Yeast-based biosensors for the specific and accessible detection of pathogens and antimicrobial resistance

Antimicrobial resistance is one of the most pressing challenges facing global health today. In particular, low- and middle-income countries bear the brunt of this crisis, where the misuse and overuse of antibiotics have accelerated the emergence of resistant strains. Among the most formidable of these are the ESKAPE pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. These bacteria are known for their ability to evade the effects of antibiotics, making infections difficult to treat and increasing the risk of severe outcomes. The AntiRYB project, an innovative research initiative, seeks to tackle this problem head-on by developing a cutting-edge yeast-based biosensor. This biosensor leverages the common yeast Saccharomyces cerevisiae, which has been genetically modified to express engineered G-protein coupled receptors (GPCRs). These GPCRs are designed to detect specific peptides that signify the presence of particular pathogens. When the GPCRs recognize these peptides, they initiate a series of reactions inside the yeast cell that ultimately lead to the production of a red pigment. The appearance of this pigment serves as a simple, visible indicator of contamination by carbapenemase-producing bacterial strains, which are among the most dangerous due to their resistance to last-resort antibiotics. The development of this biosensor is grounded in three key areas of research. First, molecular modeling and docking studies were conducted to identify and optimize the residues in the yeast GPCR Ste2, improving its sensitivity and specificity for target peptides. This work laid the foundation for creating a highly efficient receptor. Next, a sophisticated screening platform was developed. This involved constructing a yeast strain that could host a library of GPCR variants. Through high-throughput screening methods, including fluorescence-activated cell sorting (FACS), researchers identified receptor variants that responded robustly to the target peptides. This phase was crucial in ensuring that the biosensor could reliably detect the presence of antimicrobial-resistant pathogens. Finally, proteomic analysis of clinical strains of Pseudomonas aeruginosa provided deeper insights into the mechanisms of antibiotic resistance. By comparing the proteomes of resistant and non-resistant strains under various conditions, the team identified key proteins and pathways involved in resistance. This knowledge not only informed the design of the biosensors but also highlighted potential targets for future therapeutic interventions. In the face of the COVID-19 pandemic, the importance of infection control has never been more apparent. The AntiRYB project team has actively communicated the significance of combating antibiotic resistance, particularly in an era where the use of antibacterial products has surged. Their efforts include public presentations and articles aimed at raising awareness about the urgent need for rapid, effective detection methods for antimicrobial resistance. The advancements achieved by the AntiRYB project represent a significant step forward in the fight against resistant pathogens. By developing a yeast-based biosensor that is both highly sensitive and easy to use, this project offers a promising tool for improving biosecurity and disease surveillance worldwide. The ultimate goal is to deploy these biosensors in various settings, from hospitals to farms, enabling quick and accurate detection of dangerous bacteria and helping to curb the spread of antibiotic resistance.

The AntiRYB project has achieved significant technical milestones across its three main areas of focus. In molecular modeling and docking, we identified and optimized key residues in the yeast GPCR Ste2, leading to the design of receptor variants with improved sensitivity and specificity to target peptides. This advancement enabled the construction of a robust GPCR screening platform in Saccharomyces cerevisiae. Through strategic mutagenesis and high-throughput FACS screening, we identified promising receptor variants responsive to specific antimicrobial resistance markers. In parallel, the proteomics analysis of clinical strains of Pseudomonas aeruginosa provided an in-depth understanding of the proteomic changes associated with Imipenem resistance, revealing key resistance mechanisms and potential therapeutic targets. These findings facilitated the development of highly sensitive and specific yeast-based biosensors capable of detecting resistant pathogens and their resistance mechanisms. These biosensors have the potential to be deployed for rapid, on-site detection of pathogens in various settings, significantly enhancing biosecurity and disease surveillance. The technical achievements of the AntiRYB project thus contribute to advancing synthetic biology and antimicrobial resistance research while paving the way for practical applications in pathogen detection and control.

Early and specific detection of microbial infections is crucial for the containment of diseases and for reducing the dependence on the use of antibiotics. There is however a lack of reliable, cheap and easy to use detection methods for day-to-day monitoring of infection and antimicrobial resistance in samples from patients, animals and the environment. This deficiency is critical for the abuse of antibiotics and the diffusion of antimicrobial resistance. The aim of this project is to establish a method based on yeast biosensors that will detect with high specificity pathogens from different sources to develop a new, fast and specific diagnostic tool for resistant pathogens as well as specific bacterial species. We will achieve this by joining together strong research groups on antimicrobial resistance, systems biology, and strain engineering at SINTEF, Chalmers University and National Medicines Institute. Particular focus will be given to the detection of ESBL or carbapenemase-producing strains belonging to the emerging ESKAPE group of resistant pathogens. The biosensor is developed using the yeast Saccharomyces cerevisiae as host, which will be engineered to express specific receptors able to recognise unique molecules produced by the pathogens. The ligand-receptor binding initiates a cascade mechanism that activates the genes for the production of a red pigment visible to the naked eye. Using the biosensors, we aim to identify molecular markers specific for resistant pathogen strains, to enable fast, easy and inexpensive point-of-use profiling of resistant pathogens.