A new model for sensitive detection of zoonotic prions by PrP transgenic Drosophila

Abstract

Prions are transmissible protein pathogens most reliably detected by a bioassay in a suitable host, typically mice. However, the mouse bioassay is slow and cumbersome, and relatively insensitive to low titers of prion infectivity. Prions can be detected biochemically in vitro by the protein misfolding cyclic amplification (PMCA) technique, which amplifies disease-associated prion protein but does not detect bona fide prion infectivity. Here, we demonstrate that Drosophila transgenic for bovine prion protein (PrP) expression can serve as a model system for the detection of bovine prions significantly more efficiently than either the mouse prion bioassay or PMCA. Strikingly, bovine PrP transgenic Drosophila could detect bovine prion infectivity in the region of a 10^-12 dilution of classical bovine spongiform encephalopathy (BSE) inoculum, which is 10⁶-fold more sensitive than that achieved by the bovine PrP mouse bioassay. A similar level of sensitivity was observed in the detection of H-type and L-type atypical BSE and sheep-passaged BSE by bovine PrP transgenic Drosophila. Bioassays of bovine prions in Drosophila were performed within 7 weeks, whereas the mouse prion bioassay required at least a year to assess the same inoculum. In addition, bovine PrP transgenic Drosophila could detect classical BSE at a level 10⁵-fold lower than that achieved by PMCA. These data show that PrP transgenic Drosophila represent a new tractable prion bioassay for the efficient and sensitive detection of mammalian prions, including those of known zoonotic potential.

Keywords: Drosophila; PrP(Sc); bioassay; classical and atypical BSE; infectious disease; mouse; neurodegenerative disease; prion; protein misfolding.

Figure 1

Serial transmission of prion-seeding activity in classical BSE-exposed bovine PrP Drosophila.Elav x bovine PrP (Bov) or Elav x 51D (51D) Drosophila were exposed to classical BSE-infected or prion-free control bovine brain material at the larval stage. The head homogenate was prepared from 5- and 40-day-old adult Drosophila (first-passage flies) and used to inoculate fresh Elav x bovine PrP Drosophila (second-passage flies) at the larval stage. At various times after hatching, the head homogenate was prepared from harvested secondary-passage Elav x bovine PrP Drosophila and used as seed in PMCA reactions. Western blot was used to detect PK-resistant PrP27-30 in PMCA reaction products. Molecular mass markers in kilodalton are shown on the left. WB control denotes the Western blot control comprising PG127 scrapie-infected sheep brain material included to highlight the low-molecular-weight band of unglycosylated BSE PK-resistant PrP^Sc. BSE, bovine spongiform encephalopathy; C-BSE, classical BSE; PMCA, protein misfolding cyclic amplification.

Figure 2

Sensitivity of prion-seeding activity detection in classical BSE-exposed bovine PrP Drosophila. Adult Elav x bovine PrP Drosophila were assessed for prion-seeding activity after exposure at the larval stage to 10⁻² to 10⁻²⁰ dilutions of classical BSE-infected bovine brain material. The control inoculum was a 10⁻² dilution of prion-free bovine brain homogenate. At various times after hatching, the head homogenate was prepared from harvested bovine PrP Drosophila and used as seed in PMCA reactions. Western blot was used to detect PK-resistant PrP27-30 in PMCA reaction products seeded with the head homogenate from Drosophila exposed to classical BSE-infected or control bovine brain homogenate. (Data for adult Elav x bovine PrP Drosophila exposed to 10⁻² to 10⁻¹⁶ dilutions of classical BSE-infected bovine brain material are shown). Molecular mass markers in kilodalton are shown on the left. WB control denotes Western blot control comprising PG127 scrapie-infected sheep brain material included to highlight the low-molecular-weight band of unglycosylated BSE PK-resistant PrP27-30. BSE, bovine spongiform encephalopathy; PK, proteinase K; PMCA, protein misfolding cyclic amplification.

Figure 3

Accelerated loss of locomotor ability in classical BSE-exposed bovine PrP Drosophila. Adult Elav x bovine PrP Drosophila were assessed for their locomotor ability by a negative-geotaxis climbing assay after exposure at the larval stage to 10⁻² to 10⁻²⁰ dilutions of classical BSE-infected bovine brain material. Control inoculum was a 10⁻² dilution of prion-free bovine brain material (control 10⁻²). The data shown are linear regression plots of the mean performance index ± SD for three groups of flies per time point calculated as described in Experimental procedures. Statistically significant responses were demonstrated between days 15 and 40 of the locomotor ability assay when assessed by one-way ANOVA (with Dunnett’s multiple comparisons test) for classical BSE dilutions 10⁻² to 10⁻¹⁰versus prion-free control bovine brain material (p ≤ 0.029) and unpaired Student’s t test (two tailed) for classical BSE diluted 10⁻¹²versus prion-free control bovine brain material (p = 0.0016). BSE, bovine spongiform encephalopathy; C-BSE, classical BSE.

Figure 4

Accelerated loss of survival of classical BSE-exposed bovine PrP Drosophila. Adult Elav x bovine PrP Drosophila were assessed for survival after exposure at the larval stage to 10⁻² to 10⁻²⁰ dilutions of classical BSE-infected bovine brain material. The control inoculum was a 10⁻² dilution of prion-free bovine brain material (control 10⁻²). The number of surviving flies was recorded three times a week as described in Experimental procedures and the data shown as Kaplan–Meier plots. BSE, bovine spongiform encephalopathy; C-BSE, classical BSE.

Figure 5

Accelerated loss of locomotor ability in H- or L-type atypical BSE- or ovine BSE-exposed bovine PrP Drosophila. Adult Elav x bovine PrP Drosophila were assessed for their locomotor ability by a negative-geotaxis climbing assay after exposure at the larval stage to the dilutions shown of (A) H-type BSE; (B) L-type BSE; or (C) ovine BSE inoculum. The control inoculum was a 10⁻² dilution of prion-free bovine brain material (control 10⁻²). The data shown are linear regression plots of the mean performance index ± SD for three groups of flies per time point calculated as described in Experimental procedures.