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Recovery of an Acanthamoeba strain with two group I introns in the nuclear 18S rRNA gene
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ScienceDirect European Journal of Protistology 68 (2019) 88–98
Recovery of an Acanthamoeba strain with two group I introns in the nuclear 18S rRNA gene Daniele Corsaroa,∗ , Martina Köhslerb , Danielle Vendittia , Marilise B. Rottc , Julia Walochnikb a
CHLAREAS-12, Rue Du Maconnais, F-54500 Vandoeuvre-lès-Nancy, France Institute of Tropical Medicine and Specific Prophylaxis, Centre for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Kinderspitalgasse 15, 1090 Vienna, Austria c Departamento de Microbiologia, Imunologia e Parasitologia, Instituto de Ciências Básicas da Saúde, Setor de Parasitologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil b
Received 7 December 2018; received in revised form 16 January 2019; accepted 16 January 2019 Available online 28 January 2019
Abstract Nuclear group I introns are parasitic mobile genetic elements occurring in the ribosomal RNA genes of a large variety of microbial eukaryotes. In Acanthamoeba, group I introns were found occurring in the 18S rDNA at four distinct insertion sites. Introns are present as single elements in various strains belonging to four genotypes, T3 (A. griffini), T4 (A. castellanii complex), T5 (A. lenticulata) and T15 (A. jacobsi). While multiple introns can frequently be found in the rDNA of several algae, fungi and slime moulds, they are usually rare and present as single elements in amoebae. We reported herein the characterization of an A. lenticulata strain containing two introns in its 18S rDNA. They are located to already known sites and show basal relationships with respective homologous introns present in the other T5 strains. This is the first and unique reported case of multiple nuclear introns in Acanthamoeba. © 2019 Elsevier GmbH. All rights reserved. Keywords: Acanthamoeba; Acanthamoeba lenticulata; Group I intron; Nuclear IC1 introns
Introduction Free-living amoebae of the genus Acanthamoeba (Amoebozoa: Discoidea, Centramoebida) are major predators of bacteria and small eukaryotes, ubiquitous in terrestrial and aquatic environments. They are medically important as potential pathogens for humans and other vertebrates, and furthermore they can host a wide range of microbial pathogens playing a role in their transmission (Khan 2009). Molecular phylogenetic analyses based on the nuclear gene for the small subunit (SSU) ribosomal RNA (18S rDNA) have identified ∗ Corresponding
author. E-mail address: [email protected] (D. Corsaro).
https://doi.org/10.1016/j.ejop.2019.01.007 0932-4739/© 2019 Elsevier GmbH. All rights reserved.
at least 21 lineages in Acanthamoeba (genotypes T1–T21) (Corsaro and Venditti 2010, 2018a; Corsaro et al. 2015, 2017; Gast et al. 1996; Hewett et al. 2003; Magnet et al. 2014; Nuprasert et al. 2010; Qvarnstrom et al. 2013; Stothard et al. 1998), which correspond only in part to the morphologically described species (Page 1988). Sequencing of the complete 18S rRNA gene from different strains also revealed the presence of nuclear introns in Acanthamoeba. These were first identified in several strains of A. griffini and A. lenticulata (genotypes T3 and T5, respectively) (Gast et al. 1994; Ledee et al. 1996; Schroeder-Diedrich et al. 1998), subsequently in a strain of A. jacobsi (genotype T15) (Corsaro et al. 2017), and more recently in different strains of the A. castellanii complex (genotype T4), which are the sole to have remnants
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of homing endonuclease genes (HEG) (Corsaro and Venditti 2018b). Group I introns are mobile genetic elements that spread by reverse splicing (RNA level) or via an encoded endonuclease (DNA level). They are found in the viral, bacterial and nuclear and organelle genomes of a wide variety of organisms, where they disrupt the genes encoding for RNAs and proteins, while leaving the final products functional because they are spliced during transcription. Group I introns are catalytic RNAs (ribozymes) that once folded, present all a conserved series of nine base-paired regions (P1–P9). These are organized into three helical stacks referred to as the scaffold domain (P4–P6), the catalytic domain (P3, P7 and proximal P8 and P9) that hosts the binding site for the guanosine