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Expression, Purification, and Characterization of the Recombinant, Two-Component, Response Regulator ArlR from Fusobacterium nucleatum

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Abstract

Fusobacterium nucleatum is associated with the incidence and development of multiple diseases, such as periodontitis and colorectal cancer (CRC). Until now, studies have proved only a few proteins to be associated with such pathogenic diseases. The two-component system is one of the most prevalent forms of bacterial signal transduction related to intestinal diseases. Here, we report a novel, recombinant, two-component, response regulator protein ArlR from the genome of F. nucleatum strain ATCC 25,586. We optimized the expression and purification conditions of ArlR; in addition, we characterized the interaction of this response regulator protein with the corresponding histidine kinase and DNA sequence. The full-length ArlR was successfully expressed in six E. coli host strains. However, optimum expression conditions of ArlR were present only in E. coli strain BL21 CodonPlus (DE3) RIL that was later induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) for 8 h at 25 °C. The SDS-PAGE analysis revealed the molecular weight of the recombinant protein as 27.3 kDa with approximately 90% purity after gel filtration chromatography. Because ArlR was biologically active after its purification, it accepted the corresponding phosphorylated histidine kinase phosphate group and bound to the analogous DNA sequence. The binding constant between ArlR and the corresponding histidine kinase was about 2.1 μM, whereas the binding constant between ArlR and its operon was 6.4 μM. Altogether, these results illustrate an effective expression and purification method for the novel two-component system protein ArlR.

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Availability of Data and Materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

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References

  1. Liu, Y., Baba, Y., Ishimoto, T., Iwatsuki, M., Hiyoshi, Y., Miyamoto, Y., et al. (2019). Progress in characterizing the linkage between Fusobacterium nucleatum and gastrointestinal cancer. Journal of gastroenterology., 54(1), 33–41.

    Article  CAS  PubMed  Google Scholar 

  2. Luo, K., Zhang, Y., Xv, C., Ji, J., Lou, G., Guo, X., et al. (2019). Fusobacterium nucleatum, the communication with colorectal cancer. Biomedicine & Pharmacotherapy., 116, 108988.

    Article  CAS  Google Scholar 

  3. Bronzato, J. D., Bomfim, R. A., Edwards, D. H., Crouch, D., Hector, M. P., & Gomes, B. P. (2020). Detection of Fusobacterium in oral and head and neck cancer samples: A systematic review and meta-analysis. Archives of oral biology., 112, 104669.

    Article  CAS  PubMed  Google Scholar 

  4. Wang, X., Buhimschi, C. S., Temoin, S., Bhandari, V., Han, Y. W., & Buhimschi, I. A. (2013). Comparative microbial analysis of paired amniotic fluid and cord blood from pregnancies complicated by preterm birth and early-onset neonatal sepsis. PloS one., 8(2), e56131.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Weiss, E., Shaniztki, B., Dotan, M., Ganeshkumar, N., Kolenbrander, P., & Metzger, Z. (2000). Attachment of Fusobacterium nucleatum PK1594 to mammalian cells and its coaggregation with periodontopathogenic bacteria are mediated by the same galactose-binding adhesin. Oral microbiology and immunology., 15(6), 371–377.

    Article  CAS  PubMed  Google Scholar 

  6. Han, Y. W., Redline, R. W., Li, M., Yin, L., Hill, G. B., & McCormick, T. S. (2004). Fusobacterium nucleatum induces premature and term stillbirths in pregnant mice: Implication of oral bacteria in preterm birth. Infection and Immunity., 72(4), 2272–2279.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kostic, A. D., Chun, E., Robertson, L., Glickman, J. N., Gallini, C. A., Michaud, M., et al. (2013). Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment. Cell host & microbe., 14(2), 207–215.

    Article  CAS  Google Scholar 

  8. Yang, Y., Weng, W., Peng, J., Hong, L., Yang, L., Toiyama, Y., Gao, R., Liu, M., Yin, M., Pan, C., Li, H., Guo, B., Zhu, Q., Wei, Q., Moyer, M. P., Wang, P., Cai, S., Goel, A., Qin, H., & Ma, Y. (2017). Fusobacterium nucleatum increases proliferation of colorectal cancer cells and tumor development in mice by activating Toll-like receptor 4 signaling to nuclear factor-κB, and up-regulating expression of MicroRNA-21. Gastroenterology, 152(4), 851-866.e24.

    Article  CAS  PubMed  Google Scholar 

  9. Wu, J., Li, K., Peng, W., Li, H., Li, Q., Wang, X., et al. (2019). Autoinducer-2 of Fusobacterium nucleatum promotes macrophage M1 polarization via TNFSF9/IL-1β signaling. International Immunopharmacology, 74, 105724.

