Colocalization of amanitin and a candidate toxin-processing prolyl oligopeptidase in Amanita basidiocarps

Abstract

Fungi in the basidiomycetous genus Amanita owe their high mammalian toxicity to the bicyclic octapeptide amatoxins such as α-amanitin. Amatoxins and the related phallotoxins (such as the heptapeptide phalloidin) are encoded by members of the "MSDIN" gene family and are synthesized on ribosomes as short (34- to 35-amino-acid) proproteins. Antiamanitin antibodies and confocal microscopy were used to determine the cellular and subcellular localizations of amanitin accumulation in basidiocarps (mushrooms) of the Eastern North American destroying angel (Amanita bisporigera). Consistent with previous studies, amanitin is present throughout the basidiocarp (stipe, pileus, lamellae, trama, and universal veil), but it is present in only a subset of cells within these tissues. Restriction of amanitin to certain cells is especially marked in the hymenium. Several lines of evidence implicate a specific prolyl oligopeptidase, A. bisporigera POPB (AbPOPB), in the initial processing of the amanitin and phallotoxin proproteins. The gene for AbPOPB is restricted taxonomically to the amatoxin-producing species of Amanita and is clustered in the genome with at least one expressed member of the MSDIN gene family. Immunologically, amanitin and AbPOPB show a high degree of colocalization, indicating that toxin biosynthesis and accumulation occur in the same cells and possibly in the same subcellular compartments.

Fig. 1.

(A) Mature basidiocarps of Amanita bisporigera used in this study (collected in Ingham County, MI, in August, 2009). (B) Immature basidiocarps collected before emergence from the ground. Intact basidiocarps are shown on the right and longitudinal sections through the same basidiocarps on the left. Structures: a, pileus (cap); b, stipe (stem); c, universal veil, giving rise to the volva at the base of the stem as indicated in panel A; d, lamella (gill).

Fig. 2.

Amanitin immunostaining of lamellae (gills) of A. bisporigera observed by confocal laser scanning microscopy (CLSM). (A) Low-magnification CLSM view showing cross-section of two lamellae and part of a third. (B) Higher magnification of a single lamella by CLSM. (C) Same gill section as in panel B viewed by differential interference contrast (DIC). (D) A different basidiocarp, showing a cross-section of three lamellae and parts of two others by CLSM. (E) Antiamanitin immunostaining of basidia (arrows), indicated by the sterigmata (the two slender projections), sterile cells (arrowheads), and the subhymenium (CLSM superimposed on DIC). Sterigmata are the slender projections from the basidia that bear the basidiospores, which are no longer present in these sections. Sterile cells are the cells in the hymenium that, in Amanita, resemble basidia but do not bear spores. (F) Same section as in panel E viewed with DIC alone. (G) Antiamanitin immunostaining of hymenium and subhymenium showing basidia (arrows) with sterigmata and sterile cells containing amanitin (CLSM superimposed on DIC). (H) Same section as in panel G observed by DIC. (I) Basidia (arrows) showing absence of amanitin and adjacent sterile cells (arrowheads) showing presence of amanitin (CLSM superimposed on DIC).

Fig. 3.

Amanitin immunostaining of different tissues of A. bisporigera by LSM. (A) Section of the pileus of the immature basidiocarp shown in Fig. 1B. (B) Section of a mature pileus. (C) Same view as in panel B with white lines manually drawn to highlight outlines and boundaries of the cells. (D) Higher magnification of an individual hypha of a mature pileus (CLSM superimposed on DIC). (E) Section of stipe (stem) of a mature basidiocarp. (F) Section of a universal veil (volva) of a mature basidiocarp (Fig. 1A).

Fig. 4.

Dual immunostaining of A. bisporigera lamellae using antiamanitin and antiactin antibodies. (A to C) Lamella cross-sections, showing amanitin staining, actin staining, and the merge of the two, respectively. (D to F) Higher-magnification views of a cross-section of a lamella (same staining order as for panels A to C). (G to I) Higher-magnification views of a cross-section of a mature pileus.

Fig. 5.

Gene structures of AbPOPA and AbPOPB of A. bisporigera.

Fig. 6.

DNA blotting of 13 Amanita species probed with AbPOPA (A) or AbPOPB (B). Amanita species: lanes 1, A. aff. suballiacea; lanes 2, A. bisporigera; lanes 3, A. phalloides; lanes 4, A. ocreata; lanes 5, A. novinupta; lanes 6, A. flavoconia; lanes 7, A. porphyria; lanes 8, A. franchetii; lanes 9, A. muscaria; lanes 10, A. gemmata; lanes 11, A. hemibapha; lanes 12, A. velosa; lanes 13, unidentified species of Amanita section Vaginatae. Mushrooms represent sections Phalloideae (lanes 1 to 4), Validae (lanes 5 to 8), Amanita (lanes 9 and 10), Caesareae (lane 11), and Vaginatae (lanes 12 and 13). Approximately the same amount of DNA was loaded in each lane (10).

Fig. 7.

Gene maps of lambda phage clones containing AbPOPA (A) or AbPOPB (B). Arrows indicate transcriptional direction. Genes were predicted using FGENESH (Softberry Inc.).

Fig. 8.

Immunoblotting of E. coli inclusion bodies and total extracts of A. bisporigera with anti-AbPOPB antibody. Lanes: 1, BL21 empty vector (PET21) control; 2, inclusion body (1 μl = 0.05 μg) of an E. coli BL21 line expressing AbPOPB; 3 through 6, total soluble protein from A. bisporigera loaded at 24 μg, 47 μg, 94 μg, and 141 μg, respectively. All lanes were from the same gel, but some lanes between lanes 1 and 2 were removed. Size indicators (left) are in kDa.

Fig. 9.

Colocalization of amanitin and AbPOPB. (A to C) Colocalization in the pileus. The stipe is on the left. (A) Antiamanitin; (B) anti-AbPOPB; (C) merge of panels A and B. (D to F) Higher magnification of a portion of the pileus in panels A to C, respectively. (G to I) Colocalization in a single lamella, in the same order as panels A to C.