Letter to the Editor | Volume: 12, Issue: 4, April, 2022

Evacuation of yeast and fungal cells using the Sponge-Like Protocol: A new approach for fungal control

Amro Abd Al Fattah Amara Nawal Abd El-Baky   

Open Access   

Published:  Apr 05, 2022

DOI: 10.7324/JAPS.2022.120421
Abstract

No abstract available.


Keyword:     Evacuation yeast fungal Sponge


Citation:

Amara AAAF, El-Baky NA. Evacuation of yeast and fungal cells using the Sponge-Like Protocol: A new approach for fungal control. J Appl Pharm Sci, 2022; 12(04):185–186.

Copyright: © The Author(s). This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Dear Editor,

A protocol named Sponge-Like Protocol was introduced in 2013 for evacuating Escherichia coli cells (Amara et al., 2013a, 2013b, 2014a).

The first major challenge was to determine the best compounds that are able to induce pores in the bacterial cell wall and degrade their genetic material. Four main compounds were collected. They are sodium dodecyle sulphate (SDS), NaOH, CaCO3, and H2O2. SDS, NaOH, and H2O2 are active compounds that affect cell macromolecules and cell wall. CaCO3 facilitates the process of transferring these compounds into the cells.

The second major challenge was how to calculate the correct concentrations that induce evacuation without damaging bacterial cell wall or their 3D structure. The minimum inhibitory concentration (MIC) and the minimum growth concentration (MGC) of each of the used chemical compounds were calculated. For optimizing the evacuation process, successive washing using 0.5% NaCl and 60% ethanol has been conducted to remove the cytoplasmic content released due to evacuation. Gentle centrifugation process and shaking were used to enhance the evacuation process. From 12 experiments that represent the Plackett–Burman experimental randomization design using E. coli BL21, only a single colony survived. In fact, E. coli BL21 was selected because it contains a plasmid that carries the lysozyme gene. In the first design, such success was not expected, and lysozyme was expected to be complementary to the protocol. Thus, the whole cells from the experiment that showed a single surviving colony were subjected to a killing mechanism via the activation of the lysozyme gene (Amara et al., 2013a). Then, the protocol was applied to another E. coli strain, E. coli JM109 (Amara et al., 2014a). This strain was so sensitive to the protocol, and MIC/MGC values determined were different from those of E. coli BL21. Since Plackett–Burman design is a complicated statistical randomization design, the protocol was simplified and reduced to a reduced design that still involves the best two experiments obtained from the original protocol (Amara et al., 2013b). The prepared ghost cells from each experiment were examined using a spectrophotometer at 260 and 280 nm to determine the concentration of released genetic material or protein as an indication for the quality of the evacuation process. Agarose gel was used to investigate the existence of DNA, which is an indicator of the release of cytoplasmic content. The cells’ quality was investigated using both light and electron microscopes. It becomes clear that the protocol succeeded in evacuating E. coli BL21 and JM109 using the MIC/MGC combination and successive steps that avoid the interaction of the different compounds with each other and involving washing and centrifugation steps that enhance the cells’ evacuation.

The third major challenge was to investigate the quality of the surface antigens. For that purpose, a pathogenic microbe, Salmonella typhimurium ATCC 14028, was used. The prepared ghost cells showed positive interaction against S. typhimurium antibody obtained from the market. Also, the serum obtained from immunized rats showed positive results against S. typhimurium ATCC 14028 in the hemagglutination test (Amara et al., 2014b). It becomes clear that the surface antigens of ghost cells prepared using the Sponge-Like Protocol are able to show antigenicity against the specific antibody and to induce a humoral immune response in the rats (Amara et al., 2014b).

The fourth major challenge was to conduct a full immunization map for both humoral and cell-mediated immunization. Klebsiella pneumoniae was used for this purpose, and the results demonstrated that the prepared ghost cells showed immunization for humoral and cell-mediated immune response (Menisy et al., 2017). Later, the same results were obtained using Acinetobacter baumannii (Sheweita et al., 2019). Other experiments were conducted using immunization with different ghost cells and then inducing immunocompromisation in rats, followed by immunization with a virulent pathogen. The immunocompromised rats have defense ability against the virulent pathogen in the challenge test.

