Mycoplasma pneumoniae Protein-Protein Etkileşimlerinin Maya İkili-Hibrid Yöntemi ile Analizi

Öz

Protein etkileşimlerini tanımlamak, protein fonksiyonlarını belirlemede önemli bir adımdır. Bilinmeyen bir proteinin fonksiyonu, bu nedenle, anotasyonlu etkileşim partnerlerinin tanımlanmasından çıkarılabilir. Tipik olarak, maya ikili-hibrid sistemi (Y2H), protein-protein etkileşimlerini tespit etmek için en yaygın kullanılan genetik testtir. Mikoplazmalar, genomu tamamen dizilen ilk organizmalardan biridir ve minimal hücreler ile sentetik biyolojiyi çalışmak için ilginç bir modeldir. Ancak mikoplazmalarda protein-protein etkileşimlerini incelemek için kullanılan başarılı bir Y2H sistemi bulunmamaktadır. Bu çalışmada, insan patojenik bakterisi Mycoplasma pneumoniae kullanarak mikoplazmalar için kullanılabilir başarılı bir Y2H sistemi geliştirme ve uygulama hedeflenmiştir. İlk denemelerde, M. pneumoniae'nin hareket mekanizması ve hücre iskeleti bütünlüğünü korumada görevli olduğu bilinen proteinler olan P1 adhezin (MPN141), HMW1 (MPN447) ve HMW2 (MPN310) arasındaki etkileşimlere odaklanılmıştır. Başarılı bir Y2H sistemi geliştirmek için önemli iki ana unsurun olduğu bulunmuştur. İlk unsur, türler arası kodon kullanım farklılıklarına dikkat ederek kodon uyumluluğunu sağlamasıdır. UGA kodonu prokaryotlar ve ökaryotlar boyunca bir durdurma kodonu olarak kodlandığı yerde mikoplazmalarda triptofan amino asidi olarak kodlanır. İlgili hedef gen dizilerinde UGA kodonu UGG olarak düzenlenmiştir. İkinci unsur ise etkileşimleri incelenecek proteinlerin ikincil yapıları dikkate alınarak Y2H vektörlerinin tasarlanmasıdır. P1 adhezin, HMW1 ve HMW2 proteinlerinin ikincil yapıları incelenerek, Y2H vektörleri tasarlanırken hedef proteinlerin transmembran segmenti içermemeleri ve kıvrımlı-bobin, α-sarmal ve β-levhaların bozulmayacak şekilde fragmentlere ayrılmasına dikkat edilmiştir. Sonuçlarımız, P1 adhezin en uç C-terminal bölgesi ile HMW1 ve HMW2 proteinlerinin C-terminal bölgeleri arasında etkileşim olduğunu göstermiştir. Bu çalışma, M. pneumoniae'de Y2H'nin ilk başarılı denemesi olup, diğer mikoplazma türlerinde yapılacak denemeler için literatüre katkı sağlayacağı düşünülmektedir.

Anahtar Kelimeler

• mycoplasma pneumoniae
• maya ikili-hibrid
• P1-adhezin

Analysis of Mycoplasma pneumoniae Protein-Protein Interactions by Yeast Two-Hybrid Method

Abstract

Defining protein-protein interactions is a crucial step in elucidating protein functions. The function of an unknown protein can, therefore, be inferred from the identification of annotated interaction partners. Typically, the yeast two-hybrid (Y2H) system is the most widely utilized genetic assay for detecting protein-protein interactions. Mycoplasmas are among the first organisms with fully sequenced genomes and serve as intriguing models for studying minimal cells and synthetic biology. However, a successful Y2H system has not yet been established for the examination of protein-protein interactions in mycoplasmas. In this study, we aimed to develop and implement a viable Y2H system for mycoplasmas using the human pathogenic bacterium Mycoplasma pneumoniae. Initial trials focused on the interactions between proteins known to be involved in M. pneumoniae's motility and the maintenance of cytoskeletal integrity, specifically the P1 adhesin (MPN141), HMW1 (MPN447), and HMW2 (MPN310). Our findings indicate that two primary factors are essential for the successful development of a Y2H system. The first factor pertains to ensuring codon compatibility by taking into account differences in interspecies codon usage. While the UGA codon encodes a stop codon across prokaryotes and eukaryotes, in mycoplasmas, it is translated as the amino acid tryptophan. Thus, the UGA codon in the relevant target gene sequences has been modified to UGG. The second factor involves designing Y2H vectors by considering the secondary structures of the proteins to be examined for interactions. By analyzing the secondary structures of P1 adhesin, HMW1, and HMW2 proteins, it was ensured that the target proteins do not contain transmembrane segments and that the structures of the coiled-coil, α-helix, and β-sheet were preserved when fragmented. Our results demonstrate an interaction between the extreme C-terminal region of P1 adhesin and the C-terminal regions of HMW1 and HMW2 proteins. This study represents the first successful attempt at Y2H in M. pneumoniae, and it is anticipated that it will contribute to the literature for further experiments on other mycoplasmal species.

