ABSTRACT
Fungi produce various defense proteins against antagonists, including ribotoxins. These toxins cleave a single phosphodiester bond within the universally conserved sarcin-ricin loop of ribosomes and inhibit protein biosynthesis. Here, we report on the structure and function of ageritin, a previously reported ribotoxin from the edible mushroom Agrocybe aegerita. The amino acid sequence of ageritin was derived from cDNA isolated from the dikaryon A. aegerita AAE-3 and lacks, according to in silico prediction, a signal peptide for classical secretion, predicting a cytoplasmic localization of the protein. The calculated molecular weight of the protein is slightly higher than the one reported for native ageritin. The A. aegerita ageritin-encoding gene, AaeAGT1, is highly induced during fruiting, and toxicity assays with AaeAGT1 heterologously expressed in Escherichia coli showed a strong toxicity against Aedes aegypti larvae yet not against nematodes. The activity of recombinant A. aegerita ageritin toward rabbit ribosomes was confirmed in vitro. Mutagenesis studies revealed a correlation between in vivo and in vitro activities, indicating that entomotoxicity is mediated by ribonucleolytic cleavage. The strong larvicidal activity of ageritin makes this protein a promising candidate for novel biopesticide development.
IMPORTANCE Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides.
INTRODUCTION
Fungi are exposed to a large diversity of antagonists. To protect themselves, fungi mainly rely on a chemical defense mediated by secondary metabolites, peptides, and proteins (1–7). These chemicals interfere with essential biological processes or structures within the target organisms. Ribosomes are essential molecular machines present in all living cells, making them ideal targets for defense effectors (8, 9). Previous studies have revealed three main classes of proteins with ribonucleolytic activity toward rRNA. The first class comprises classical RNases that catalyze the breakdown of RNA molecules into smaller components. These RNases have a rather low specificity for rRNAs and include also nontoxic RNases (10, 11). The second class of proteins is represented by ribosome-inactivating proteins (RIPs) (12). RIPs are N-glycosidases that depurinate a specific adenine residue located in the highly conserved sarcin-ricin loop (SRL) of the large subunit of eukaryotic and prokaryotic ribosomes (13, 14). Depurination of this adenine residue disrupts the binding of translation elongation factors (15), inhibits protein synthesis, and leads to the death of the cells (16, 17). High effectiveness and specificity make RIPs part of the defense systems of many organisms, including bacteria (18), algae (19), fungi (20), and plants (21).
The third class of proteins with ribonucleolytic activity toward rRNA comprises the ribotoxins. Ribotoxins are highly specific, small (10 to 20 kDa), and highly toxic fungal endonucleases that cleave a single phosphodiester bond at a universally conserved GAGA tetraloop of the SRL loop (5, 22, 23). Similar to RIPs, the damage of the SRL loop inhibits the binding of translation elongation factors and, thus, halts protein biosynthesis, ultimately resulting in the death of the target cells (24). Until recently, fungal ribotoxins were exclusively known from members of the phylum Ascomycota. Two of the best-studied ribotoxins are α-sarcin (25) and restrictocin (26), produced by Aspergillus giganteus and Aspergillus restrictus, respectively. Hirsutellin A (27), from the fungal pathogen of mites, Hirsutella thompsonii, and anisoplin (28), from the entomopathogenic fungus Metarhizium anisopliae, are other, recently discovered ribotoxins. Lately, a protein with ribonucleolytic activity toward rRNA, named ageritin, was purified by Landi et al. (29) from southern Italian mushroom specimens and by Landi et al. (30) and Ruggiero et al. (31) from specimens of apparently unspecified origin, all classified as Agrocybe aegerita, a cultivated edible mushroom from the phylum Basidiomycota. In addition to demonstrating its RNase activity, the authors also provided evidence for the cytotoxicity- and cell death-promoting effects of ageritin toward tumor cell lines of the human central nervous system (CNS).
In the present study, we made use of the recently published genome sequence of the dikaryon A. aegerita AAE-3 (32) to identify the A. aegerita ageritin-encoding gene, AaeAGT1, and the neighboring gene, AaeAGT2, which encodes a paralog with high sequence homology to ageritin. Assessing the transcription of both genes during vegetative growth and mushroom formation revealed that AaeAGT1, but not AaeAGT2, is induced during this developmental process. The predicted amino acid sequence of ageritin lacks homology to the amino acid sequences of all other fungal ribotoxins described so far. Still, we detected predicted homologs in a wide variety of basidiomycetes, including some plant-pathogenic species. Employing heterologous expression of AaeAGT1 in Escherichia coli, we tested wild-type and mutagenized ageritin for insect and nematode toxicity as well as for in vitro ribonucleolytic activity. In addition, we functionally characterized the AaeAGT2-encoded ageritin paralog, which we had identified based on its high sequence similarity to AaeAgt1.
RESULTS
Identification of the ageritin-encoding gene in the A. aegerita genome sequence.By using the published 25 N-terminal residues of ageritin (29) as a query sequence for a BLAST search against the predicted proteome of A. aegerita, we were able to identify the ageritin-encoding gene in the genome sequence of the fungus. It is referred to here as AaeAGT1 (gene identifier [ID], AAE3_01767 [www.thines-lab.senckenberg.de/agrocybe_genome]). The respective cDNA, derived from the total RNA of fruiting-primed mycelium, was cloned and sequenced. The cDNA-derived coding sequence of AAE3_01767 was ultimately deposited in GenBank under the accession number MK411345. In contrast to the predicted coding sequence of AaeAGT1 (gene ID, AAE3_01767 [www.thines-lab.senckenberg.de/agrocybe_genome]), the experimentally verified coding sequence with GenBank accession number MK411345 lacks 60 nucleotides due to a nonrecognized intron within the first piece of the predicted coding sequence. The encoded protein comprises 156 amino acid residues and contains N-terminal (20 residues) and C-terminal (9 residues) extensions compared to the ageritin amino acid sequence recently published by Landi et al. (30). In addition to AaeAGT1, we found a neighboring gene on the chromosomal DNA of A. aegerita encoding a paralogous protein, which is referred to as AaeAGT2 (gene ID, AAE3_01768 [www.thines-lab.senckenberg.de/agrocybe_genome]). The deduced amino acid sequence of AaeAgt2 shows 63% sequence identity to the ageritin sequence (Fig. 1A). Interestingly, the predicted amino acid sequence of AaeAgt1 neither reveals sequence similarity to any of the known ascomycete ribotoxins, nor does it contain, according to in silico prediction, a signal sequence for secretion, suggesting that it represents a novel type of ribotoxin.