The fifth major challenge was to apply this protocol to Gram-positive microbes. The same concept was used, but instead of using chemical compounds, a white egg that contains the lysozyme was used against a Gram-positive microbe that lives in harsh environments. Lysozyme and proteinase K have been used to evacuate Bacillus stearothermophilus (spore-forming bacteria) (Amara, 2016). The protocol succeeded in evacuating B. stearothermophilus. Another report has used the original protocol to prepare ghost cells from Listeria monocytogenes (Wu et al., 2017).

The sixth major challenge was to apply the protocol or part of it to evacuate microbes outside the prokaryotic kingdom. The first was Newcastle disease virus (Lasota strain) (El-Baky and Amara, 2014), and the second was Pestikal Lasota viruses (Amara, 2020). The virus, which has an acellular structure, was treated only by H2O2 at concentrations used in E. coli evacuation. The study proved successful evacuation of the virus from its RNA (El-Baky and Amara, 2014). The yeast cell represented by Saccharomyces cerevisiae was also evacuated (Amara, 2015b), and the evacuated cells were further processed to be used as a drug delivery system for the gossypol acetic acid (Amara, 2015a). The main change in protocol was using NaHCO3 instead of CaCO3. NaHCO3 is more suitable for eukaryotic microbial cell wall. The evacuation of S. cerevisiae encourages the application of the protocol on filamentous fungi.

Recently, the protocol has shown success to evacuate fungal cells of Aspergillus flavus and Aspergillus niger (El-Baky et al., 2021, 2018a, 2018b), in addition to the spores of oyster mushrooms, which were evacuated as well (Haddad et al., 2019). The randomization of the conditions highlights the effect of each of the used chemicals at either MIC or MGC value. SDS is responsible for the destabilization of cell wall/plasma membrane and pore formation in the fungal cell wall, while H2O2 is responsible for DNA degradation. The protocol was investigated to control the growth of both A. flavus and A. niger on jojoba tissue culture as a preliminary trial to control fungal growth on plants (El-Baky et al., 2021). This was the first trial on plant using this protocol. The Sponge-Like Protocol has succeeded in killing the fungal cells that have been sprayed on the jojoba tissue culture as well as on the surface of growth media (compared with the control plants).

This protocol can be applied to control human fungal pathogens and diseases associated with fungal spores and protect in vitro tissue culture of plants from fungal infections or even may be developed to formulate chemical sprays to treat plants in field to control different plant fungal diseases.

Other scientific groups have found the protocol interesting and used it to evacuate or to kill some targeted microbes (Vinod et al., 2014; Wu et al., 2017). Others considered it one of the suggested protocols to develop vaccine trials (Batah and Ahmad, 2020) and to be used in drug delivery (Alanazi et al., 2020).

We want to draw the attention of the scientific community to the application of the Sponge-Like Protocol to evacuate and control fungal pathogens.


REFERENCES

Alanazi FK, Alsuwyeh AA, Haq N, Salem-Bekhit MM, Al-Dhfyan A, Shakeel F. Vision of bacterial ghosts as drug carriers mandates accepting the effect of cell membrane on drug loading. Drug Dev Ind Pharm, 2020; 46(10):1716–25. CrossRef

Amara AA. Bacterial and yeast ghosts: E. coli and Saccharomyces cerevisiae preparation as drug delivery model. Int Sci Invest J, 2015a; 4(7):11–22.

Amara AA. Saccharomyces cerevisiae ghosts using the sponge-like re-reduced protocol. SOJ Biochem, 2015b; 2(1):4. CrossRef

Amara AA. The critical activity for the cell wall degrading enzymes: could the use of the lysozyme for microbial ghosts preparation establish emergence oral vaccination protocol? Int Sci Invest J, 2016; 5(2):351–69.

