasitic worm.
  • These findings are significant because previously there has been a lack of scalable, practical techniques for purifying these proteins. The method demonstrated here paves the way for a new form of Bt Cry proteins that can be useful for pharmaceutical and agricultural applications.

Future Directions

  • This study suggests that purified Bt crystals could be an essential pharmaceutical ingredient to cure parasitic nematode diseases, as well as a tool for agricultural pest control.
  • The resultant product, called purified cytosolic crystals (PCC), has a strong potential for ingestible drug delivery and formulation.

Cite This Article

APA
Chicca J, Cazeault NR, Rus F, Abraham A, Garceau C, Li H, Atwa SM, Flanagan K, Soto ER, Morrison MS, Gazzola D, Hu Y, Liu DR, Nielsen MK, Urban JF, Ostroff GR, Aroian RV. (2022). Efficient and Scalable Process to Produce Novel and Highly Bioactive Purified Cytosolic Crystals from Bacillus thuringiensis. Microbiol Spectr, 10(4), e0235622. https://doi.org/10.1128/spectrum.02356-22

Publication

Microbiology spectrum
ISSN: 2165-0497
NlmUniqueID: 101634614
Country: United States
Language: English
Volume: 10
Issue: 4
Pages: e0235622
PII: e02356-22

Researcher Affiliations

Chicca, Jeffrey
  • Program in Molecular Medicine, UMASS Chan Medical School, Worcester, Massachusetts, USA.
Cazeault, Nicholas R
  • Program in Molecular Medicine, UMASS Chan Medical School, Worcester, Massachusetts, USA.
Rus, Florentina
  • Program in Molecular Medicine, UMASS Chan Medical School, Worcester, Massachusetts, USA.
Abraham, Ambily
  • Program in Molecular Medicine, UMASS Chan Medical School, Worcester, Massachusetts, USA.
Garceau, Carli
  • Program in Molecular Medicine, UMASS Chan Medical School, Worcester, Massachusetts, USA.
Li, Hanchen
  • Program in Molecular Medicine, UMASS Chan Medical School, Worcester, Massachusetts, USA.
Atwa, Samar M
  • M.H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, USA.
Flanagan, Kelly
  • Program in Molecular Medicine, UMASS Chan Medical School, Worcester, Massachusetts, USA.
Soto, Ernesto R
  • Program in Molecular Medicine, UMASS Chan Medical School, Worcester, Massachusetts, USA.
Morrison, Mary S
  • Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
Gazzola, David
  • Program in Molecular Medicine, UMASS Chan Medical School, Worcester, Massachusetts, USA.
Hu, Yan
  • Program in Molecular Medicine, UMASS Chan Medical School, Worcester, Massachusetts, USA.
Liu, David R
  • Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
Nielsen, Martin K
  • M.H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, USA.
Urban, Joseph F
  • U.S. Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal and Parasitic Diseases Laboratory, Beltsville, Maryland, USA.
  • U.S. Department of Agriculture, Agricultural Research Service, Beltsville Human Nutrition Research Center, Diet, Genomics and Immunology Laboratory, Beltsville, Maryland, USA.
Ostroff, Gary R
  • Program in Molecular Medicine, UMASS Chan Medical School, Worcester, Massachusetts, USA.
Aroian, Raffi V
  • Program in Molecular Medicine, UMASS Chan Medical School, Worcester, Massachusetts, USA.

MeSH Terms

  • Animals
  • Anthelmintics / therapeutic use
  • Bacillus thuringiensis
  • Bacterial Proteins
  • Cytosol
  • Horses
  • Humans
  • Nematoda
  • Pilot Projects

Grant Funding

  • R01 AI056189 / NIAID NIH HHS
  • R01 AI150866 / NIAID NIH HHS

Conflict of Interest Statement

The authors declare no conflict of interest.

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Citations

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