    Article  CAS  PubMed  Google Scholar 

  10. Yu, T., Guo, F., Yu, Y., Sun, T., Ma, D., Han, J., Qian, Y., Kryczek, I., Sun, D., Nagarsheth, N., Chen, Y., Chen, H., Hong, J., Zou, W., & Fang, J. Y. (2017). Fusobacterium nucleatum promotes chemoresistance to colorectal cancer by modulating autophagy. Cell, 170(3), 548-563.e16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kolenbrander, P. E., Palmer, R. J., Periasamy, S., & Jakubovics, N. S. (2010). Oral multispecies biofilm development and the key role of cell–cell distance. Nature Reviews Microbiology., 8(7), 471–480.

    Article  CAS  PubMed  Google Scholar 

  12. Han, Y. W., Shi, W., Huang, G.T.-J., Haake, S. K., Park, N.-H., Kuramitsu, H., et al. (2000). Interactions between periodontal bacteria and human oral epithelial cells: Fusobacterium nucleatum adheres to and invades epithelial cells. Infection and Immunity, 68(6), 3140–3146.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Alvarez, A. F., Barba-Ostria, C., Silva-Jiménez, H., & Georgellis, D. (2016). Organization and mode of action of two component system signaling circuits from the various kingdoms of life. Environmental Microbiology., 18(10), 3210–3226.

    Article  CAS  PubMed  Google Scholar 

  14. Tierney, A. R., & Rather, P. N. (2019). Roles of two-component regulatory systems in antibiotic resistance. Future Microbiology., 14(6), 533–552.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Mattos-Graner, R. O., & Duncan, M. J. (2017). Two-component signal transduction systems in oral bacteria. Journal of oral microbiology., 9(1), 1400858.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Li, X., Lv, X., Lin, Y., Zhen, J., Ruan, C., Duan, W., et al. (2019). Role of two-component regulatory systems in intracellular survival of Mycobacterium tuberculosis. Journal of cellular biochemistry., 120(8), 12197–12207.

    Article  CAS  PubMed  Google Scholar 

  17. Zschiedrich, C. P., Keidel, V., & Szurmant, H. (2016). Molecular mechanisms of two-component signal transduction. Journal of Molecular Biology, 428(19), 3752–3775.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Jacob-Dubuisson, F., Mechaly, A., Betton, J.-M., & Antoine, R. (2018). Structural insights into the signalling mechanisms of two-component systems. Nature Reviews Microbiology., 16(10), 585–593.

    Article  CAS  PubMed  Google Scholar 

  19. Sidote, D. J., Barbieri, C. M., Wu, T., & Stock, A. M. (2008). Structure of the Staphylococcus aureus AgrA LytTR domain bound to DNA reveals a beta fold with an unusual mode of binding. Structure, 16(5), 727–735.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sun, F., Li, C., Jeong, D., Sohn, C., He, C., & Bae, T. (2010). In the Staphylococcus aureus two-component system sae, the response regulator SaeR binds to a direct repeat sequence and DNA binding requires phosphorylation by the sensor kinase SaeS. Journal of Bacteriology., 192(8), 2111–2127.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Fan, R., Shi, X., Guo, B., Zhao, J., Liu, J., Quan, C., et al. (2021). The effects of L-arginine on protein stability and DNA binding ability of SaeR, a transcription factor in Staphylococcus aureus. Protein Expression and Purification, 177, 105765.

    Article  CAS  PubMed  Google Scholar 

  22. Rooks, M. G., Veiga, P., Reeves, A. Z., Lavoie, S., Yasuda, K., Asano, Y., et al. (2017). QseC inhibition as an antivirulence approach for colitis-associated bacteria. Proceedings of the National Academy of Sciences, 114(1), 142–147.

    Article  CAS  Google Scholar 

  23. Massmig, M., Reijerse, E., Krausze, J., Laurich, C., Lubitz, W., Jahn, D., et al. (2020). Carnitine metabolism in the human gut: Characterization of the two-component carnitine monooxygenase CntAB from Acinetobacter baumannii. Journal of Biological Chemistry., 295(37), 13065–13078.

    Article  CAS  Google Scholar 

  24. Chen, J., Ma, M., Uzal, F. A., & McClane, B. A. (2014). Host cell-induced signaling causes Clostridium perfringens to upregulate production of toxins important for intestinal infections. Gut Microbes, 5(1), 96–107.

