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

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.