In this study, Naegleria fowleri, a pathogenic amoeba and the causative agent of Primary Amebic Meningoencephalitis (PAM), was utilized to determine the presence or absence of classically conserved Golgi molecules featured in the expression of a Golgi apparatus. Previous studies concluded no Golgi expression via light microscopy and transmission electron microscopy, but a recent report on Naegleria gruberi indicated the presence of dispersed Golgi tubules. Non-pathogenic species of the Naegleria genus such as Naegleria gruberi 30540 and Naegleria lovaniensis 30569 were utilized in Western immunoblot analysis compared to reduced whole-cell lysate proteins of two strains of N. fowleri and Vero CCL-81, Chlorocebus sp. kidney epithelial cells, which were utilized as a positive control for Golgi expression. N. fowleri and N. lovaniensis whole-cell lysates had indications of a 110 kDa reduced protein, associated with the predicted molecular weights of the beta-COPI subunit of the COPI cis-Golgi vesicular transport complex with further Western immunoblot indication of a weak band around 25 kDa corresponding to rabbit polyclonal antibodies specific for ARF1. Serial Dilutions of Wheat Germ Agglutinin Alexa Fluor 488TM were performed on Vero cells, Naegleria fowleri 30894, and N. gruberi 30540 with 1:100 dilution of recommended stock dilution of WGA 488 determined for utilization in sequential immunofluorescence. Sequential immunofluorescence with Wheat Germ Agglutinin Alexa Fluor 488TM and then blocked with 3% BSA:PBS [wt/vol] dilution with subsequent incubation in rabbit anti-beta-COPI primary 1:250, and 1:1000 of Alexa Fluor 594 goat anti-rabbit secondary antibody exposure showed strong indications of organized cis- and trans-punctate Golgi body markers in close association in individual and dividing cells of Naegleria fowleri and conserved Golgi expression in the positive control Vero cells, but further experiments are necessary to verify this finding with N. fowleri.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-7104 |
| Date | 01 January 2019 |
| Creators | Poe, Tyler M, Marciano-Cabral, Francine |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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