Charting the fitness landscape of a penicillin-binding protein

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The rapid rise of antimicrobial resistance (AMR) is one of the most pressing problems affecting the world. AMR is directly causing 1.3 million deaths a year, and is expected to plunge 24 million people into extreme poverty by 2030 (WHO and the UN Interagency Coordinating Group on Antimicrobial Resistance). The need to combat AMR is extremely urgent.A common way for microbes to evolve AMR is through mutations. For instance, mutations can accumulate and affect the function of bacterial genes that mediate antibiotic resistance. Beta-lactam (BL) antibiotics are one of the most widely prescribed antibiotics, and they kill bacteria by binding and inhibiting their penicillin-binding proteins. As such, mutations that cause amino acid substitutions in penicillin-binding proteins can allow a bacterium to escape the killing effect of BL antibiotics.Group A Streptococcus (GAS) is a notable human pathogen that was deemed highly susceptible to BL antibiotics. However, clinical isolates of GAS are found to be less susceptible to BL antibiotics in recent years, and there is an emerging concern that reduced antibiotic susceptibility will pave the way to full-blown antibiotic resistance in GAS. The decreasing susceptibility to BL antibiotics in GAS is primarily driven by substitutions in a particular penicillin-binding protein called PBP2x. In the absence of prior knowledge, it is nearly impossible to understand the impact of these individual substitutions and their numerous combinations on PBP2x and the bacterial susceptibility to BL antibiotics, let alone predict which substitutions will lead to further evolution of resistance to BL antibiotics.The questions that my study will address are: Which substitutions in PBP2x will contribute directly to the evolution of BL antibiotic resistance in GAS, which ones are redundant? Which substitutions are more evolutionarily accessible? How do genetic and environmental factors mediate or constrain the acquisition of these substitutions?Here, I will dissect and predict the impact of PBP2x substitutions on the bacterial susceptibility to BL antibiotics using a combination of experimental and computational approaches. First, I will quantify the effects of pbp2x mutations comprehensively under the selection of three types of BL antibiotics and two non-BL antibiotics. This will allow me to capture the mutational effects of thousands and millions of pbp2x mutations in parallel, and map the relationship between pbp2x mutations and bacterial susceptibilities to the various types of antibiotics. Next, I will also investigate the mutational effects of these pbp2x mutations in bacterial hosts with different genetic backgrounds. This line of study will reveal unexpected genetic interactions between mutations that would otherwise be difficult to predict. An example of such a scenario is when a pbp2x mutation does not make a bacterium less susceptible to an antibiotic, but its presence is absolutely essential for the accumulation of other more deleterious mutations. On the whole, the outcome of this research will allow us to distinguish which pbp2x mutations are directly relevant to GAS resistance to BL antibiotics, and/or which ones are important for the evolution of BL antibiotic resistance. Insights derived from this research will help us to predict bacterial variants of concern ahead of time, which can lead to further implications for combatting AMR.