Files
Abstract
The opportunistically pathogenic free-living amoeba, Naegleria fowleri, is highly understudied and causes primary amoebic meningoencephalitis (PAM) in humans. PAM is a devastating neurological infection that is caused when amoeba-contaminated water is forced up the nose, allowing the pathogen to adhere to the nasal cavity and migrate to the brain. Once in the brain, N. fowleri induces massive inflammation and damage, leading to death in >97% of its victims in only 5-12 days. The currently recommended drug regimens consist of empirically derived cocktails of toxic antimicrobial compounds with very low success rates when implemented to treat PAM. Additionally, the early symptoms of PAM are unremarkable while the late symptoms closely resemble more common bacterial/viral meningitis, leading to incorrect and late diagnoses in most cases. Current diagnoses of PAM target the cerebrospinal fluid (CSF) and rely on invasivespinal taps followed by subjective microscopic visualization of amoebae with definitive diagnoses in the U.S. requiring shipment to one of two off-site diagnostic centers for qPCR. Thus, there is a need for further studies on the universal effectiveness of therapeutics to multiple isolates of N. fowleri, a deeper understanding of amoebic pathogenesis, and the development of more sensitive diagnostics targeting other biofluids that are applicable in point-of-care situations.
In this study, we tested multiple N. fowleri clinical isolates and discovered significant differences in growth rate and susceptibility to drugs in the currently recommended treatment regimen. Furthermore, we characterized the ultrastructure of multiple axenically cultured N. fowleri clinical isolates versus those actively feeding on mammalian cells as well as N. fowleri-secreted extracellular vesicles (Nf-EVs) via scanning electron microscopy. Additionally, we identified possible routes of Nf-EV secretion, characterized the Nf-EV protein contents, and demonstrated that Nf-EVs are actively taken up by multiple mammalian cell lines and other amoebae. Moreover, we sequenced the RNA contents of Nf-EVs and identified several highly prevalent small RNA biomarkers that can be detected in the blood and urine of infected mice and in the CSF and blood of infected humans. Lastly, we tested several clinical isolates in the mouse model of PAM and determined that they significantly differ in virulence as well as minimal infectious dose.