Yeast-mediated display: probing Helicobacter pylori HopQ and CEACAM1 interaction
,
Abstract
Background and Objectives: Helicobacter pylori (H. pylori), as a Gram-negative pathogen plays a key role in causing gastritis, peptic ulcer disease, and gastric malignancies. The bacterial adhesin HopQ binds human CEACAM1, promoting adherence and CagA oncoprotein translocation. This study aimed to establish a yeast-based surface expression platform to investigate the HopQ–CEACAM1 interaction as a basis for future inhibitor screening.
Materials and Methods: The N-terminal domain of human CEACAM1 (C1ND) was displayed on the surface of Saccharomyces cerevisiae BY4741 as C1ND or C1ND–EGFP via Aga2 fusion. Constructs were introduced by electroporation and confirmed by PCR. Protein expression and localization were validated by western blot, confocal microscopy, and flow cytometry. Binding assays involved GFP-tagged HopQ and GFP-expressing H. pylori.
Results: Western blot confirmed surface expression of C1ND and C1ND–EGFP. Confocal microscopy and flow cytometry showed strong fluorescence signals, with significantly higher mean fluorescence intensity and anti-GFP–positive yeast compared to controls (P < 0.01). Yeast-displayed C1ND specifically bound HopQ–GFP and GFP-expressing H. pylori.
Conclusion: Yeast surface display of CEACAM1’s N-domain is an effective model for studying HopQ–CEACAM1 binding and offers potential for identifying inhibitors to block H. pylori adhesion and associated disorders.
1. Hooi JK, Lai WY, Ng WK, Suen MM, Underwood FE, Tanyingoh D, et al. Global prevalence of Helicobacter pylori infection: systematic review and meta-analysis. Gastroenterology 2017; 153: 420-429.
2. Zamani M, Ebrahimtabar F, Zamani V, Miller W, Alizadeh-Navaei R, Shokri-Shirvani J, et al. Systematic review with meta‐analysis: the worldwide prevalence of Helicobacter pylori infection. Aliment Pharmacol Ther 2018; 47: 868-876.
3. Salama NR, Hartung ML, Müller A. Life in the human stomach: persistence strategies of the bacterial pathogen Helicobacter pylori. Nat Rev Microbiol 2013; 11: 385-399.
4. Wroblewski LE, Peek Jr RM, Wilson KT. Helicobacter pylori and gastric cancer: factors that modulate disease risk. Clin Microbiol Rev 2010; 23: 713-739.
5. Gur C, Maalouf N, Gerhard M, Singer BB, Emgård J, Temper V, et al. The Helicobacter pylori HopQ outermembrane protein inhibits immune cell activities. Oncoimmunology 2019; 8(4): e1553487.
6. Hatakeyama M. Helicobacter pylori oncoprotein CagA and bacterial EPIYA effector family. Seikagaku 2014; 86: 744-754.
7. Backert S, Moese S, Selbach M, Brinkmann V, Meyer TF. Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells. Mol Microbiol 2001; 42: 631-644.
8. Backert S, Tegtmeyer N, Selbach M. The versatility of Helicobacter pylori CagA effector protein functions: The master key hypothesis. Helicobacter 2010; 15: 163-176.
9. Zhao Q, Busch B, Jiménez-Soto LF, Ishikawa-Ankerhold H, Massberg S, Terradot L, et al. Integrin but not CEACAM receptors are dispensable for Helicobacter pylori CagA translocation. PLoS Pathog 2018; 14(10): e1007359.
10. Hatakeyama M, Higashi H. Helicobacter pylori CagA: a new paradigm for bacterial carcinogenesis. Cancer Sci 2005; 96: 835-843.
11. Matsuo Y, Kido Y, Yamaoka Y. Helicobacter pylori outer membrane protein-related pathogenesis. Toxins (Basel) 2017; 9: 101.
12. Nguyen QA, Schmitt L, Mejías-Luque R, Gerhard M. Effects of Helicobacter pylori adhesin HopQ binding to CEACAM receptors in the human stomach. Front Immunol 2023; 14: 1113478.