cofactor for splicing, and the substrate domain (P1 and P2) made up of both intron and exon sequences (Hedberg and Johansen 2013). Based on the overall structural motifs and sequence variations in the core pairs (P3–P7), group I introns are classified into five major classes IA, IB, IC, ID and IE, and fourteen subclasses (IA1–IA3, IB1–IB4, IC1–IC3, ID, IE1–IE3) (Li and Zhang 2005; Michel and Westhof 1990; Suh et al. 1999). Nuclear group I introns are IC1 and IE introns found only in the nuclear rRNA genes of various microbial eukaryotes. They are especially frequent in Fungi, green and red algae (Plantae), and myxogastrid slime moulds (syn. myxomycetes) (Amoebozoa: Mycetozoa), and often the same rDNA operon may contain multiple introns at different insertion sites that are not related to each other and may also belong to distinct subclasses (Hedberg and Johansen 2013; Haugen et al. 2005). Algae and fungi usually contain two to three introns in their SSU rDNA, while myxogastrids may contain five or more introns and up to 20 introns considering also the LSU rDNA. In other organisms such as amoebae, nuclear group I introns are rarely reported, usually as single elements. In Acanthamoeba, nuclear introns occur at one of four sites in the 18S rDNA (Corsaro and Venditti 2018b; Corsaro et al. 2017; Gast et al. 1994; SchroederDiedrich et al. 1998) (Fig. 1), and recent phylogenetic analyses indicated that they form distinct lineages within subclass IC1 (Corsaro and Venditti 2018b; Corsaro et al. 2017). Our study reports on an unusual strain of A. lenticulata carrying two introns in its 18S rDNA. These occur at previously recorded positions and have low similarities with respective homologous introns found in other Acanthamoeba strains although sharing common origins. We present herein the characterization of this Acanthamoeba strain and its two nuclear introns as well as the first comprehensive phylogenetic analysis including all known nuclear introns in the genus (Table 1).
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Material and Methods Sample origin and amoeba culture The Acanthamoeba strain dog-T5/D8 was isolated from nasal mucosa of an injured dog, in Brazil. The strain was previously genotyped as T5 by partial sequencing (ASA.S1 fragment) and characterized for temperature growth, mannitol osmo-tolerance and ability to induce cytopathogenic effect (CPE) on mammalian culture cells (Carlesso et al. 2014). Strain D8 was cultivated on non-nutrient agar (NNA) plates with Escherichia coli as food bacteria, and plates were evaluated daily by microscopy. Morphology of trophozoites and cysts was studied by light microscopy, according to Page (1988).
18S rDNA molecular analysis Amoebae were harvested from agar plates, washed and resuspended in sterile saline solution. Whole cell DNA was extracted with the QIAamp DNA mini kit (Qiagen GmbH, Germany), according to the manufacturer’s recommendations. The amoebal 18S rDNA was amplified employing the SSU1 and SSU2 primers complementary to the 5 and the 3 ends of the 18S rRNA gene and a series of internal primers p1fw–p3rev (Walochnik et al. 2004) and additionally primers 892/1262 (Wilcox et al. 1992). Fragments were amplified with a standard PCR program (1 min at 95 ◦ C, 1 min at 54 ◦ C, and 2 min at 72 ◦ C) for 35 cycles. PCR products were ® sequenced in both directions using the AB BigDye sequenc® ing kit and an ABI PRISM automated sequencer (AB Life Technologies, Vienna, Austria). The final sequence was carefully assembled and edited using BioEdit, and then was deposited in GenBank (ID. MK248592). 18S rDNA molecular analyses were carried out as described previously (Corsaro et al. 2015). Briefly, sequences were aligned using MAFFT, thus ambiguous sites were excluded manually using BioEdit. Maximum Likelihood (ML) tree (GTR + + I:4 model, 1000 replicates) was built with TREEFINDER (Jobb et al. 2004). Pair-wise similarity values were calculated with BioEdit.
Intron characterization The secondary structures of introns were inferred by manually identifying the conserved core paired helices P3–P7, using as guide the reference intron sequence alignment (Michel and Westhof 1990) and previous works on Acanthamoeba introns (Corsaro and Venditti 2018b; Corsaro et al. 2017; Gast et al. 1994; Schroeder-Diedrich et al. 1998). The remaining portions of the molecules were determined using Mfold.