Expression pattern and in vitro rRNA cleavage activity of ageritin. (A) The amino acid sequences of ageritin and its paralog were aligned using the ClustalW algorithm (version 2.1). Black shading indicates conservation of the residues. (B) Expression level for the ageritin-encoding gene (AaeAGT1) and its paralog (AaeAGT2) at different stages of fruiting body development relative to that of vegetative mycelium (developmental stage I). The dotted horizontal line represents the mycelial expression level of both AaeAGT1 and AaeAGT2 and is used as a baseline value (developmental stage I) for comparison with the other developmental stages of A. aegerita AAE-3: stage II, fruiting-primed mycelium 24 h to 48 h before emergence of visible fruiting body initials; stage III, fruiting body initials; stage IV, entire fruiting body primordia; stage Vs, young fruiting body stipe; stage Vc, young fruiting body cap. The error bars represent the standard deviation from three biological replicates. (C) Ribonucleolytic activity of ageritin assayed with 400 nM purified ageritin toward ribosomes of rabbit reticulocyte lysate. The ribotoxin α-sarcin and buffer were used as positive and negative controls, respectively. rRNAs and the classical ribotoxin cleavage product, the α fragment, are indicated. The numbers to the left of the gel are in nucleotides (nt).
Expression of the ageritin gene during fruiting body formation of A. aegerita.In order to assess the expression of both the ageritin-encoding gene (AaeAGT1) and the paralogous gene (AaeAGT2), quantitative real-time reverse transcription-PCR (qRT-PCR) analyses were carried out using RNA from different dikaryotic developmental stages of A. aegerita AAE-3. Using vegetative mycelium as a reference, a strong upregulation of the expression of AaeAGT1, which encodes ageritin, was found throughout fruiting body development of A. aegerita AAE-3. No upregulation was found in the case of the paralog, AaeAGT2 (Fig. 1B). The results are shown in Fig. 1B as the relative expression compared to the expression by the vegetative mycelium (stage I). AaeAGT1 expression was highly upregulated (by a factor of 87) during the shift from vegetative mycelium to the ready-to-fruit mycelium (stage II). The expression level peaked at 160-fold upregulation when the first macroscopically visible complex multicellular plectenchymatic structures of fruiting body development, which are referred to as fruiting body (FB) initials (stage III), could be spotted. Although a less pronounced expression increase was recorded both in subsequently emerging FB primordia (stage IV, in which AaeAGT1 expression was upregulated by a factor of 19) and in the stipe plectenchyme of the thereafter developing young FBs (stage Vs, in which AaeAGT1 expression was upregulated by a factor of 5), increased expression was again observed within the cap plectenchyme of young FBs (stage Vc, 100-fold upregulated AaeAGT1 expression).
AaeAGT2 showed no differential expression during fruiting body development, varying from 0.4-fold in stage IV to 2.3-fold in stage III. The quantification cycle (Cq) values of both genes in the baseline stage (stage I) were similar, with an average of 25.55 for the AaeAGT1 amplicon and an average of 25.73 for its paralog, AaeAGT2, indicating comparable template amounts for a 1-fold expression for the two genes in this cDNA pool.
Heterologous expression and purification of ageritin from E. coli cells.In contrast to all other ribotoxins, AaeAgt1 does not contain a signal peptide for classical secretion according to in silico prediction and is thus predicted to be localized in the cytoplasm. Based on this prediction, we expressed ageritin in the cytoplasm of E. coli. Both untagged and N-terminally His8-tagged ageritin were expressed in soluble form in the bacterial cytoplasm (see Fig. S2A in the supplemental material). His8-ageritin could be purified over Ni-nitrilotriacetic acid (NTA) affinity columns to homogeneity (Fig. S2E) and be used for an in vitro ribonucleolytic cleavage assay and for a toxicity assay employing the insect cell line S. frugiperda Sf21.
rRNA cleavage activity of recombinant ageritin.The ribonucleolytic activity of recombinant, His8-tagged ageritin against ribosomes of rabbit reticulocyte lysate was assayed as was done for the protein isolated from the fungus (29, 31). The results indicate that recombinant ageritin, similar to α-sarcin (33), acts on the 28S rRNA subunit of ribosomes and releases the α fragment as a classical ribotoxin cleavage product (Fig. 1C). These results confirm previous results obtained with ageritin isolated from the fungus (29, 31). The rRNA patterns of ribosomes that were treated only with α-sarcin or simultaneously with both α-sarcin and recombinant ageritin were almost identical, suggesting that the cleavage site for ageritin is either very close to or identical to the one for α-sarcin (Fig. 1C).
Entomo- and nematotoxicity of recombinant ageritin.E. coli cells expressing untagged or His8-tagged recombinant ageritin were tested for entomotoxic activity against third-stage (L3) larvae of Aedes aegypti mosquitoes. E. coli cells expressing the entomo- and nematotoxic galectin Cgl2 from the mushroom Coprinopsis cinerea were used as a positive control (3). After 4 days, larval mortality was recorded by counting the surviving larvae (Fig. 2A). Concomitantly, feeding inhibition was measured by determining the reduction in the optical density at 600 nm (OD600) of the respective bacterial suspensions (Fig. 2B). In these assays, larvae feeding on cells expressing the empty vector (negative control) were not reduced in number over the examined time period and reduced the bacterial cell density in the corresponding suspension, resulting in a significant drop of the OD600. In contrast, both the number of larvae and the drop in bacterial cell density were significantly reduced in the case of Cgl2-, ageritin-, or His8-ageritin-expressing bacteria (Fig. 2A and B).