13. Kalali B, Mejías-Luque R, Javaheri A, Gerhard M. H. pylori virulence factors: influence on immune system and pathology. Mediators Inflamm 2014; 2014: 426309.
14. Ansari S, Yamaoka Y. Helicobacter pylori virulence factors exploiting gastric colonization and its pathogenicity. Toxins (Basel) 2019; 11: 677.
15. Cao P, Cover TL. Two different families of hopQ alleles in Helicobacter pylori. J Clin Microbiol 2002; 40: 4504-4511.
16. Königer V, Holsten L, Harrison U, Busch B, Loell E, Zhao Q, et al. Erratum: Helicobacter pylori exploits human CEACAMs via HopQ for adherence and translocation of CagA. Nat Microbiol 2016; 2: 16233.
17. Bonsor DA, Zhao Q, Schmidinger B, Weiss E, Wang J, Deredge D, et al. The Helicobacter pylori adhesin protein HopQ exploits the dimer interface of human CEACAMs to facilitate translocation of the oncoprotein CagA. EMBO J 2018; 37(13): e98664.
18. Zhuo Y, Yang J-Y, Moremen KW, Prestegard JH. Correction: Glycosylation alters dimerization properties of a cell-surface signaling protein, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). J Biol Chem 2020; 295: 3748.
19. Ru G-Q, Han Y, Wang W, Chen Y, Wang H-J, Xu W-J, et al. CEACAM6 is a prognostic biomarker and potential therapeutic target for gastric carcinoma. Oncotarget 2017; 8: 83673-83683.
20. Hall C, Clarke L, Pal A, Buchwald P, Eglinton T, Wakeman C, et al. A review of the role of carcinoembryonic antigen in clinical practice. Ann Coloproctol 2019; 35: 294-305.
21. Najjar SM. Regulation of insulin action by CEACAM1. Trends Endocrinol Metab 2002; 13: 240-245.
22. Beauchemin N, Arabzadeh A. Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) in cancer progression and metastasis. Cancer Metastasis Rev 2013; 32: 643-671.
23. Javaheri A, Kruse T, Moonens K, Mejías-Luque R, Debraekeleer A, Asche CI, et al. Helicobacter pylori adhesin HopQ engages in a virulence-enhancing interaction with human CEACAMs. Nat Microbiol 2016; 2: 16189.
24. Moonens K, Hamway Y, Neddermann M, Reschke M, Tegtmeyer N, Kruse T, et al. Helicobacter pylori adhesin HopQ disrupts trans dimerization in human CEACAM s. EMBO J 2018; 37(13): e98665.
25. Huang Y-H, Zhu C, Kondo Y, Anderson AC, Gandhi A, Russell A, et al. CEACAM1 regulates TIM-3-mediated tolerance and exhaustion. Nature 2015; 517: 386-390.
26. Lee SY, Choi JH, Xu Z. Microbial cell-surface display. Trends Biotechnol 2003; 21: 45-52.
27. Löfblom J. Bacterial display in combinatorial protein engineering. Biotechnol J 2011; 6: 1115-1129.
28. Ueda M, Tanaka A. Cell surface engineering of yeast: construction of arming yeast with biocatalyst. J Biosci Bioeng 2000; 90: 125-136.
29. Kondo A, Ueda M. Yeast cell-surface display—applications of molecular display. Appl Microbiol Biotechnol 2004; 64: 28-40.
30. Lopez-Morales J, Vanella R, Appelt EA, Whillock S, Paulk AM, Shusta EV, et al. Protein engineering and High‐Throughput screening by yeast surface display: survey of current methods. Small Sci 2023; 3: 2300095.
31. Yang F, Cao M, Jin Y, Yang X, Tian S. Construction of a novel а-agglutinin expression system on the surface of wild-type Saccharomyces cerevisiae Y5 and genetic immobilization of β-glucosidase1. BioEnergy Res 2013; 6: 1205-1211.
32. Nayebhashemi M, Enayati S, Zahmatkesh M, Madanchi H, Saberi S, Mostafavi E, et al. Surface display of pancreatic lipase inhibitor peptides by engineered Saccharomyces boulardii: Potential as an anti-obesity probiotic. J Funct Foods 2023; 102: 105458.
