Talaromyces marneffei Cu, Zn Superoxide Dismutase Recombinant Protein Expression in Pichia pastoris, Enzymatic Activity and Its Resistance to Oxidative Stress

Sophit Khanthawong; Kanruethai Wongsawan; Ronachai Pratanaphon; Nongnuch Vanittanakom

doi:10.69650/ahstr.2024.1570

Authors

Sophit Khanthawong Department of Microbiology and Parasitology, Faculty of Medical Science, Centre of Excellence in Fungal Research (CEFR) and Center of Excellence in Medical Biotechnology (CEMB) Naresuan University, Phitsanulok, 65000, Thailand
Kanruethai Wongsawan Division of Veterinary Paraclinic, Department of Veterinary Bioscience and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, 50100, Thailand
Ronachai Pratanaphon Division of Biotechnology, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai, 50100, Thailand
Nongnuch Vanittanakom Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand

DOI:

https://doi.org/10.69650/ahstr.2024.1570

Keywords:

Talaromyces marneffei, Cu, Zn SOD, recombinant protein, antioxidant activity, oxidative stress

Abstract

Talaromyces marneffei is a dimorphic fungus that is known to cause a disease called talaromycosis, also known as penicilliosis, in immunocompromised individuals. The fungal pathogenicity and virulence factors remain unclear. Superoxide dismutase (SOD) is a neutralizing enzyme through reactive oxygen species generated by the host and has been proven to contribute to the virulence of many pathogenic bacteria and fungi. In this study, full-length sodA gene encoding T. marneffei Cu, Zn SOD was amplified, cloned into pPICzαB vector and successfully integrated into the Pichia pastoris yeast genome. The selected positive clone was induced for protein expression by methanol. An approximately 23 kDa molecular mass of secreted recombinant Cu, Zn SOD is enzymatically active which is like the native and standard enzyme. A rabbit polyclonal antibody raised against recombinant Cu, Zn SOD was proved to be reactive to the native enzyme by using Western blot analysis. pPICzαB/sodA also appeared to be more resistant than the control pPICzαB recombinant yeast in the oxidative stress conditions. This is the first study of the expression of recombinant T. marneffei Cu, Zn SOD protein and its enzyme activity determination. This enzyme is an important virulence factor and targeting this enzyme may be a promising strategy for developing new therapeutics.

References

Amsri, A., Jeenkeawpieam, J., Sukantamala, P., & Pongpom, M. (2021). Role of acuK in control of iron acquisition and gluconeogenesis in Talaromyces marneffei. Journal of Fungi, 7(10), 798. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8539426/pdf/jof-07-00798.pdf

Bertrand, R. L., & Eze, M. O. (2014). Modifying polyacrylamide background color for the nitroblue tetrazolium-based superoxide dismutase staining assay. Advances in Enzyme Research, 2(2), 77-81. http://dx.doi.org/10.4236/aer.2014.22008

Chang, W. S., & So, J. S. (1999). Characterization of superoxide dismutase in Lactococcus lactis. Journal of Microbiology and Biotechnology , 9(6), 732-736. https://koreascience.kr/article/JAKO199911922246881.pdf

Cooper, C. R. Jr., & Haycocks, N. G. (2000) Penicillium marneffei: an insurgent species among the penicillia. Journal of Eukaryotic Microbiology, 47(1), 24-28. https://doi.org/10.1111/j.1550-7408.2000.tb00006.x

Ellett, F., Pazhakh, V., Pase, L., Benard, E. L, Weerasinghe, H., Azabdaftari, D., Alasmari, S., Andrianopoulos, A., & Lieschke, G. J. (2018). Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo. PLoS Pathogens, 14(6), e1007063. https://doi.org/10.1371/journal.ppat.1007063

Fridovich, I. (1995). Superoxide radical and superoxide dismutases. Annual Review of Biochemistry, 64, 97-112. https://www.annualreviews.org/doi/pdf/10.1146/annurev.bi.64.070195.000525