    Article  PubMed  Google Scholar 

  25. Goto, R., Miki, T., Nakamura, N., Fujimoto, M., & Okada, N. (2017). Salmonella Typhimurium PagP-and UgtL-dependent resistance to antimicrobial peptides contributes to the gut colonization. PloS one, 12(12), e0190095.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Wakimoto, S., Nakayama-Imaohji, H., Ichimura, M., Morita, H., Hirakawa, H., Hayashi, T., et al. (2013). PhoB regulates the survival of Bacteroides fragilis in peritoneal abscesses. PloS one, 8(1), e53829.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. van der Stel, A. X., van Mourik, A., Heijmen-van Dijk, L., Parker, C. T., Kelly, D. J., van de Lest, C. H., et al. (2015). The C ampylobacter jejuni RacRS system regulates fumarate utilization in a low oxygen environment. Environmental Microbiology., 17(4), 1049–1064.

    Article  PubMed  CAS  Google Scholar 

  28. Giannakopoulou, N., Mendis, N., Zhu, L., Gruenheid, S., Faucher, S. P., & Le Moual, H. (2018). The virulence effect of CpxRA in Citrobacter rodentium is independent of the auxiliary proteins NlpE and CpxP. Frontiers in Cellular and Infection Microbiology, 8, 320.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Pacheco, A. R., Curtis, M. M., Ritchie, J. M., Munera, D., Waldor, M. K., Moreira, C. G., et al. (2012). Fucose sensing regulates bacterial intestinal colonization. Nature, 492(7427), 113–117.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Fujimoto, M., Goto, R., Haneda, T., Okada, N., & Miki, T. (2018). Salmonella enterica serovar Typhimurium CpxRA two-component system contributes to gut colonization in salmonella-induced colitis. Infection and Immunity., 86(7), e00280.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Liu, M., Hao, G., Li, Z., Zhou, Y., Garcia-Sillas, R., Li, J., Wang, H., Kan, B., & Zhu, J. (2019). CitAB two-component system-regulated citrate utilization contributes to Vibrio cholerae competitiveness with the gut microbiota. Infection and Immunity, 87(3), e00746-e818.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sonnenburg, E. D., Sonnenburg, J. L., Manchester, J. K., Hansen, E. E., Chiang, H. C., & Gordon, J. I. (2006). A hybrid two-component system protein of a prominent human gut symbiont couples glycan sensing in vivo to carbohydrate metabolism. Proceedings of the National Academy of Sciences, 103(23), 8834–8839.

    Article  CAS  Google Scholar 

  33. Lowe, E. C., Baslé, A., Czjzek, M., Firbank, S. J., & Bolam, D. N. (2012). A scissor blade-like closing mechanism implicated in transmembrane signaling in a Bacteroides hybrid two-component system. Proceedings of the National Academy of Sciences, 109(19), 7298–7303.

    Article  CAS  Google Scholar 

  34. Wu, C., Chen, Y. W., Scheible, M., Chang, C., Wittchen, M., Lee, J. H., et al. (2021). Genetic and molecular determinants of polymicrobial interactions in Fusobacterium nucleatum. Proceeding of the National Academy of Science U S A, 118(23), e2006482118.

    Article  CAS  Google Scholar 

  35. Hirakawa, H., Kurushima, J., Hashimoto, Y., & Tomita, H. (2020). Progress overview of bacterial two-component regulatory systems as potential targets for antimicrobial chemotherapy. Antibiotics (Basel), 9(10), 635.

    Article  CAS  Google Scholar 

  36. Rosales-Hurtado, M., Meffre, P., Szurmant, H., & Benfodda, Z. (2020). Synthesis of histidine kinase inhibitors and their biological properties. Medicinal Research Reviews, 40(4), 1440–1495.

    Article  CAS  PubMed  Google Scholar 

  37. Gumerov, V. M., Ortega, D. R., Adebali, O., Ulrich, L. E., & Zhulin, I. B. (2020). MiST 3.0: an updated microbial signal transduction database with an emphasis on chemosensory systems. Nucleic Acids Research, 48(D1), D459–D64.

    Article  CAS  PubMed  Google Scholar 

  38. Robert, X., & Gouet, P. (2014). Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Research, 42(W1), W320–W324.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Letunic, I., Khedkar, S., & Bork, P. (2021). SMART: Recent updates, new developments and status in 2020. Nucleic Acids Research., 49(D1), D458–D460.

    Article  CAS  PubMed  Google Scholar 

  40. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., & Sternberg, M. J. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols, 10(6), 845–858.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Solovyev, V., & Salamov, A. (2011). Automatic annotation of microbial genomes and metagenomic sequences. In R. W. Li (Ed.), Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies (pp. 61–78). Nova Science Publishers.

    Google Scholar 

  42. Unger, T., Jacobovitch, Y., Dantes, A., Bernheim, R., & Peleg, Y. (2010). Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. Journal of Structural Biology, 172(1), 34–44.

    Article  CAS  PubMed  Google Scholar 

  43. Zhang, C., Li, A., Wang, R., Cao, Y., Jiang, H., Ouyang, S., et al. (2019). Recombinant expression, purification and bioactivity characterization of extracellular domain of human tumor necrosis factor receptor 1. Protein Expression and Purification, 155, 21–26.