33. Benatuil L, Perez JM, Belk J, Hsieh C-M. An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng Des Sel 2010; 23: 155-159.
34. Uchański T, Zögg T, Yin J, Yuan D, Wohlkönig A, Fischer B, et al. An improved yeast surface display platform for the screening of nanobody immune libraries. Sci Rep 2019; 9: 382.
35. Sing CN, Yang EJ, Higuchi-Sanabria R, Pon LA, Boldogh IR, Swayne TC. Imaging the actin cytoskeleton in fixed budding yeast cells. Methods Mol Biol 2022; 2364: 81-100.
36. Prole DL, Chinnery PF, Jones NS. Visualizing, quantifying, and manipulating mitochondrial DNA in vivo. J Biol Chem 2020; 295: 17588-17601.
37. Teymennet-Ramírez KV, Martínez-Morales F, Trejo-Hernández MR. Yeast surface display system: Strategies for improvement and biotechnological applications. Front Bioeng Biotechnol 2022; 9: 794742.
38. Tanaka T, Yamada R, Ogino C, Kondo A. Recent developments in yeast cell surface display toward extended applications in biotechnology. Appl Microbiol Biotechnol 2012; 95: 577-591.
39. Wang S, Cho YK. Yeast surface display of full‐length human microtubule‐associated protein tau. Biotechnol Prog 2020; 36(1): e2920.
40. Mathew E, Zhu H, Connelly SM, Sullivan MA, Brewer MG, Piepenbrink MS, et al. Display of the HIV envelope protein at the yeast cell surface for immunogen development. PLoS One 2018; 13(10): e0205756.
41. Maneira C, Bermejo PM, Pereira GAG, de Mello FDSB. Exploring G protein-coupled receptors and yeast surface display strategies for viral detection in baker's yeast: SARS-CoV-2 as a case study. FEMS Yeast Res 2021; 21: foab004.
42. Wellner A, McMahon C, Gilman MS, Clements JR, Clark S, Nguyen KM, et al. Rapid generation of potent antibodies by autonomous hypermutation in yeast. Nat Chem Biol 2021; 17: 1057-1064.
43. Vogt S, Stadlmayr G, Stadlbauer K, Stracke F, Bobbili MR, Grillari J, et al. Construction of Yeast Display Libraries for Selection of Antigen-Binding Variants of Large Extracellular Loop of CD81, a Major Surface Marker Protein of Extracellular Vesicles. Methods Mol Biol 2022; 2491: 561-592.
44. Peek RM Jr, Blaser MJ. Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nat Rev Cancer 2002; 2: 28-37.
45. Wu L, Li H, Tang T. A novel yeast surface display method for large-scale screen inhibitors of sortase A. Bioengineering (Basel) 2017; 4: 6.
2. Zamani M, Ebrahimtabar F, Zamani V, Miller W, Alizadeh-Navaei R, Shokri-Shirvani J, et al. Systematic review with meta‐analysis: the worldwide prevalence of Helicobacter pylori infection. Aliment Pharmacol Ther 2018; 47: 868-876.
3. Salama NR, Hartung ML, Müller A. Life in the human stomach: persistence strategies of the bacterial pathogen Helicobacter pylori. Nat Rev Microbiol 2013; 11: 385-399.
4. Wroblewski LE, Peek Jr RM, Wilson KT. Helicobacter pylori and gastric cancer: factors that modulate disease risk. Clin Microbiol Rev 2010; 23: 713-739.
5. Gur C, Maalouf N, Gerhard M, Singer BB, Emgård J, Temper V, et al. The Helicobacter pylori HopQ outermembrane protein inhibits immune cell activities. Oncoimmunology 2019; 8(4): e1553487.
6. Hatakeyama M. Helicobacter pylori oncoprotein CagA and bacterial EPIYA effector family. Seikagaku 2014; 86: 744-754.
7. Backert S, Moese S, Selbach M, Brinkmann V, Meyer TF. Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells. Mol Microbiol 2001; 42: 631-644.
8. Backert S, Tegtmeyer N, Selbach M. The versatility of Helicobacter pylori CagA effector protein functions: The master key hypothesis. Helicobacter 2010; 15: 163-176.