Entomotoxicity of ageritin toward Aedes aegypti larvae and Spodoptera frugiperda Sf21 cells. (A and B) The toxicity of ageritin and its tagged version toward mosquito larvae was tested by recording the mortality (A) and by measuring the consumption of E. coli by determination of the reduction of the OD600 of a respective E. coli suspension (B). Mosquito larvae were fed for 96 h on IPTG-induced E. coli BL21 expressing proteins Cgl2, ageritin, and His8-ageritin. E. coli BL21 cells either expressing the previously characterized entomotoxic protein Cgl2 or carrying the empty vector (EV) were used as the positive and the negative controls, respectively. Statistical analysis was done using Dunnett’s multiple-comparison test. The error bars represent the standard deviation from five biological replicates. ****, P < 0.0001 versus the empty vector. (C) Ageritin toxicity against the insect cell line S. frugiperda Sf21 was tested. Different concentrations of purified ageritin were incubated with S. frugiperda Sf21 cells for 72 h. The number of viable cells was counted for each sample. DMSO and the ribotoxin α-sarcin (α-Sar) were used as positive controls, and PBS buffer was used as a negative control. Dunnett’s multiple-comparison test was used for statistical analysis. The error bars represent the standard deviation from six biological replicates. Symbols and abbreviations: ns, not significant versus PBS; ***, P < 0.001 versus PBS; ****, P < 0.0001 versus PBS.
In agreement with the mosquitocidal activity of recombinant ageritin, purified His8-tagged ageritin also showed toxicity against S. frugiperda Sf21 insect cells in a concentration-dependent manner (Fig. 2C).
After confirming the entomotoxic activity of recombinant ageritin, we tested its activity against five different bacterivorous nematode species (Table S1). Synchronized L1 larvae of the nematodes Caenorhabditis elegans, C. briggsae, C. tropicalis, Distolabrellus veechi, and Pristionchus pacificus were fed bacteria expressing untagged ageritin, and their development was assessed after 2 days. Interestingly, recombinant ageritin was not active against any of the tested nematode species, which indicates its specificity for insects (Fig. S3).
Correlation between rRNA cleavage and toxicity.The amino acid sequence of ageritin was aligned to the amino acid sequences of the 10 most homologous proteins predicted from other Basidiomycota. Six conserved residues were selected and mutated to alanine in the expression construct for His8-tagged ageritin (Fig. 3A). All constructs yielded a highly soluble protein in the cytoplasm of E. coli (Fig. S2B). In order to assess the importance of the mutated residues for the entomotoxicity of ageritin, we performed toxicity assays against A. aegypti larvae. No toxicity was detected for three mutants with conserved residues (the R87A, D89A, and H98A mutants), while the toxicity of the other three mutants (the Y57A, D91A, and K110A mutants) was similar to that of wild-type ageritin (Fig. 3B). As a second approach, we measured the reduction in OD600 to check larval feeding inhibition. The toxic constructs inhibited feeding, whereas the reduction in OD600 by nonentomotoxic bacterial strains was similar to that for the control (Fig. 3C). In order to differentiate between the three point mutants (the R87A, D89A, and H98A mutants) which had shown a significant reduction in ageritin toxicity, the mosquito larvae were allowed to develop into adults. The D89A mutant showed a complete loss of toxicity, as the proportion of fully developed larvae was comparable to that for the negative-control samples (Fig. 3D). An average of 40% of the larvae developed into adults when the other two conserved-site mutants (the R87A and H98A mutants) were assayed. This indicates that these two mutants were still partially toxic (Fig. 3D).
Effect of mutations in conserved residues on the in vivo and in vitro activities of ageritin. (A) The amino acid sequence of ageritin was used as a query sequence for a BLAST search against the JGI MycoCosm fungal database. The hit regions of the top 10 sequences with the highest homology were aligned using the ClustalW algorithm (version 2.1). Numbering refers to the ageritin amino acid sequence. Gray shading indicates the degree of conservation from dark to light. Individually mutated conserved regions are indicated by red line boxes and asterisks. The numerical values, the data sets used, and the origin of the species of the genes used in these alignments are given in Table S4 in the supplemental material. (B to D) The entomotoxic activity of the wild-type (wt) and mutant ageritin versions was monitored by feeding A. aegypti larvae E. coli BL21 cells expressing the respective His8-tagged protein of interest. Toxicity was assessed by counting the number of surviving larvae (B) and measuring the OD600-based bacterial consumption (C) every day for 4 days and by counting the number of larvae that reached the adult stage by the end of day 7 for the larvae feeding on bacteria expressing one of the corresponding proteins (D). E. coli BL21 cells expressing either the previously characterized entomotoxic protein Cgl2 or carrying the empty vector (EV) were used as a positive control and a negative control, respectively. Statistical analysis was done using Dunnett’s multiple-comparison test. The error bars represent the standard deviation from five biological replicates. ns, not significant versus E. coli BL21 cells carrying the empty vector; ****, P < 0.0001 versus E. coli BL21 cells carrying the empty vector. (E) The ribonucleolytic activity of the ageritin wild-type and mutant proteins was assessed by exposing ribosomes of rabbit reticulocyte lysate to 400 nM the respective purified His8-tagged protein. α-Sarcin and PBS buffer (Buf.) were used as positive and negative controls, respectively. rRNAs and the ribotoxin cleavage product, i.e., the α fragment, are indicated. Lanes M, molecular mass markers (the numbers to the left of the gel are in nucleotides).