Holdom, M. D., Hay, R. J., & Hamilton, A. J. (1995). Purification, N-terminal amino acid sequence and partial characterization of a Cu, Zn superoxide dismutase from the pathogenic fungus Aspergillus fumigatus. Free Radical Research, 22(6), 519–531. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC227976/pdf/330495.pdf

Holdom, M. D., Lechenne, B., Hay, R. J., Hamilton, A. J., & Monod, M. (2000). Production and characterization of recombinant Aspergillus fumigatus Cu, Zn superoxide dismutase and its recognition by immune human sera. Journal of Clinical Microbiology, 38(2), 558-562. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC86148/pdf/jm000558.pdf

Kudeken, N., Kawakami, K., & Saito, A. (1998). Different susceptibilities of yeasts and conidia of Penicillium marneffei to nitric oxide (NO)-mediated fungicidal activity of murine macrophages.Clinical and Experimental Immunology, 112, 287-293. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1904956/pdf/cei0112-0287.pdf

Kudeken, N., Kawakami, K., & Saito, A. (1999). Role of superoxide anion in the fungicidal activity of murine peritoneal exudate macrophages against Penicillium marneffei. Microbiology and Immunology, 43, 323-330. https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1574-695X.1999.tb01378.x

Kummasook, A., Tzarphmaag, A., Thirach, S., Pongpom, M., Cooper, C. R. Jr., & Vanittanakom, N. (2011). Penicillium marneffei actin expression during phase transition, oxidative stress, and macrophage infection. Molecular Biology Reports, 38, 2813–2819. https://doi.org/10.1007/s11033-010-0427-1

Li, J. R., & Yu, P. (2007). Expression of Cu, Zn-superoxide dismutase gene from Saccharomyces cerevisiae in Pichia pastoris and its resistance to oxidative stress. Applied Biochemistry and Biotechnology, 136, 127–139. https://doi.org/10.1007/BF02685943

Lu, S., Hu, Y., Lu, C., Zhang, J., Li, X., & Xi, L. (2013). Development of in vitro macrophage system to evaluate phagocytosis and intracellular fate of Penicillium marneffei conidia. Mycopathologia, 176, 11–22. https://doi.org/10.1007/s11046-013-9650-3

Macauley-Patrick, S., Fazenda, M. L., McNeil, B., & Harvey, L. M. (2005). Heterologous protein production using the Pichia pastoris expression system. Yeast, 22, 249–270. https://onlinelibrary.wiley.com/doi/epdf/10.1002/yea.1208

Maurya, R., & Namdeo, M. (2021). Superoxide dismutase: A key enzyme for the survival of intracellular pathogens in host. In A. Rizwan (Ed.), Reactive oxygen species. IntechOpen. https://doi.org/10.5772/intechopen.100322

Sapmak, A., Kaewmalakul, J., Nosanchuk, J. D., Vanittanakom, N., Andrianopoulos, A., Pruksaphon, K., & Youngchim, S. (2016). Talaromyces marneffei laccase modifies THP-1 macrophage responses. Virulence, 17, 702-717. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991349/pdf/kvir-07-06-1193275.pdf

Thirach, S., Cooper, C. R. Jr., Vanittanakom, P., & Vanittanakom, N. (2007). The copper, zinc superoxide dismutase gene of Penicillium marneffei: cloning, characterization, and differential expression during phase transition and macrophage infection. Medical Mycology, 45(5), 409-417. https://doi.org/10.1080/13693780701381271

Warrisa, A., & Balloub, E. R. (2019). Oxidative responses and fungal infection biology. Seminars in Cell and Developmental Biology, 89, 34–46. https://doi.org/10.1016/j.semcdb.2018.03.004

Zhang, L. Q., Guo, F. X., Xian, H. Q., Wang, X. J., Li, A. N., & Li, D. C. (2011). Expression of a novel thermostable Cu, Zn-superoxide dismutase from Chaetomium thermophilum in Pichia pastoris and its antioxidant properties. Biotechnology Letters, 33(6), 1127–1132. https://doi.org/10.1007/s10529-011-0543-6