9. Zhao Q, Busch B, Jiménez-Soto LF, Ishikawa-Ankerhold H, Massberg S, Terradot L, et al. Integrin but not CEACAM receptors are dispensable for Helicobacter pylori CagA translocation. PLoS Pathog 2018; 14(10): e1007359.
10. Hatakeyama M, Higashi H. Helicobacter pylori CagA: a new paradigm for bacterial carcinogenesis. Cancer Sci 2005; 96: 835-843.
11. Matsuo Y, Kido Y, Yamaoka Y. Helicobacter pylori outer membrane protein-related pathogenesis. Toxins (Basel) 2017; 9: 101.
12. Nguyen QA, Schmitt L, Mejías-Luque R, Gerhard M. Effects of Helicobacter pylori adhesin HopQ binding to CEACAM receptors in the human stomach. Front Immunol 2023; 14: 1113478.
13. Kalali B, Mejías-Luque R, Javaheri A, Gerhard M. H. pylori virulence factors: influence on immune system and pathology. Mediators Inflamm 2014; 2014: 426309.
14. Ansari S, Yamaoka Y. Helicobacter pylori virulence factors exploiting gastric colonization and its pathogenicity. Toxins (Basel) 2019; 11: 677.
15. Cao P, Cover TL. Two different families of hopQ alleles in Helicobacter pylori. J Clin Microbiol 2002; 40: 4504-4511.
16. Königer V, Holsten L, Harrison U, Busch B, Loell E, Zhao Q, et al. Erratum: Helicobacter pylori exploits human CEACAMs via HopQ for adherence and translocation of CagA. Nat Microbiol 2016; 2: 16233.
17. Bonsor DA, Zhao Q, Schmidinger B, Weiss E, Wang J, Deredge D, et al. The Helicobacter pylori adhesin protein HopQ exploits the dimer interface of human CEACAMs to facilitate translocation of the oncoprotein CagA. EMBO J 2018; 37(13): e98664.
18. Zhuo Y, Yang J-Y, Moremen KW, Prestegard JH. Correction: Glycosylation alters dimerization properties of a cell-surface signaling protein, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). J Biol Chem 2020; 295: 3748.
19. Ru G-Q, Han Y, Wang W, Chen Y, Wang H-J, Xu W-J, et al. CEACAM6 is a prognostic biomarker and potential therapeutic target for gastric carcinoma. Oncotarget 2017; 8: 83673-83683.
20. Hall C, Clarke L, Pal A, Buchwald P, Eglinton T, Wakeman C, et al. A review of the role of carcinoembryonic antigen in clinical practice. Ann Coloproctol 2019; 35: 294-305.
21. Najjar SM. Regulation of insulin action by CEACAM1. Trends Endocrinol Metab 2002; 13: 240-245.
22. Beauchemin N, Arabzadeh A. Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) in cancer progression and metastasis. Cancer Metastasis Rev 2013; 32: 643-671.
23. Javaheri A, Kruse T, Moonens K, Mejías-Luque R, Debraekeleer A, Asche CI, et al. Helicobacter pylori adhesin HopQ engages in a virulence-enhancing interaction with human CEACAMs. Nat Microbiol 2016; 2: 16189.
24. Moonens K, Hamway Y, Neddermann M, Reschke M, Tegtmeyer N, Kruse T, et al. Helicobacter pylori adhesin HopQ disrupts trans dimerization in human CEACAM s. EMBO J 2018; 37(13): e98665.
25. Huang Y-H, Zhu C, Kondo Y, Anderson AC, Gandhi A, Russell A, et al. CEACAM1 regulates TIM-3-mediated tolerance and exhaustion. Nature 2015; 517: 386-390.
26. Lee SY, Choi JH, Xu Z. Microbial cell-surface display. Trends Biotechnol 2003; 21: 45-52.
27. Löfblom J. Bacterial display in combinatorial protein engineering. Biotechnol J 2011; 6: 1115-1129.
28. Ueda M, Tanaka A. Cell surface engineering of yeast: construction of arming yeast with biocatalyst. J Biosci Bioeng 2000; 90: 125-136.
