Browsing by Subject "Screening Assays"

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    Elucidating Sequence-Function Relationships of AMPs via Advancement of Screening Assays
    (2024-01) Collins, Jonathan
    Antimicrobial resistance is drastically increasing worldwide as fewer treatment options are being developed. Antimicrobial proteins (AMPs) have the potential to address the root causes of this impending crisis. However, to effectively develop AMPs with therapeutic efficacy, platforms used to characterize and evolve existing AMPs require drastic increases in throughput. This work seeks to implement the technological and scientific aims of 1) advancing AMP screening assays to increase high-integrity throughput multiple orders of magnitude and 2) use them to elucidate sequence-function landscapes of AMPs and identify potent variants. We pursued two platform technologies to efficiently link antimicrobial activity to the AMP-encoding gene: self-depletion of a host cell expressing AMP intracellularly and physical encapsulation of an AMP-expressing host cell and its target pathogen with a fluorescent metric for cell viability.We advanced a nascent technology from the Hackel lab, Sequence Activity Mapping via Depletion (SAMP-Dep), to characterize proline-rich AMPs (PrAMPs) that target protein production through ribosomal inhibition. All single and localized double mutants of three PrAMPs derived from insects and mammals were internally produced in E. coli bacteria. Depletion from the population, quantified by deep sequencing, determined relative potency of PrAMP variants, elucidating sequence-function relationships. External treatment with purified variants, selected from platform screening, corroborated activity for insect-derived PrAMPs. In contrast, mammalian-derived PrAMPs showed high potency for all variants selected, even the most statistically inactive variant from SAMP-Dep screening. This work expands the breadth of SAMP-Dep to PrAMPs while highlighting its focus on intracellular mechanisms of action. In the second platform, co-encapsulation of both target pathogens and AMP-secreting bacterial host within an agarose microdroplet exhibited differential fluorescence – with a cellular viability dye – of active and inactive AMPs in clonal experiments. However, deep sequencing upon sorting mixtures of droplets showed no enrichment of active AMP secretors. Long-term staining of secreting host bacteria within droplets confirmed crosstalk between droplets caused the experimental inability to distinguish droplets encapsulating active vs. inactive secreting hosts. Overall, this work advanced knowledge of AMP sequence-function relationships, identified potent AMP variants with capacity for therapeutic utility, and identified and expanded the bounds of implementation for high-integrity, high-throughput AMP screening methods.