Keywords

• mycoplasma pneumoniae
• yeast two-hybrid
• P1-adhesin

Kaynakça

1. Ana Y., Gerngross D., Serrano L. Heterologous protein exposure and secretion optimization in Mycoplasma pneumoniae. Microbial Cell Factories 2024; 23(1): 306.
2. Bajantri B., Venkatram S., Diaz-Fuentes G. Mycoplasma pneumoniae: A potentially severe infection. Journal of Clinical Medicine Research 2018; 10(7): 535.
3. Balish MF., Santurri RT., Ricci AM., Lee KK., Krause DC. Localization of Mycoplasma pneumoniae cytadherence‐associated protein HMW2 by fusion with green fluorescent protein: implications for attachment organelle structure. Molecular Microbiology 2003; 47(1): 49-60.
4. Baseman JB., Cole RM., Krause DC., Leith DK. Molecular basis for cytadsorption of Mycoplasma pneumoniae. Journal of Bacteriology 1982; 151(3): 1514-1522.
5. Bose SR., Balish MF., Krause DC. Mycoplasma pneumoniae cytoskeletal protein HMW2 and the architecture of the terminal organelle. Journal of Bacteriology 2009; 191(21): 6741-6748.
6. Feldner J., Gobel U., Bredt W. Mycoplasma pneumoniae adhesin localized to tip structure by monoclonal antibody. Nature 1982; 298: 765-767.
7. Gietz RD., Woods RA. Yeast transformation by the LiAc/SS carrier DNA/PEG method. In: Xiao W. (ed.) Yeast Protocol. Methods in Molecular Biology. NJ: Humana Press 2006; 313.
8. Hahn TW., Willby MJ., Krause DC. HMW1 is required for cytadhesin P1 trafficking to the attachment organelle in Mycoplasma pneumoniae. Journal of Bacteriology 1998; 180: 1270-1276.