Amara AA. E. coli BL21 bacterial and Newcastle Pestikal lasota viral ghosts preparation, DNA, RNA elimination/purification/isolation/quantification using agarose gel entrapment bubble method. J Protein Res Bioinf, 2020; 2:010 CrossRef

Amara AA, Salem-Bekhit MM, Alanazi FK. Preparation of bacterial ghosts for E. coli JM109 using sponge-like reduced protocol. Asian J Biol Sci, 2013a; 6(8):363–9. CrossRef

Amara AA, Salem-Bekhit MM, Alanazi FK. Sponge-like: a new protocol for preparing bacterial ghosts. Sci World J, 2013b; 2013:545741. CrossRef

Amara AA, Salem-Bekhit MM, Alanazi FK. Plackett–Burman randomization method for bacterial ghosts preparation form E. coli JM109. Saudi Pharm J, 2014a; 22(3):273–9. CrossRef

Amara AA, Neama AJ, Hussein A, Hashish EA, Sheweita SA. Evaluation the surface antigen of the Salmonella typhimurium ATCC 14028 ghosts prepared by “SLRP.” Sci World J, 2014b; 2014:840863. CrossRef

Batah AM, Ahmad TA. The development of ghost vaccines trials. Expert Rev Vaccines, 2020; 19(6):549–62. CrossRef

El-Baky NA, Abdel Rahman RA, Sharaf MM., Amara AA. The development of a phytopathogenic fungi control trial: Aspergillus flavus and Aspergillus niger infection in Jojoba tissue culture as a model. Sci World J, 2021; 2021:Article ID 6639850. CrossRef

El-Baky NA, Amara AA. Newcastle disease virus (LaSota strain) as a model for virus ghosts preparation using H2O2 bio-critical concentration. Int Sci Invest J, 2014; 3(5):38–50.

El-Baky NA, Sharaf MM, Amer E, Kholef HR, Hussain MZ, Abd El Rahman R, Amara AA. The minimum inhibition and growth concentration for controlling fungal infection as well as for ghost cells preparation: Aspergillus flavus as a model. Biomed J Sci Tech Res, 2018a; 10(2):1–5.

El-Baky NA, Sharaf MM, Amer E, Kholef HR, Hussain MZ, Amara AA. Protein and DNA isolation from Aspergillus niger as well as ghost cells formation. SOJ Biochem, 2018b; 4(1):1–7. CrossRef

Haddad A, Sharaf MM, Kenawy AMA, Amara AA. Oyster mushroom spores ghost preparation for medicinal, biotechnological and forensic applications. Biomed J Sci Tech Res, 2019; 24(1):2019. CrossRef

Menisy MM, Hussein A, Ghazy AA, Sheweita S, Amara AA. Klebsiella pneumoniae ghosts as vaccine using sponge like reduced protocol. Cell Mol Med, 2017; 3(2):11.

Sheweita SA, Batah AM, Ghazy AA, Hussein A, Amara AA. A new strain of Acinetobacter baumannii and characterization of its ghost as a candidate vaccine. J Infect Public Health, 2019; 12(6):831–42. CrossRef

Vinod N, Oh S, Kim S, Choi CW, Kim SC, Jang CH. Chemically induced Salmonella enteritidis ghosts as a novel vaccine candidate against virulent challenge in a rat model. Vaccine, 2014; 32(26):3249–55. CrossRef

Wu X, Ju X, Du L, Yuan J, Wang L, He R, Chen Z. Production of bacterial ghosts from Gram-positive pathogen Listeria monocytogenes. Foodborne Pathog Dis, 2017; 14(1):1–7. CrossRef

Reference

Alanazi FK, Alsuwyeh AA, Haq N, Salem-Bekhit MM, Al- Dhfyan A, Shakeel F. Vision of bacterial ghosts as drug carriers mandates accepting the effect of cell membrane on drug loading. Drug Dev Ind Pharm, 2020; 46(10):1716-25. https://doi.org/10.1080/03639045.2020.1820039

Amara AA. Bacterial and yeast ghosts: E. coli and Saccharomyces cerevisiae preparation as drug delivery model. Int Sci Invest J, 2015a; 4(7):11-22.

Amara AA. Saccharomyces cerevisiae ghosts using the sponge-like re-reduced protocol. SOJ Biochem, 2015b; 2(1):4. https://doi.org/10.15226/2376-4589/2/1/00107

Amara AA. The critical activity for the cell wall degrading enzymes: could the use of the lysozyme for microbial ghosts preparation establish emergence oral vaccination protocol? Int Sci Invest J, 2016; 5(2):351-69.

