Authors:

Lucas Riley Hendershot

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About Paper:

Acanthamoeba, a genus of pathogenic free-living amoeba, are capable of inflicting significant diseases in humans and animals alike. Species belonging to the Acanthamoeba are responsible for causing granulomatous amoebic encephalitis (GAE), Acanthamoeba keratitis, skin and systemic diseases. Currently, the best treatment for these infections involves a chemical combinational therapy made up of non- specific anti-biotics, anti-fungals, and anti-parasitics. However, recent studies have suggested Azithromycin, a macrolide antibiotic, to be a frontline therapeutic against diseases caused by Acanthamoeba and other free-living amoeba. Azithromycin has been proven to work by binding to the 50S subunit, specifically the 23S rRNA subunit in bacterial ribosomes. Upon binding, azithromycin reduces protein synthesis and prevents cell growth. Unfortunately, this mechanism has only been validated in bacteria and not in any amoeba species. This study aims to develop azithromycin resistant Acanthamoeba mutants to potentially yield the phenotypical mechanism of action or resistance pathway(s). Developing resistant mutants capable of reaching a 100-fold shift in its IC50 towards a compound is the first step in discovering a mechanical mode of action (MMOA) within Acanthamoeba. Mutants were developed using a step-wise approach and now we have mutants growing in 60x- 80x concentrations of the original azithromycin IC50. We will use specific target sequencing and potentially whole genome sequencing of mutants to identify azithromycin's MOA. If successful, this study would be the first to utilize drug resistant Acanthamoeba for studying molecular modes of action. Understanding the MMOA of Acanthamoeba is vital to developing enhanced therapeutic drugs that can effectively mitigate the diseases caused by this organism. Keywords: Acanthamoeba; Free-Living Amoeba; GAE; Mechanism of Action; Drug Resistance

Source:

Purdue University / 2025

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Co-authors:

Lucas Riley Hendershot