All of the above-described His8-ageritin variants were produced in E. coli BL21 and purified over Ni-NTA columns (Fig. S2D). Subsequently, the ribonucleolytic activities of the purified proteins against ribosomes of rabbit reticulocyte lysate were assessed. Interestingly, all three single-site mutants which were nontoxic in vivo against A. aegypti larvae were also inactive in the ribosome cleavage assay (Fig. 3E). The single-site mutants that were still toxic in vivo performed similarly to wild-type ageritin, in that they all released the 400-nucleotide-long ribotoxin cleavage product, the α fragment (Fig. 3E). In combination with our data on insecticidal activity, the in vitro results demonstrate that the toxicity is dependent on the ribonucleolytic activity of ageritin.
Functional comparison between ageritin and its paralog.Given the different expression dynamics of AaeAGT2 and AaeAGT1 throughout the developmental stages of the fungus and the bioactivity of AaeAgt1, we investigated the in vitro and in vivo function of AaeAgt2. For this purpose, AaeAGT2 was expressed in the cytoplasm of E. coli BL21 in the same manner as AaeAGT1. Bacterial expression of both untagged and His8-tagged AaeAgt2 yielded soluble protein, albeit with a rather low expression of the untagged form (Fig. S2D and E). In order to address the function of this protein, we tested the ribonucleolytic activity of purified His8-tagged AaeAgt2 toward the ribosomes of rabbit reticulocyte lysate. The results of these assays showed that AaeAgt2 was as active as ageritin, in agreement with the conservation of the essential amino acid residues in the paralogous protein (Fig. 1A and 4A). We then tested E. coli cells expressing untagged and His8-tagged AaeAgt2 for toxicity against A. aegypti mosquitoes. Interestingly, the ageritin paralog was similarly active against A. aegypti larvae, suggesting the conservation of both the in vitro and in vivo functions between the two paralogous proteins (Fig. 4B). The weaker entomotoxic activity of the untagged versus His8-tagged version of the AaeAgt2 protein can be explained by its significantly lower expression in E. coli (Fig. S2D).
Functional analysis of the identified ageritin paralog. (A) Ribonucleolytic activity of ageritin and its paralog, assayed with 400 nM purified protein, toward ribosomes of rabbit reticulocyte lysate. The ribotoxin α-sarcin and buffer were used as the positive and the negative controls, respectively. rRNAs and the classical ribotoxin cleavage product, the α fragment, are indicated. Numbers to the left of the gel are in nucleotides. (B) Entomotoxic activity of the ageritin paralog against A. aegypti larvae. L3 mosquito larvae were fed E. coli BL21 bacteria expressing untagged and His8-tagged versions of the ageritin paralog. Bacteria either expressing the previously characterized entomotoxic protein Cgl2 or carrying the empty vector (EV) were used as the positive and the negative controls, respectively. The error bars represent the standard deviation from three biological replicates.
Distribution of ageritin homologs in the fungal kingdom.Fungal ageritin homologs were identified using the JGI MycoCosm portal (34). A high degree of sequence conservation was found among homologs from different fungal species. The top 30 hits were retrieved from the MycoCosm portal, and their phylogenetic relationships are presented as a circular cladogram (Fig. 5; Table S4). All 30 hits originated from the phylum Basidiomycota, including several plant-pathogenic species, such as Rhizoctonia solani, Moniliophthora perniciosa, Rigidoporus microporus, and Pleurotus eryngii. Interestingly, although ageritin is likely a cytoplasmic protein, according to our in silico analysis (no classical secretion signal was detected), several of its potential homologs possess signal sequences for classical secretion, implying the existence of both secreted and nonsecreted versions of this novel type of ribotoxin in the phylum Basidiomycota.
Rooted circular cladogram of ageritin homologs. The amino acid sequence of ageritin was used as a query sequence for a BLAST search against the database of the Gene Catalog Proteins (GCP) at the JGI MycoCosm fungal database. The complete amino acid sequences of the top 30 hits were aligned using the ClustalW algorithm (version 2.1), and the phylogenetic relationships among the sequences were depicted via a circular cladogram. The hit with the lowest homology to ageritin among those 30 hits had an E value of 7.2E−16, an identity of 44.8%, and a subject coverage of 64.8%. The numerical values and data sets for the genes used in these alignments can be found in Table S4 in the supplemental material. The branch lengths are relative and not to scale. Maximum likelihood bootstrap support values are indicated next to each node, if the bootstrap support values exceeded 50%. Different potential ageritin homologs within the genome of a given species are labeled by consecutive numbers.
DISCUSSION
Ribotoxins have been suggested to be part of the fungal defense system against insect predators (23, 35, 36). This is corroborated by the results of the present work, in which we report the gene, cDNA, and amino acid sequences of a novel type of ribotoxin, termed ageritin, from the edible mushroom A. aegerita, which belongs to the fungal phylum Basidiomycota. The amino acid sequence of ageritin is remarkably different from the amino acid sequences of the well-characterized ribotoxins from the Ascomycota. Besides the lack of significant overall sequence similarity, in silico prediction indicates the lack of a classical secretion signal, which suggests that this ribotoxin resides in the cytoplasm. The reported amino acid sequence of ageritin, which was derived from the cDNA sequence isolated from A. aegerita AAE-3 mushrooms, differs from the one reported by two previous studies (30, 31). The isolated cDNA sequence revealed an unpredicted intron which leads to a 5′ extension of the predicted coding sequence of ageritin in the A. aegerita AAE-3 genome sequence (32) (www.thines-lab.senckenberg.de/agrocybe_genome) and to an extension of the ageritin N terminus by 20 amino acid residues. The N terminus and calculated molecular weight of the isolated cDNA sequence did not correlate with the experimentally determined N terminus and molecular weight of native ageritin isolated from the fungus (29–31). We hypothesize that this extension may play a role in protection of the fungal ribosomes from the cytoplasmic fungal protein toxin and may be cleaved off from this proageritin by an unknown fungal protease. The observed higher expression levels of N-terminally His8-tagged versus untagged ageritin (and its paralog) in E. coli support this hypothesis. The inhibition by the N-terminal extensions appeared to be only partial, however, since both versions of recombinant ageritin showed clear in vitro and/or in vivo activity. Alternative explanations for the apparent difference in molecular weight between the native and recombinant ageritins are partial degradation of the native protein during purification from the mushroom or strain-specific differences between the ageritin-encoding genes, since native ageritin was isolated from the fruiting bodies of A. aegerita strains different from A. aegerita AAE-3.