29. Kondo A, Ueda M. Yeast cell-surface display—applications of molecular display. Appl Microbiol Biotechnol 2004; 64: 28-40.
30. Lopez-Morales J, Vanella R, Appelt EA, Whillock S, Paulk AM, Shusta EV, et al. Protein engineering and High‐Throughput screening by yeast surface display: survey of current methods. Small Sci 2023; 3: 2300095.
31. Yang F, Cao M, Jin Y, Yang X, Tian S. Construction of a novel а-agglutinin expression system on the surface of wild-type Saccharomyces cerevisiae Y5 and genetic immobilization of β-glucosidase1. BioEnergy Res 2013; 6: 1205-1211.
32. Nayebhashemi M, Enayati S, Zahmatkesh M, Madanchi H, Saberi S, Mostafavi E, et al. Surface display of pancreatic lipase inhibitor peptides by engineered Saccharomyces boulardii: Potential as an anti-obesity probiotic. J Funct Foods 2023; 102: 105458.
33. Benatuil L, Perez JM, Belk J, Hsieh C-M. An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng Des Sel 2010; 23: 155-159.
34. Uchański T, Zögg T, Yin J, Yuan D, Wohlkönig A, Fischer B, et al. An improved yeast surface display platform for the screening of nanobody immune libraries. Sci Rep 2019; 9: 382.
35. Sing CN, Yang EJ, Higuchi-Sanabria R, Pon LA, Boldogh IR, Swayne TC. Imaging the actin cytoskeleton in fixed budding yeast cells. Methods Mol Biol 2022; 2364: 81-100.
36. Prole DL, Chinnery PF, Jones NS. Visualizing, quantifying, and manipulating mitochondrial DNA in vivo. J Biol Chem 2020; 295: 17588-17601.
37. Teymennet-Ramírez KV, Martínez-Morales F, Trejo-Hernández MR. Yeast surface display system: Strategies for improvement and biotechnological applications. Front Bioeng Biotechnol 2022; 9: 794742.
38. Tanaka T, Yamada R, Ogino C, Kondo A. Recent developments in yeast cell surface display toward extended applications in biotechnology. Appl Microbiol Biotechnol 2012; 95: 577-591.
39. Wang S, Cho YK. Yeast surface display of full‐length human microtubule‐associated protein tau. Biotechnol Prog 2020; 36(1): e2920.
40. Mathew E, Zhu H, Connelly SM, Sullivan MA, Brewer MG, Piepenbrink MS, et al. Display of the HIV envelope protein at the yeast cell surface for immunogen development. PLoS One 2018; 13(10): e0205756.
41. Maneira C, Bermejo PM, Pereira GAG, de Mello FDSB. Exploring G protein-coupled receptors and yeast surface display strategies for viral detection in baker's yeast: SARS-CoV-2 as a case study. FEMS Yeast Res 2021; 21: foab004.
42. Wellner A, McMahon C, Gilman MS, Clements JR, Clark S, Nguyen KM, et al. Rapid generation of potent antibodies by autonomous hypermutation in yeast. Nat Chem Biol 2021; 17: 1057-1064.
43. Vogt S, Stadlmayr G, Stadlbauer K, Stracke F, Bobbili MR, Grillari J, et al. Construction of Yeast Display Libraries for Selection of Antigen-Binding Variants of Large Extracellular Loop of CD81, a Major Surface Marker Protein of Extracellular Vesicles. Methods Mol Biol 2022; 2491: 561-592.
44. Peek RM Jr, Blaser MJ. Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nat Rev Cancer 2002; 2: 28-37.
45. Wu L, Li H, Tang T. A novel yeast surface display method for large-scale screen inhibitors of sortase A. Bioengineering (Basel) 2017; 4: 6.
| Files | ||
| Issue | Vol 17 No 6 (2025) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v17i6.20366 | |
| Keywords | ||
| Helicobacter pylori HopQ CEACAM1 C1ND | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Mofarrah N, Larypoor M, Norozi J. Yeast-mediated display: probing Helicobacter pylori HopQ and CEACAM1 interaction. Iran J Microbiol. 2025;17(6):977-990.
