Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of ...these targets, T6SS potency can vary across recipient species. To understand the functional basis of intrinsic T6SS susceptibility, we screened for essential Escherichia coli (Eco) genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin from Pseudomonas aeruginosa, Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, lipopolysaccharide, that modulate Tae1 toxicity in vivo. Disruption of genes in early lipopolysaccharide biosynthesis provided Eco with novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistant Eco exhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, our study reveals the complex functional underpinnings of susceptibility to Tae1 and T6SS which regulate the impact of toxin-substrate interactions in vivo.
Insertions and deletions (indels) enable evolution and cause disease. Due to technical challenges, indels are left out of most mutational scans, limiting our understanding of them in disease, ...biology, and evolution. We develop a low cost and bias method, DIMPLE, for systematically generating deletions, insertions, and missense mutations in genes, which we test on a range of targets, including Kir2.1. We use DIMPLE to study how indels impact potassium channel structure, disease, and evolution. We find deletions are most disruptive overall, beta sheets are most sensitive to indels, and flexible loops are sensitive to deletions yet tolerate insertions.
Mutations in transporters can impact an individual’s response to drugs and cause many diseases. Few variants in transporters have been evaluated for their functional impact. Here, we combine ...saturation mutagenesis and multi-phenotypic screening to dissect the impact of 11,213 missense single-amino-acid deletions, and synonymous variants across the 554 residues of OCT1, a key liver xenobiotic transporter. By quantifying in parallel expression and substrate uptake, we find that most variants exert their primary effect on protein abundance, a phenotype not commonly measured alongside function. Using our mutagenesis results combined with structure prediction and molecular dynamic simulations, we develop accurate structure-function models of the entire transport cycle, providing biophysical characterization of all known and possible human OCT1 polymorphisms. This work provides a complete functional map of OCT1 variants along with a framework for integrating functional genomics, biophysical modeling, and human genetics to predict variant effects on disease and drug efficacy.
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•Deep mutational scan of OCT1 reveals the determinants of biogenesis and substrate uptake•Discovery of a conserved motif, the stability helix, essential to SLC22 biogenesis•AI structure prediction and mutational data for accurate structure-function modeling•Integrating genomic-health records reveals variants effect on human physiology
Yee et al. create a complete functional map of how genetic variants in the OCT1 transporter alter biogenesis and substrate uptake. This work provides a framework for biophysically informed precision medicine by integrating functional genomics, mechanistic modeling, and human genetics to predict variant effects on disease and drug efficacy.