We detected a high toxicity of ageritin against A. aegypti larvae. In nature, ageritin might thus function in the natural defense of A. aegerita against insect antagonists of mushrooms, like fungus gnats. In cultivation facilities of the button mushroom (Agaricus bisporus), fungus gnats like Lycoriella ingenua (Sciaridae) can cause severe damages, e.g., by larval feeding on the mushroom cultures (37). Entomotoxic activity is a common feature of fungal ribotoxins (38), such as the two ribotoxins anisopilin (28) and hirsutellin A (27), which are both toxic against insect cells. Accordingly, ageritin showed cytotoxicity against the insect cell line S. frugiperda Sf21, albeit at a higher concentration than the well-studied α-sarcin, suggesting that ageritin is less active than α-sarcin. Similar results were obtained in the in vitro ribonucleolytic assay with ribosomes of rabbit reticulocyte lysate, where the cleavage of 28S rRNA was still incomplete for ageritin (in addition to cleaved 28S rRNA, some uncut 28S rRNA was also still present; Fig. 1C), in contrast to α-sarcin, after 30 min of incubation. Since the ribonucleolytic activity of native ageritin was reported to be comparable to that of α-sarcin (31), these results are in line with a partial inhibition of the catalytic function of the protein by the N-terminal extension, as discussed in the previous paragraph.
Furthermore, we investigated whether the entomotoxic activity of ageritin depends on its ribonucleolytic activity. Although the lack of sequence similarity with the previously characterized ascomycete ribotoxins prevented identification of the putative catalytic residues of ageritin, previous studies have shown that the catalytic residues of ribotoxins mostly consist of acidic and basic residues (39). These charged residues have been found to support ribotoxin binding to the negatively charged rRNA, facilitating interactions with target ribosomes (39, 40). For instance, the active site of α-sarcin consists of two histidine residues and one glutamic acid residue (H50, E96, and H137) (25, 41). Therefore, we mutated the conserved charged amino acid residues (R87, D89, D91, H98, and K110) and tyrosine (Y57) in the ageritin amino acid sequence. Tyrosine-48 (42), along with arginine-121 (43) and leucine-145 (44), was shown to contribute to the biological activity of α-sarcin. Three of the six mutations (R87A, D89A, and H98A) removed both entomotoxic activity and in vitro ribonucleolytic cleavage, while the other three mutations (Y57A, D91A, and K110A) did not affect either the in vivo or the in vitro activity of ageritin. Thus, these mutant studies indicate that the entomotoxic activity of ageritin depends on the rRNA cleavage activity and that residues R87, D89, and H98 are part of the catalytic site of ageritin. This supports the claim by Landi et al. (30), who also discussed this very His residue (by counting with acknowledgment that their ageritin lacks 20 amino acid residues at the N terminus in comparison to our AaeAgt1 sequence) to be involved in the catalytic activity of ageritin.
While nematodes are important predators of fungi (45), no activity of ribotoxins against these organisms has been reported so far. As ageritin structurally differs from other ribotoxins, we tested the susceptibility of five nematode species (C. elegans, C. briggsae, C. tropicalis, D. veechi, and P. pacificus) toward ageritin. The nematotoxicity results, however, showed that ageritin is inactive, at least against these five species, suggesting that their ribosomes are either resistant to or not accessible to ageritin. These findings are in line with those of previous studies on the activity spectrum of ribotoxins, where these proteins exhibited specificity for insect cells due to the special structure and composition of the insect cell membrane (23, 36). So far, no specific receptors supporting the entry of ribotoxins into the target cell have been identified. Still, the low molecular weight and high number of positively charged residues were found to be crucial for the binding of ribotoxins to the cell membrane of target cells (46). The capacity to bind to different kinds of cell membranes and the diffusion efficiency of ribotoxins are presumed to be the main factors for the toxicity of ribotoxins (47). Thus, the permeability and the composition of the target cell membrane are crucial for the susceptibility of cells to ribotoxins. The insect cell membrane has a significantly lower cholesterol/phospholipid ratio that makes it thinner and more fluidic with higher permeability (38, 48). Furthermore, it contains more acidic phospholipids, which facilitates the binding of basic ribotoxins and their subsequent entry into the cell through the plasma membrane (46, 49). Hence, it is suggested that fungal ribotoxins are specific to insects and act as biological insecticides (23, 27, 36). In agreement with this view, the entomotoxic activity is conserved between ageritin and its paralog.
Interestingly, the gene expression patterns of the genes AaeAGT1 and AaeAGT2 differed from each other. While both genes were hardly expressed in vegetative A. aegerita mycelium and AaeAGT2 expression levels remained unchanged after fruiting induction, AaeAGT1 transcription was turned on in a switch-like manner at this developmental stage. AaeAGT1 expression first peaked when the first multicellular plectenchymatic structures, i.e., the fruiting body initials, emerged. A second, slightly lower expression maximum was reached within the cap of the already well-differentiated young fruiting bodies. This may relate to the evolutionary survival strategy of mushrooms, which requires protection of the developing meiospores and the surrounding plectenchyme (tissue) from adverse conditions, including biotic stresses like fungivory, e.g., by accumulation of defense proteins within such fruiting body tissues, as pointed out previously (3, 50, 51). The drop in the AaeAGT1 expression increase within primordia can be explained by the sampling procedure applied: while RNA from young fruiting bodies was extracted from separately sampled caps and stipes, the primordia were extracted in their entirety.
