The understanding of the molecular mechanisms by which pathogens like viruses, bacteria, fungi, and parasites operate, particularly in relation to their host interactions, is essential to effectively develop therapeutic lead candidates. Protein–protein interactions serve as fundamental components of cellular protein complexes and pathways and play a crucial role in determining protein functionality. The yeast two-hybrid (Y2H) screening method remains a valuable tool for analyzing the interactomes of various model organisms and pathogens, as well as for investigating host–pathogen interactions.
In order to achieve reproducible and comprehensive Y2H outcomes, we developed a patented mating protocol that allows 83 million interactions to be tested, on average, per screen. Additionally, we used a domain-based strategy to create highly intricate, random-primed cDNA/genomics libraries with several million independent clones in yeast derived from several bacteria and viruses, such as Caulobacter crescentus, Escherichia coli, and Streptococcus pneumoniae, as well as human tissues, rodents, flies, and plants. The use of protein domain libraries allows for the isolation of multiple independent fragments for each interactant, facilitating the immediate identification of minimally interacting domains and the calculation of confidence scores.
We used this methodology to investigate biological pathways in Staphylococcus aureus. A total of 26 essential proteins from S. aureus were screened against our highly complex genomic library of the same organism. Among the identified proteins are critical genes associated with cell wall biosynthesis, cell division, and antibiotic resistance to allow for an understanding of the underlying molecular pathways and the development of therapeutic targeting and antimicrobial resistance research. The resulting protein interaction map identified 167 CDSs that are connected by 191 protein–protein interactions (PPIs), and, on average, every bait protein has 9.6 PPIs and can therefore can serve as a foundational resource for identifying potential new targets for antimicrobial drug development and future treatments.