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Fig. 1. Schematic draw of the Acanthamoeba 18S rDNA showing (upper) the insertion sites of the introns (black triangles) and their distribution among genotypes, and (down) the relative positions of some PCR primers (arrows) and amplimers (solid lines). Numbers on the 18S rDNA refer to A. castellanii (Genbank ID U07413) positions. Hypervariable regions are indicated by gray boxes. Note that there is no relationship between the S943 introns of A. lenticulata and A. jacobsi, which although located at the same site, form two distinct types. Table 1. Summary of Acanthamoeba strains harboring nuclear 18S rDNA group I introns. GT
T3
T4
T5
T15 a Partial
Species/strain
18S rDNA
Intron
bp
bp
2812 2781 1976a 1974a 2812
518 494 523 523 518b
Acanthamoeba castellanii complexc AcaVN14 Salt cave, Slovakia KA/E4 Human keratitis, Korea KA/E5 Human keratitis, Korea KA/E21 Human keratitis, Korea KA/MSS2 Ocean sediment, Korea KA/MSS6 Ocean sediment, Korea KA/MSS7 Ocean sediment, Korea KA/MSG23 Ocean sediment, Korea
2395a 3242 3236 3262 3275 3256 3275 3583
933 957 959 982 969 959 967 1275
Acanthamoeba lenticulata PD2ST ATCC 30841 NJSP-3-2 ATCC 50429 E18-2 ATCC 50690 118 ATCC 50706 53-2 ATCC 50691 Egy3 25-1 ATCC 50707 Jc-1 ATCC 50428 TUMSJ-341
Swimming pool, France Sewage plant, New Jersey, USA Sewage dump site, Atlantic Ocean, USA Human nasal mucosa, Germany Sewage dump site, New York Bight, USA Human corneal scraping, Egypt Human nasal mucosa, Germany Freshwater stream, New York, USA Eutrophic lake sediment, Malaysia
2950 2936 2935 2934 2930 2950 2973 2992 2990d
Dog D8
Injured dog, Brazil
3449
407-3a ATCC 50692
Waste dump site, Atlantic Ocean, USA
3012
656 642 641 640 636 656b 679 699 639 663 503 721
Acanthamoeba jacobsi Pool-4-37
Thermal pond, Italy
2891
558
Acanthamoeba griffini S7T ATCC 30731 H37 ATCC 50702 AcaVN06 AcaVNAK05 Egy4
Origin
Beach bottom, Connecticut, USA Human keratitis, Scotland, UK Air conditioner, Slovakia Human keratitis, Czech Republic Human corneal scraping, Egypt
sequences (GTSA.B1 fragment). identical to type strains S7 and PD2S. c All introns in T4 strains contain endonuclease pseudogenes. d 18S rDNA likely chimeric. b Sequences
GenBank ID Site
S516
S516
S1389
S943
S956 S943 S956 S1389 S943
U07412 S81337 GQ397468 GQ905499 MF350347 GQ397476 AF349045 AY148954 EF140633 AY173013 AY173014 AY173015 AY176047 U94741 U94738 U94735 U94736 U94737 MF350346 U94740 U94739 AF352391 MK248592 U94734 KY513796
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Fig. 2. LM pictures of strain D8; a: trophozoite; b: cysts. Scale bar = 10 m.
All available Acanthamoeba nuclear introns were already been retrieved from GenBank (Corsaro and Venditti 2018b). Sequences were thus aligned with a large set of nuclear IC1 introns based on secondary structure. The conserved core pairs (P3–P8) were selected to infer phylogeny as described previously (Corsaro et al. 2017), using maximum likelihood (GTR + model) (1000 replicates) with TREEFINDER (Jobb et al. 2004), and neighbour joining (NJ) (Jukes–Cantor model) (2000 replicates) and maximum parsimony (MP) (1000 replicates) with TBR (tree-bisectionreconnection, search level 1 with 10 random additions) with MEGA7 (Kumar et al. 2016). Nuclear IE introns were used as outgroup. In parallel, to retrace evolutionary history of introns within amoebae, UPGMA (Unweighted Pair-Group Method with Arithmetic Mean) and ML (GTR + + I:4) cladograms for introns and 18S rDNA, respectively, were build and compared. Trees were based on full-length sequences of the introns without HEG and intron-less 18S rDNA of Acanthamoeba, using MEGA7 and TREEFINDER, respectively, with 1000 replicates, as described previously (Corsaro and Venditti 2018b; Corsaro et al. 2015).
Fig. 3. Maximum likelihood tree of A. lenticulata. Strains for which complete 18S rDNA sequence is available are noted as “Alent”. For the remaining strains partial sequences are included, mostly the GTSA.B1 fragment. Lateral bars along the tree indicate possible subgroups (cf. Fig. 4). A. pyriformis (T21) was used as outgroup (not shown). Bootstraps values (>50%) after 1000 replicates are indicated at the nodes.