Ribotoxins cleave the rRNA in the universally conserved GAGA tetraloop within the sarcin-ricin loop of the large ribosomal subunit. Therefore, all known ribosomes are potentially susceptible, and a ribotoxin-producing fungus has to find means to protect itself from its own toxin. Protection can be achieved by preventing the active ribotoxin from getting in touch with the ribosomes of the producer, e.g., by compartmentalization and efficient secretion systems (52). However, unlike in all previously described ribotoxins, ageritin does not contain a signal peptide, according to in silico prediction, and is thus either retained in the cytoplasm or secreted by a yet unknown secretion pathway similar to the one described for Cts1 in the more basal basidiomycete Ustilago maydis (53). Interestingly, we could express ageritin in a highly soluble form in the cytoplasm of E. coli, although the same protein proved to be active in in vitro ribonucleolytic assays toward rabbit ribosomes. These results suggest that ageritin may have evolved specificity toward ribosomes of certain organisms, despite the high conservation of its target. Such a specificity would be an alternative means to the above-discussed inhibition by the N-terminal extension for self-protection of the fungus against cytoplasmic ageritin. Agrocybe aegerita could, for example, shield the cleavage site in its ribosomes by a minor sequence variation in its SRL region. Furthermore, the ribosomal proteins in proximity of the SRL or a secondary structure modification in the region could make the GAGA tetraloop inaccessible for ageritin. We will address this highly interesting issue in future experiments.
Our results imply that ageritin homologs are widely distributed among diverse fungi, including several plant-pathogenic ones, such as R. solani, M. perniciosa, R. microporus, and P. eryngii. Since Kettles et al. (54) described the Zt6 protein from the wheat pathogen Zymoseptoria tritici as an effector with RNase activity in the antagonistic interactions between the host and the local microbial community and/or the plant host (55), it might be worthwhile to functionally characterize ageritin homologs for activities other than entomotoxic activities. Accordingly, α-sarcin was recently reported to have antifungal activity (56). On the other hand, several of the identified ageritin homologs possess predicted signal sequences for classical secretion, indicating that some of these proteins are secreted. One should keep in mind, however, that most of these protein sequences have not been verified experimentally.
The high toxicity against mosquito larvae makes ageritin a candidate molecule for the development of new biological insecticides. Mosquitoes are vectors of some of the most important infectious diseases globally, most notably, malaria, dengue, and filariosis (57), and the increasing spread of invasive Aedes mosquitoes leads to the emergence of arboviral diseases, such as dengue and chikungunya, in more temperate regions (58, 59). One of the key challenges is insecticide resistance to currently available insecticides (60, 61). Protective interventions, such as insecticide-treated bed nets and indoor residual spraying, have become more and more ineffective. We therefore urgently need new agents for mosquito vector control, and ageritin could potentially expand our current repertoire of such agents.
MATERIALS AND METHODS
Strains, cultivation conditions, and sampling of fruiting body development stages.Cultivation and strain maintenance of A. aegerita AAE-3 were performed as described previously (62). The bacterial strains Escherichia coli DH5α and E. coli BL21(DE3) were used for cloning and protein expression, respectively. The other organisms, including microorganisms, used in this study are listed in Table S1 in the supplemental material.
Fruiting induction for the sake of sampling different developmental stages of A. aegerita AAE-3 fruiting bodies was essentially performed as described by Herzog et al. (62). In brief, 1.5% (wt/vol) malt extract agar (MEA) plates were inoculated centrally with a 0.5-cm-diameter agar plug originating from the growing edge of a still growing A. aegerita AAE-3 culture. Before fruiting induction (at 20°C with a 12-h light and 12-h dark cycle, saturated humidity, aeration, and local injury of the mycelium by punching out a 0.5-cm-diameter mycelium-overgrown MEA plug), the fungal plates were incubated for 10 days at 25°C in the dark.
Samples consisting of at least three independent replicates from each of the following stages were collected: stage I, vegetative mycelium prior to fruiting induction at day 10 postinoculation (p.i.); stage II, fruiting-primed mycelium 24 h to 48 h before the emergence of fruiting body initials at day 14 p.i.; stage III, fruiting body initials at day 15 or 16 p.i.; stage IV, fruiting body primordia at days 17 to 19 p.i.; and stages Vs and Vc, young fruiting bodies separated into stipe (stage Vs) and cap (stage Vc) plectenchymes at days 19 to 21 p.i. Mature fruiting bodies exhibiting full cap expansion and a spore print emerging by day 22 p.i. were not sampled. Samples of stages I and II were obtained by gently scraping off the outermost 1 cm of mycelium from three replicate agar plates with a sterile spatula. Samples were immediately transferred to a 2-ml microcentrifuge tube containing 1 ml of RNAlater (product ID, R0901; Sigma-Aldrich GmbH, Munich, Germany), which was transferred to 4°C for a maximum of 3 days before freezing at −80°C until total RNA extraction.
Isolation of total RNA from A. aegerita for assessing expression of AaeAGT1 and AaeAGT2.For total RNA extraction, a NucleoSpin RNA plant kit (product ID, 740949; Macherey-Nagel GmbH & Co. KG, Düren, Germany) was used, with a modified cell homogenization and lysis procedure being applied. First, the RNAlater (product ID, R0901; Sigma-Aldrich GmbH) was removed from each pooled sample and 350 μl lysis buffer RA1 was added per 100 mg fungal biomass. One 4-mm-diameter and about ten 1-mm-diameter acetone-cleaned stainless steel beads (product IDs, G40 and G10, respectively; KRS-Trading GmbH, Barchfeld-Immelborn, Germany) were then added to each tube. Homogenization was achieved using a mixer mill (model MM 200; Retsch, Haan, Germany) set to 8 min at 25 Hz. Then, the protocol followed the manufacturer’s recommendations for RNA extraction from filamentous fungi, including a DNA digestion step with the ribosomal DNase from the kit, beginning with the filtrate lysate step.
