Understanding how penicillin resistance develops in Streptococcus pneumoniae clinical populations

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Project BackgroundStreptococcus pneumoniae is a successful human pathogen and a leading cause of pneumonia and death in the world. Penicillins (and similar drugs) are commonly used to treat these infections; however a growing number of antibiotic resistant strains are emerging globally which threaten patients. To improve disease management, we must understand how S.pneumoniae cells deal with penicillin stress and how this selection shapes bacterial populations.Recent work in the Fenton lab has revealed loss-of-function mutations in the gene called pde1 leads to penicillin resistance in S.pneumoniae clinical populations. Pde1 is part of the second messenger bacterial stress response and to study its function we carried out whole-genome profiling (Tn-seq), identifying four genes required for pde1-dependent penicillin resistance. We hypothesise these proteins form a complex, linking second messenger signalling with cell wall biology and penicillin drug targets.Objectives1) Characterise the functions of genes required for Pde1-dependent penicillin resistance in S.pneumoniae 2) Measure the effect each locus has on penicillin resistance across clinical strains 3) Develop a molecular model linking genetic variation in these genes to penicillin resistance in clinical populations.TimelinessSet against the backdrop of increasing antimicrobial resistance there is a clear and timely need to develop new control regimes for S.pneumoniae infections. This project builds on our labs recent findings that pde1-dependent penicillin resistance is an important 'gateway' towards penicillin resistance and will develop a molecular understanding for how this biology works.This project combines the expertise of multiple labs with complementary experience in: molecular genetics, bacterial cell signalling and evolutionary biology. This formidable collaboration will generate fundamental understanding of S.pneumoniae biology, using this towards improving treatment and surveillance approaches.Research ApproachOur work has identified genes required for pde1-dependant penicillin resistance in S.pneumoniae. We hypothesise two are involved in cell wall stress, whilst two are involved in sugar-phosphate metabolism. To meet our objectives, Objective1 will involve purifying each protein and carrying out protein binding assays using second messenger signalling-nucleotides and cell wall substrates. Structural modelling suggests identified proteins form a membrane-spanning complex and we will test this using split-luciferase assays and live-cell co-localisation microscopy, modelling the putative complex using AlphaFold. Objective2, will use our clinical isolate collection to test the impact these genes have on penicillin resistance across the species. Objective3, will use our comparative genomics methods to study genetic variation within the four genes across penicillin resistant isolates. Combined, this data will generate a model for how penicillin resistance emerges in S.pneumoniae clinical populations.