    Article  CAS  PubMed  Google Scholar 

  44. Mueller, A. M., Breitsprecher, D., Duhr, S., Baaske, P., Schubert, T., & Längst, G. (2017). Microscale thermophoresis: A rapid and precise method to quantify protein–nucleic acid interactions in solution. Methods in Molecular Biology, 1654, 151–164.

    Article  CAS  PubMed  Google Scholar 

  45. Walker, J. N., Crosby, H. A., Spaulding, A. R., Salgado-Pabón, W., Malone, C. L., Rosenthal, C. B., et al. (2013). The Staphylococcus aureus ArlRS two-component system is a novel regulator of agglutination and pathogenesis. PLoS Pathog., 9(12), e1003819.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Fournier, B., & Hooper, D. C. (2000). A new two-component regulatory system involved in adhesion, autolysis, and extracellular proteolytic activity of Staphylococcus aureus. Journal of Bacteriology, 182(14), 3955–3964.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Nguyen, M.-P., Yoon, J.-M., Cho, M.-H., & Lee, S.-W. (2015). Prokaryotic 2-component systems and the OmpR/PhoB superfamily. Canadian Journal of Microbiology., 61(11), 799–810.

    Article  CAS  PubMed  Google Scholar 

  48. Bai, J., Zhu, X., Zhao, K., Yan, Y., Xu, T., Wang, J., et al. (2019). The role of ArlRS in regulating oxacillin susceptibility in methicillin-resistant Staphylococcus aureus indicates it is a potential target for antimicrobial resistance breakers. Emerging Microbes & Infections., 8(1), 503–515.

    Article  CAS  Google Scholar 

  49. Radin, J. N., Kelliher, J. L., Párraga Solórzano, P. K., & Kehl-Fie, T. E. (2016). The two-component system ArlRS and alterations in metabolism enable Staphylococcus aureus to resist calprotectin-induced manganese starvation. PLoS Pathogens., 12(11), e1006040.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Casali, N. (2003). Escherichia coli host strains. Methods in Molecular Biology, 235, 27–48.

    CAS  PubMed  Google Scholar 

  51. Willett, J. W., Tiwari, N., Müller, S., Hummels, K. R., Houtman, J. C., Fuentes, E. J., & Kirby, J. R. (2013). Specificity residues determine binding affinity for two-component signal transduction systems. mBio., 4(6), e00420-13.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Sun, F., Li, C., Jeong, D., Sohn, C., He, C., & Bae, T. (2010). In the Staphylococcus aureus two-component system sae, the response regulator SaeR binds to a direct repeat sequence and DNA binding requires phosphorylation by the sensor kinase SaeS. Journal of Bacteriology, 192(8), 2111–2127.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Rajasree, K., Fasim, A., & Gopal, B. (2016). Conformational features of the Staphylococcus aureus AgrA-promoter interactions rationalize quorum-sensing triggered gene expression. Biochemistry and Biophysics Reports, 23(6), 124–134.

    Article  Google Scholar 

  54. Yan, H., Wang, Q., Teng, M., & Li, X. (2019). The DNA-binding mechanism of the TCS response regulator ArlR from Staphylococcus aureus. J Struct Biol, 208(3), 107388.

    Article  CAS  PubMed  Google Scholar 

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Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 21272031) and the Fundamental Research Funds for the Central Universities (Grant No. wd01190).

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Authors and Affiliations

  1. School of Bioengineering, Dalian University of Technology, Dalian, China

    Ruochen Fan, Lulu Wang & Yuesheng Dong

  2. Key Laboratory of Biotechnology and Bioresources Utilization (Ministry of Education), College of Life Science, Dalian Minzu University, Dalian, China

    Ruochen Fan, Zhuting Li, Xian Shi, Lulu Wang, Xuqiang Zhang & Chunshan Quan

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  1. Ruochen Fan
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Contributions

Ruochen Fan participated in formal analysis, writing the original draft, conducting experiments, acquiring data, analyzing data, and writing the paper. Zhuting Li conducted experiments and acquired data. Xian Shi conducted experiments and acquired data. Lulu Wang conducted experiments and acquired data. Xuqiang Zhang conducted experiments and acquired data. Chunshan Quan is involved in funding acquisition. Yuesheng Dong is involved in funding acquisition.

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Correspondence to Chunshan Quan.

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Fan, R., Li, Z., Shi, X. et al. Expression, Purification, and Characterization of the Recombinant, Two-Component, Response Regulator ArlR from Fusobacterium nucleatum. Appl Biochem Biotechnol 194, 2093–2107 (2022). https://doi.org/10.1007/s12010-021-03785-5

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