Total RNA was eluted in nuclease-free water (product ID, T143; Carl Roth GmbH & Co. KG, Karlsruhe, Germany), and the RNA concentration was measured spectrophotometrically with a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA quality was visually assessed by checking the integrity of the major rRNA bands in a denaturing polyacrylamide gel. One microgram of total RNA was loaded onto each lane of precast Tris-borate-EDTA (TBE)-urea 6% polyacrylamide gels (product ID, EC68652BOX; Thermo Fisher Scientific) and separated for 1 h at 180 V. For detection, SYBR Gold (product ID, S11494; Thermo Fisher Scientific) was used to stain the gel following the manufacturer’s recommendations. The respective sample was further processed only if no degradation of the RNA was observed and the major rRNA bands were intact. Total RNAs were stored at −80°C.
One-step qRT-PCR using total RNA samples.Primers and quantitative real-time reverse transcription-PCR (qRT-PCR) conditions were designed according to the general recommendations of the MIQE guidelines (63). The software Geneious R11 (Geneious), described by Kearse et al. (64), was used to design the primers for the genes encoding ageritin (AaeAGT1; gene ID, AAE3_01767) and its paralog (AaeAGT2; gene ID, AAE3_01768) as well as the two reference genes AaeTIF1 (gene ID, AAE3_07669) and AaeIMP1 (gene ID, AAE3_02268) from the A. aegerita AAE-3 genome sequence (32) (www.thines-lab.senckenberg.de/agrocybe_genome). The primers for these genes are listed in Table S2. All qRT-PCRs were performed on an AriaMX real-time PCR system (Agilent Technologies, La Jolla, CA, USA) in optically clear 96-well plates with 8-cap strips using Brilliant Ultra-Fast SYBR green qRT-PCR master mix (product ID, 600886; Agilent Technologies). This one-step master mix included reverse transcriptase, the reaction buffer, the DNA polymerase, and SYBR green. The reverse transcription reaction was performed in each well prior to the start of the qRT-PCR program. All samples were run in three biological replicates with 25 ng total template RNA per reaction mixture. The final concentration for each primer was 250 nM. A melting curve analysis was done at the end of the qRT-PCR to determine amplicon purity. See Table S3 for the details of the qRT-PCR program used.
Relative differential expression analysis.Data analysis was based on Cq values calculated from raw fluorescence intensities. The baseline correction and determination of the Cq and mean PCR efficiency (E) for each amplicon were done as described by Ruijter et al. (65) using the LinRegPCR program (version 2017.1).
Relative expression ratios were calculated using the software REST (version REST2009) (66). This software employs the Pfaffl method (67) to calculate the E-corrected relative gene expression ratios, allowing for the simultaneous use of multiple reference genes for normalization, based on the work of Vandesompele et al. (68). The 95% confidence intervals around the mean relative expression ratios were calculated on the basis of 2,000 iterations. Vegetative mycelium that was not induced for fruiting (developmental stage I) was chosen as a reference sample.
cDNA generation from A. aegerita RNA.The cDNA was synthesized from total RNA of fruiting-primed mycelium using a RevertAid first-strand cDNA synthesis kit (product ID, K1621; Thermo Fisher Scientific) and oligo(dT)18 primers. First-strand total cDNA was then directly used as a template to produce the specific cDNA of the ageritin-encoding gene AaeAGT1 (gene ID, AAE3_01767) with the primer pair cds01767-f and cds01767-r (Table S2) in a standard 3-step PCR using Phusion polymerase (product ID, F530; Thermo Fisher Scientific) with an annealing temperature of 62°C. DNA sequence information for primer design was obtained from the genome sequence of A. aegerita AAE-3 (32) (www.thines-lab.senckenberg.de/agrocybe_genome).
Construction of ageritin expression vectors.The coding sequence of ageritin was identified by BLAST analysis using the published 25 N-terminal residues of ageritin as a query sequence against the predicted proteome of A. aegerita (29, 32). The sequence was amplified with the primer pair pAGT1-Nd and pAGT1-N (Table S2) from the AaeAGT1 cDNA and cloned into a pET24b(+) expression vector. The sequence of the cloned cDNA was confirmed by DNA sequencing. The plasmid was transformed into E. coli BL21 cells. For expression of ageritin, E. coli BL21 transformants were precultivated in Luria-Bertani (LB) medium supplemented with 50 mg/liter kanamycin at 37°C. At an OD600 of about 0.5, the cells were induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG; product ID, I8000; BioSynth AG, Switzerland) and cultivated overnight at 16°C. The expression and solubility of ageritin were checked as previously described (69).
Toxicity assays against mosquito larvae and nematodes.Egg masses of the yellow fever mosquito (Aedes aegypti) were harvested on filter papers of the Rockefeller Laboratory colony reared at the Swiss Tropical and Public Health Institute (Basel, Switzerland). For the experiments, mosquito larvae were reared by placing 2- to 5-cm2 small pieces of the egg paper, depending on the density of the eggs, into glass petri dishes containing tap water at 28°C. The larvae hatched within a few hours. They were fed finely ground commercially available food for ornamental fish. The toxicity assays against the mosquito larvae were performed as described previously (69). In brief, larvae in their third stage (L3) were used for the toxicity assays. The food source was changed from fish food to E. coli, adjusted in all the bioassays to an OD600 of 0.4. The mosquito larvae readily fed on the E. coli bacteria and were able to develop to the adult stage. The consumption of E. coli bacteria carrying the empty vector by the mosquito larvae could be tracked by the reduction in the optical density. The bioassay was performed by transferring 10 L3 larvae to 100-ml Schott bottles containing 99 ml of tap water and 1 ml of a suspension with E. coli cells expressing the desired protein. The mosquito larvae were kept in the dark at 28°C, and the toxicity of the protein was assessed by the larval mortality and by the reduction in the optical density. In addition, the number of adult mosquitoes which were able to develop from the larvae was counted after 7 days. The previously characterized entomo- and nematotoxic lectin Cgl2 was used as a positive control in all toxicity assays (3). Starvation controls, in which the mosquitoes received no food at all, were used to check whether the death of the larvae was due to toxicity or refrainment of the larvae from consuming the bacteria. Nematotoxicity assays against five different species of nematodes, Caenorhabditis elegans, C. briggsae, C. tropicalis, Distolabrellus veechi, and Pristionchus pacificus, were performed as described before (50, 69). Dunnett’s multiple-comparison test was used to calculate the statistically significant differences between the mean values for the treatment and the control groups.
