Propagation of RML Prions in Mice Expressing PrP Devoid of GPI Anchor Leads to Formation of a Novel, Stable Prion Strain

PrPC, a host protein which in prion-infected animals is converted to PrPSc, is linked to the cell membrane by a GPI anchor. Mice expressing PrPC without GPI anchor (tgGPI- mice), are susceptible to prion infection but accumulate anchorless PrPSc extra-, rather than intracellularly. We investigated whether tgGPI− mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrPSc. We found that RML and ME7, but not 22L prions propagated in tgGPI− brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA). Surprisingly, the levels of proteinase K-resistant PrPSc (PrPres) in RML- or ME7-infected tgGPI− brain were 25–50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrPSc and SFL PrPSc are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPI− mice

PrP^res in RML- and ME7-infected wild-type and tgGPI^- mouse brains, quantified by Western blot and sandwich ELISA.

<p>(A) Western blot of RML⁻ and ME7-infected brain homogenates before and after PK treatment. “µg”, total protein loaded. ‘r’, 2.5 ng recombinant murine PrP (recPrP). GPI⁻[RML] (lanes 5, 7) and GPI⁻/GPI⁻[ME7] (lane 17) brain homogenates gave rise to ladders reflecting multimers with molecular weights extending to >250 kDa, which were reduced to monomers by PK digestion, while GPI⁻[ME7] homogenate gave no detectable signals. (B) Western blot of PK-digested brain homogenates treated or not with PNGase. Lanes 3, 5, 7, 9 show, from top to bottom, diglycosylated, monoglycosylated and unglycosylated, truncated PrP; PNGase-treated samples (lanes 4, 6, 8, 10) show a single band corresponding to unglycosylated, truncated PrP. Samples from tgGPI⁻ mice (lanes 1, 11) show two bands, corresponding to truncated, monoglycosylated (top) and unglycosylated PrP, and, after PNGase treatment, a single band corresponding to truncated, deglycosylated PrP (lanes 2, 12). Because anchorless PrP is retained inefficiently by PVDF membranes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002746#ppat.1002746-Nishina2" target="_blank">[69]</a>, 24 times more total protein was loaded for GPI⁻ than for C57 samples, to give about the same signal strength. (<b>C</b>) Sandwich ELISA of PK-treated samples. Absorbance of quadruplicate samples is plotted against log[input protein]. Samples a to g are identified in panel D; h, uninfected C57 brain homogenate. The abundance of a sample relative to that of RML can be read off by comparing the amounts of protein required to give the same absorbance. For example, an absorbance of 0.1 is given by 0.01 µg GPI⁻[RML] and 0.5 µg C57[RML] brain homogenate (total protein prior to PK treatment), therefore the abundance of GPI⁻[RML] is about 50 times higher than that of C57[RML] PrP^res. In <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002746#ppat.1002746.s002" target="_blank">Figure S2</a> absorbance of the same samples is plotted against input protein on a linear scale, to show that the response is almost linear up to a protein input of 1.5 µg/well. (D) The PrP^res signals (“pix”) from the western blots of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002746#ppat-1002746-g001" target="_blank">Figure 1A</a> were quantified relative to C57[RML] and the log of the ratio was plotted (red bars). For the sandwich ELISA, the plot shows the log of the absorbance (A) relative to that of C57[RML] (blue bars).</p

The cell tropism of various prion strains changes after propagation in tgGPI⁻ mice.

<p>Homogenates of GPI⁻ or C57 brains infected with the strains indicated were subjected to the CPA. (<b>A</b>) The patterns elicited by 22L from both sources were very similar, however RML, 79A and 139A prions from wild-type brain were swa sensitive on PK1 cells and R33^2H11 incompetent, while those from tgGPI⁻ brain were swa resistant and R33^2H1 competent. The RI₆₀₀ (Response Index for 600 spots) on CAD, PK1, PK1_+swa and R33^2H11 cells is given within the graphs (left upper corner) and the logarithm ± SD of the ratios RI_CAD/RI_PK1 (blue) and RI_PK1/RI_PK1+swa (red) is plotted in the bar graph (B). The matrix (C) gives the p values for the pairwise comparison of two strains on the basis of their log[RI_CAD/RI_PK1] (blue) and log[RI_PK1/RI_PK1+swa] (red) values. The framed “ns” indicates p values>0.1 for <i>both</i> log[ratios]. For example, C57[RML] and GPI⁻[RML] prions are significantly different (p = 0.0097 for log[RI_CAD/RI_PK1] and p = 0.0001 for log[RI_PK1/RI_PK1+swa]), as are C57[79A] and GPI⁻[79A] prions, whereas C57[22L] and GPI⁻[22L] prions do not show a significant difference (framed “ns”; p>0.1) for both logRI ratios. By the same token C57[79A] and C57[RML], and GPI⁻[139A] and GPI⁻[RML] prions are not distinguishable, while C57[139A] and C57[RML] prions differ.</p

High-throughput identification of DNA-encoded IgG ligands that distinguish active and latent Mycobacterium tuberculosis infections

The circulating antibody repertoire encodes a patient's health status and pathogen exposure history, but identifying antibodies with diagnostic potential usually requires knowledge of the antigen(s). We previously circumvented this problem by screening libraries of bead-displayed small molecules against case and control serum samples to discover "epitope surrogates" (Ligands of IgGs enriched in the case sample). Here, we describe an improved version of this technology that employs DNA-encoded libraries and high-throughput FACS-based screening to discover epitope surrogates that differentiate noninfectious/latent (LTB) patients from infectious/active TB (ATB) patients, which is imperative for proper treatment selection and antibiotic stewardship. Normal control/LTB (10 patients each, NCL) and ATB (10 patients) serum pools were screened against a library (5 × 10 beads, 448 000 unique compounds) using fluorescent antihuman IgG to label hit compound beads for FACS. Deep sequencing decoded all hit structures and each hit's occurrence frequencies. ATB hits were pruned of NCL hits and prioritized for resynthesis based on occurrence and homology. Several structurally homologous families were identified and 16/21 resynthesized representative hits validated as selective ligands of ATB serum IgGs (p < 0.005). The native secreted TB protein Ag85B (though not the E. coli recombinant form) competed with one of the validated ligands for binding to antibodies, suggesting that it mimics a native Ag85B epitope. The use of DNA-encoded libraries and FACS-based screening in epitope surrogate discovery reveals thousands of potential hit structures. Distilling this list down to several consensus chemical structures yielded a diagnostic panel for ATB composed of thermally stable and economically produced small molecule ligands in place of protein antigens