Thursday, November 21, 2024
11/21/2024
Project Summary Bacteriophage, or phage, infect and kill bacterial cells. At a programmed time, phage use a tailored suite of proteins to release newly built virions by lysing the host cell. As the most common cell lysis event on the planet, phage-induced lysis drives both microbial biogeochemical nutrient cycling and population dynamics in cellular life. Host cell lysis in tailed phages is an active process carried out by phage proteins that specifically target each cell envelope layer. In Gram-negative bacteria, phage holin proteins compromise the inner membrane, endolysins degrade the peptidoglycan, and spanins disrupt the outer membrane. Molecular studies in a few classic systems, such as phages T4 and lambda, exposed common functional principles governing lysis. However, striking differences in their mechanisms of action also taught us about bacterial envelope layers and their regulation. In addition, bacteria often co-opt phage lysis proteins to accomplish diverse behaviors beneficial to a population such as seeding biofilm establishment and releasing toxins. Therefore, studying novel phage lysis proteins will advance our understanding of both phage and fundamental principles in their hosts. Further, we and others have demonstrated that identifying or predicting the function of novel lysis proteins based on sequence analysis alone is ineffective. Therefore, the studies proposed here focus on experimental characterization of novel phage lysis protein mechanisms. For example, in E. coli phage Mu, where bioinformatics predicted a canonical lysis pathway, we demonstrated that Mu lyses cells using a protein of unknown mechanism in place of a holin. In a second example, we showed that E. coli phage phiKT produces an antimicrobial peptide-like protein instead of spanins to disrupt the outer membrane, akin to eukaryotic antimicrobial peptides. Finally, we have identified for study lysis protein candidates that are significantly different from known proteins in phages infecting abundant and understudied human gut microbiota. Our goals for the next five years center on in-depth molecular characterization of two specific novel lysis proteins and diverse candidates from less-studied phages infecting gut microbiota. We will use genetic, biochemical, and microscopy approaches to elucidate mechanisms of individual proteins. Direct competition assays will probe the contributions of lysis proteins to phage persistence and fitness, characteristics that drive environmental microbial population composition. Overall, these projects are expected to uncover divergent mechanisms of action used by phage in E. coli model systems and other prevalent gut microbiota. Broadly, this foundation can be leveraged to study lysis protein types active across many different bacteria, which is critical to understanding global microbial population fitness and turnover. The new molecular strategies uncovered here may lead to more efficacious medical treatments since lysis is also the basis of successful phage therapy, which aspires to use phages as alternative treatments for antibiotic-resistant bacterial infections.
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