Amara AA. E. coli BL21 bacterial and Newcastle Pestikal lasota viral ghosts preparation, DNA, RNA elimination/purification/isolation/ quantification using agarose gel entrapment bubble method. J Protein Res Bioinf, 2020; 2:010 https://doi.org/10.24966/PRB-1545/100010

Amara AA, Salem-Bekhit MM, Alanazi FK. Preparation of bacterial ghosts for E. coli JM109 using sponge-like reduced protocol. Asian J Biol Sci, 2013a; 6(8):363-9. https://doi.org/10.3923/ajbs.2013.363.369

Amara AA, Salem-Bekhit MM, Alanazi FK. Sponge-like: a new protocol for preparing bacterial ghosts. Sci World J, 2013b; 2013:545741. https://doi.org/10.1155/2013/545741

Amara AA, Salem-Bekhit MM, Alanazi FK. Plackett-Burman randomization method for bacterial ghosts preparation form E. coli JM109. Saudi Pharm J, 2014a; 22(3):273-9. https://doi.org/10.1016/j.jsps.2013.06.002

Amara AA, Neama AJ, Hussein A, Hashish EA, Sheweita SA. Evaluation the surface antigen of the Salmonella typhimurium ATCC 14028 ghosts prepared by "SLRP." Sci World J, 2014b; 2014:840863. https://doi.org/10.1155/2014/840863

Batah AM, Ahmad TA. The development of ghost vaccines trials. Expert Rev Vaccines, 2020; 19(6):549-62. https://doi.org/10.1080/14760584.2020.1777862

El-Baky NA, Abdel Rahman RA, Sharaf MM., Amara AA. The development of a phytopathogenic fungi control trial: Aspergillus flavus and Aspergillus niger infection in Jojoba tissue culture as a model. Sci World J, 2021; 2021:Article ID 6639850. https://doi.org/10.1155/2021/6639850

El-Baky NA, Amara AA. Newcastle disease virus (LaSota strain) as a model for virus ghosts preparation using H2O2 bio-critical concentration. Int Sci Invest J, 2014; 3(5):38-50.

El-Baky NA, Sharaf MM, Amer E, Kholef HR, Hussain MZ, Abd El Rahman R, Amara AA. The minimum inhibition and growth concentration for controlling fungal infection as well as for ghost cells preparation: Aspergillus flavus as a model. Biomed J Sci Tech Res, 2018a; 10(2):1-5.

El-Baky NA, Sharaf MM, Amer E, Kholef HR, Hussain MZ, Amara AA. Protein and DNA isolation from Aspergillus niger as well as ghost cells formation. SOJ Biochem, 2018b; 4(1):1-7. https://doi.org/10.15226/2376-4589/4/1/00129

Haddad A, Sharaf MM, Kenawy AMA, Amara AA. Oyster mushroom spores ghost preparation for medicinal, biotechnological and forensic applications. Biomed J Sci Tech Res, 2019; 24(1):2019. https://doi.org/10.26717/BJSTR.2019.24.003994

Menisy MM, Hussein A, Ghazy AA, Sheweita S, Amara AA. Klebsiella pneumoniae ghosts as vaccine using sponge like reduced protocol. Cell Mol Med, 2017; 3(2):11.

Sheweita SA, Batah AM, Ghazy AA, Hussein A, Amara AA. A new strain of Acinetobacter baumannii and characterization of its ghost as a candidate vaccine. J Infect Public Health, 2019; 12(6):831-42. https://doi.org/10.1016/j.jiph.2019.05.009

Vinod N, Oh S, Kim S, Choi CW, Kim SC, Jang CH. Chemically induced Salmonella enteritidis ghosts as a novel vaccine candidate against virulent challenge in a rat model. Vaccine, 2014; 32(26):3249-55. https://doi.org/10.1016/j.vaccine.2014.03.090

Wu X, Ju X, Du L, Yuan J, Wang L, He R, Chen Z. Production of bacterial ghosts from Gram-positive pathogen Listeria monocytogenes. Foodborne Pathog Dis, 2017; 14(1):1-7. https://doi.org/10.1089/fpd.2016.2184

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