9. Hashemifar S., Neyshabur B., Khan AA., Xu J. Predicting protein–protein interactions through sequence-based deep learning. Bioinformatics 2018; 34(17): 802-810.
10. Hegermann J., Herrmann R., Mayer F. Cytoskeletal elements in the bacterium Mycoplasma pneumoniae. Naturwissenschaften 2002; 89: 453-458.
11. Hu PC., Collier AM., Baseman JB. Surface parasitism by Mycoplasma pneumoniae of respiratory epithelium. Journal of Experimental Medicine 1977; 145: 1328-1343.
12. Inamine JM., Ho KC., Loechel S., Hu PC. Evidence that UGA is read as a tryptophan codon rather than as a stop codon by Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma gallisepticum. Journal of Bacteriology 1990; 172(1): 504-506.
13. Inamine JM., Loechel S., Hu PC. Analysis of the nucleotide sequence of the P1 operon of Mycoplasma pneumoniae. Gene 1988; 73: 175-183.
14. Jumper J., Evans R., Pritzel A., Green T., Figurnov M., Ronneberger O., Tunyasuvunakool K., Bates R., Zidek A., Potapenko A., Bridgland A. Highly accurate protein structure prediction with AlphaFold. Nature 2021; 596(7873): 583-589.
15. Krause DC. Mycoplasma pneumoniae cytadherence: unravelling the tie that binds. Molecular Microbiology 1996; 20: 247-253.
16. Krause DC., Proft T., Hedreyda CT., Hilbert H., Plagens H., Herrmann R. Transposon mutagenesis reinforces the correlation between Mycoplasma pneumoniae cytoskeletal protein HMW2 and cytadherence. Journal of Bacteriology 1997; 179(8): 2668-77.
17. Kuzmanov U., Emili A. Protein-protein interaction networks: probing disease mechanisms using model systems. Genome Medicine 2013; 5: 1-12.
18. Liu Y., Li J., Lu X., Zhen S., Huo J. Toll‐like receptor 4 exacerbates Mycoplasma pneumoniae via promoting transcription factor EB‐Mediated autophagy. Contrast Media and Molecular Imaging 2022; 1: 3357694.
19. Makuch L. Yeast two-hybrid screen. Methods in Enzymology. MD: Academic Press; 2014; 539, 31-51.
20. Mehla J., Caufield JH., Uetz P. Mapping protein–protein interactions using yeast two-hybrid assays. Cold Spring Harbor Protocols 2015; 5: pdb-prot086157.
21. Meng KE., Pfister RM. Intracellular structures of Mycoplasma pneumoniae revealed after membrane removal. Journal of Bacteriology 1980; 144: 390-399.
22. Ori A., Iskar M., Buczak K., Kastritis P., Parca L., Andres-Pons A., Singer S., Bork P., Beck M. Spatiotemporal variation of mammalian protein complex stoichiometries. Genome Biology 2016; 17: 1-15.
23. Popham PL., Hahn TW., Krebes KA., Krause DC. Loss of HMW1 and HMW3 in noncytadhering mutants of Mycoplasma pneumoniae occurs post-translationally. Proceedings of the National Academy of Sciences 1997; 94: 13979- 13984.
24. Radestock U., Bredt W. Motility of Mycoplasma pneumoniae. Journal of Bacteriology 1977; 129: 1495-1501.
25. Razin S., Yogev D., Naot Y. Molecular biology and pathogenicity of mycoplasmas. Microbiology and Molecular Biology Reviews 1998; 62(4): 1094-1156.
26. Sambrook J., Fritsch EF., Maniatis T. Molecular cloning: a laboratory manual. NY: Cold Spring Harbor Laboratory Press; 1989.
27. Schurwanz N., Jacobs E., Dumke R. Strategy to create chimeric proteins derived from functional adhesin regions of Mycoplasma pneumoniae for vaccine development. Infection and Immunity 2009; 77(11): 5007-15.
28. Stevens MK., Krause DC. Localization of the Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW4 in the cytoskeleton-like Triton shell. Journal of Bacteriology 1991; 173(3): 1041-1050.
29. Sun T., Zhou B., Lai L., Pei J. Sequence-based prediction of protein protein interaction using a deep-learning algorithm. BMC Bioinformatics 2017; 18: 1-8.
30. Szklarczyk D., Franceschini A., Kuhn M., Simonovic M., Roth A., Minguez P., Doerks T., Stark M., Muller J., Bork P., Jensen LJ. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Research 2010; 39(suppl_1): D561-D568.
31. Titeca K., Lemmens I., Tavernier J., Eyckerman S. Discovering cellular protein-protein interactions: Technological strategies and opportunities. Mass Spectrometry Reviews 2019; 38(1): 79-111.
32. Torres A., Cilloniz C., Niederman MS., Menendez R., Chalmers JD., Wunderink RG., van der Poll T. Pneumonia. Nature Reviews Disease Primers 2021; 7: 25.
33. Tully JG. New laboratory techniques for isolation of Mycoplasma pneumoniae. Yale Journal of Biology and Medicine 1983; 56: 511-515.
34. Wang Y., Ma C., Hao X., Wang W., Luo H., Li M. Identification of Mycoplasma pneumoniae proteins interacting with NOD2 and their role in macrophage inflammatory response. Frontiers in Microbiology 2024; 15: 1391453.
35. Willby MJ., Balish MF., Ross SM., Lee KK., Jordan JL., Krause DC. HMW1 is required for stability and localization of HMW2 to the attachment organelle of Mycoplasma pneumoniae. Journal of Bacteriology 200; 186(24): 8221-8228.
36. Yu Y., Zhang L., Chen Y., Li Y., Wang Z., Li G., Wang G., Xin J. GroEL protein (heat shock protein 60) of Mycoplasma gallisepticum induces apoptosis in host cells by interacting with Annexin A2. Infection and Immunity 2019; 87(9): 10-128.
37. Zhang J., Guan M., Wang Q., Zhang J., Zhou T., Sun X. Single-cell transcriptome-based multilayer network biomarker for predicting prognosis and therapeutic response of gliomas. Briefings in Bioinformatics 2020; 21(3): 1080-1097.