Tagging and purification of ageritin.For the purification of recombinant ageritin over Ni-NTA columns (Macherey-Nagel), the protein was tagged with a polyhistidine (His8) tag at its N terminus. A plasmid was constructed by PCR using the pF_8His-Ag and pR_8His-Ag primer pair listed in Table S2. His8-ageritin was expressed as described above for untagged ageritin. Protein purification was performed as described previously (70) but by using Tris-HCl, pH 7.5, as the lysis, purification, and storage buffer.
In vitro rRNA cleavage assay.To detect rRNA cleavage activity, 20 μl of untreated rabbit reticulocyte lysate (product ID, L4151; Promega, WI, USA) was mixed with purified recombinant, wild-type, or mutant ageritin or its paralog to a final concentration of 400 nM in a reaction buffer containing 15 mM Tris-HCl, 15 mM NaCl, 50 mM KCl, and 2.5 mM EDTA at a pH of 7.6 at a final volume of 30 μl (71). The reaction mixture was incubated for 1 h at 30°C, and the reaction was stopped by adding 3 μl of 10% SDS. RNA was isolated from the reaction mixture by phenol-chloroform extraction. The RNA was mixed with 2× RNA loading dye (Thermo Fisher Scientific) and denatured for 5 min at 65°C, cooled on wet ice, and run on a 2% native agarose gel in cold TBE buffer for 30 min at 100 V. α-Sarcin (product ID, BCO-5005-1; Axxora, USA) and the reaction buffer were used as positive and negative controls, respectively.
Toxicity assay toward insect cells.The cytotoxicity of ageritin against the insect cell line Spodoptera frugiperda Sf21 (the IPLB-Sf21-AE cell line) was tested. The insect cells were precultivated in Sf-900 II serum-free medium (SFM; Invitrogen, CA, USA) supplemented with streptomycin (100 μg/ml) and penicillin (100 μg/ml). The cells were diluted to a final density of 0.29 × 106/ml, and 500 μl was dispensed into each well of 24-well plates. The insect cells were challenged with different concentrations of recombinant ageritin (0.1 μM, 1 μM, and 10 μM) dissolved in phosphate-buffered saline (PBS). The well plates were incubated for 3 days at 27°C. The liquid medium was removed, and the cells were stained with 15 μl of 0.4% trypan blue solution. The number of alive and thus unstained cells was determined under a microscope. α-Sarcin (Axxora) or 5% dimethyl sulfoxide (DMSO) was used as a positive control, whereas the PBS buffer served as a negative control. Dunnett’s multiple-comparison test was used to test whether the observed differences between the mean values for the treatment and the control groups were statistically significant.
Alignment and phylogenetic tree.The complete amino acid sequence of ageritin was used as a query in a BLAST search against the database of the Gene Catalog Proteins (GCP) at the JGI MycoCosm fungal database (34). The hit regions of the top 10 sequences with the highest homology were aligned using the ClustalW algorithm (version 2.1) at CLC Genomics Workbench (72).
For the analysis of the phylogenetic relationship among the top homologs of ageritin, the complete amino acid sequences of 30 hits were aligned using the ClustalW algorithm (version 2.1) (72). A phylogenetic tree was constructed by employing the maximum likelihood algorithm (73). The tree was designed as a rooted circular cladogram. The hit with the lowest homology to ageritin among the 30 hits had an E value of 7.2E−16. Detailed information about the 30 hits is provided in Table S4.
Creation and expression of mutant versions of ageritin.Six residues (Y57, R87, D89, D91, H98, and K110) of ageritin that were conserved among the various homologs from other fungi were mutated individually to alanine using the site-specific primers listed in Table S2. The plasmid carrying His8-ageritin was used as the template for the construction of single-site mutants. Expression and purification of the mutant ageritin variants were done as described above for wild-type His8-ageritin.
Expression and purification of the ageritin paralog.The predicted coding sequence for the paralog of ageritin (63% sequence identity) was ordered in a codon-optimized version (for E. coli) from GenScript (Piscataway, NJ, USA) (Fig. S4). Expression and purification of the paralogous protein were done as described above for ageritin. The primers for PCR-mediated construction of the sequence encoding the His8-tagged version of the protein, HAgP-FW and AgP-RV, are described in Table S2.
Data availability.The cDNA-derived coding sequence of AAE3_01767 was deposited in GenBank under accession number MK411345.
ACKNOWLEDGMENTS
We thank Markus Aebi for inspiring discussions, Magdalena Martinovic for excellent technical support, and the insectary team of Swiss TPH for keeping the mosquito colonies.
A.T., P.L., F.H., and M.K. are inventors on European patent application no. EP18215370.0, filed by ETH Zürich and Senckenberg Gesellschaft für Naturforschung (74).
This work was supported by the Swiss National Science Foundation (grant no. 31003A-173097), ETH Zürich, and the Senckenberg Gesellschaft für Naturforschung.
FOOTNOTES
- Received 11 July 2019.
- Accepted 21 August 2019.
- Accepted manuscript posted online 23 August 2019.
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.01549-19.
- Copyright © 2019 American Society for Microbiology.
