Establishing Gene Expression System in Naegleria

Abstract

By Kyla Doan, Biological Sciences

Advisor: Yoshi Odaka

Award: Excellence in Research Communication

Presentation ID: 261

Abstract: Naegleria fowleri is a pathogenic amoeba responsible for primary amebic meningoencephalitis (PAM), a rare but highly fatal infectious disease of the central nervous system with over a 97% fatality. To better understand the biology of Naegleria and to identify potential drug targets, we aim to establish a gene expression system in non-pathogenic Naegleria gruberi. Previously, we reported that N. gruberi transforms to a metabolically dormant cyst-like form when treated with TOR (target of rapamycin) enzyme inhibitor, Torin-1, suggesting that TOR may influence the transformation through the direct or indirect phosphorylation of its target proteins. Further analysis identified that 228 phosphoproteins were highly sensitive to TOR activities. The enzyme, enolase, one of the identified phosphoproteins that participates in cellular respiration, is of particular interest because it has been implicated in cyst formation in Naegleria and another pathogenic amoeba, Entamoeba. Therefore, we hypothesize that TOR controls the transformation of amoeba through regulating enolase function. The promoter region of the Naegleria ubiquitin (Ubi) gene was cloned and amplified by polymerase chain reaction (PCR) and placed in the upstream region of the RFP (red fluorescent protein) gene in the mammalian gene expression vector. Using the expression system, the Ubi promoter-driven chimeric gene, encoding GFP (green fluorescent protein)-fused enolase, was also generated. We are currently investigating whether the system can express these genes and where GFP-enolase localizes within the amoeba.