Results Morphology and physiological features The strain D8 shows characteristic morphological traits of Acanthamoeba, with trophozoites (approx. 25 m in length and 18 m in breadth) bearing multiple acanthopodia. Cysts (15 m in diameter) exhibit group III morphology, with an almost circular endocyst and a slightly undulate ectocyst (Fig. 2). The strain exhibits growth at 37 ◦ C and exhibited CPE on mammalian culture cells (Carlesso et al. 2014). Mor-
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Fig. 4. Distribution of group I introns in the Acanthamoeba tree. At left, maximum likelihood phylogeny of A. lenticulata (genotype T5) and representatives of other intron-bearing genotypes (T3, T4, and T15) and their close relatives, based on full 18S rDNA sequences without introns. A. pyriformis (T21) and A. comandoni (T9) were used as outgroup (not shown). At right, UPGMA cladogram based on complete sequences of the introns without HEG insertions. For both trees, bootstrap values >50% (1000 replicates) are shown at the nodes. Monophyletic groups/subgroups of sequences are highlighted by shaded areas. Solid and dotted lines connecting host and intron phylogenies indicate good correspondence or incongruence, respectively. The dog strain D8 investigated herein is in bold and correspondences with its introns are highlighted by a thick solid line.
phological and biological characters correspond well to those of A. lenticulata within the thermophilic acanthamoebae of group III (Molet and Ermolieff-Braun 1976; Page 1988), which are potentially pathogenic (De Jonckheere and Michel 1988).
18S rDNA analysis The full 18S rDNA of strain D8 is 3449 bp in length, being >1000 bp longer compared to the usual 2300-bp sequences of Acanthamoeba, and it shows similarity values of 97.1% (96.4–97.2%) with the other strains of A. lenticulata when complete sequences were compared after intron removal. The 18S rDNA sequence of the TUMSJ-341 strain was excluded because of its chimeric nature (Corsaro and Venditti 2019). In
phylogenetic reconstruction (Fig. 3), strain D8 clusters with a few environmental strains characterized by partial sequencing (GTSA.B1 fragment), isolated either from dust in Brazil (Possamai et al. 2018) or soil in Thailand (Chusattayanond et al. unpubl.), forming a basal subgroup of the species. Analyses performed by including the T5-type 5 -end sequence of TUMSJ-341 indicated that this strain also belongs to this subgroup (not shown). Most of the sequence variations within A. lenticulata are found in some regions of rDNA, notably the helices E23-1 and H10, and their distribution is congruent with the different subgroups and subtypes identified here (Supplementary Fig. 1). Phylogeny based on complete 18S rDNA sequences (Fig. 4, left panel) confirms that strain D8 branches early as sister to all other T5 strains, which are >99% similar each other and cluster into four tight subgroups, i.e.,
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Fig. 5. Predicted secondary structures of the S943 and S956 introns present in the nuclear 18S rDNA of A. lenticulata. The two intron types occurring together in the dog D8 strain (a, c) are compared with homologous introns found separately in distinct amoeba strains, e.g. PD2S (b) and Jc-1 (d). Sequences of the introns are in uppercase, those of the flanking exons are in lowercase. Paired elements (P1–P10) and the 5 and 3 splice sites (black arrowheads) are indicated. Structures of PD2S and Jc-1 introns modified from Schroeder-Diedrich et al. (1998).
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Fig. 6. Phylogenetic relationships between IC1 subclass introns located in the nuclear rDNA operon of eukaryotes, with emphasis on those found in the Acanthamoeba 18S rDNA. The alignment is based on the conserved core P3 to P8. The two introns carried by the strain of A. lenticulata characterized in this study are in bold. Nuclear introns of the IE class are used as outgroup. Bootstrap values (BV) for ML/NJ/MP
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Jc-1, 45, PD2S, and 25-1. As expected, the complete 18S rDNA-based tree provides better resolution and higher bootstrap values, recovering the main subgroups as defined by variations in the E23-1, H10 and 45-1 helices. The slight difference with the partial tree in the internal cluster is probably due to the few variations in helices E10-1, E23-5 and 29-1, which could differentiate subtypes within the main groups.
Identification of introns The extraordinary length of the 18S rDNA of strain D8, even exceeding the sequences of the A. lenticulata strains known to contain introns by ∼500 bp also, strongly indicated the presence of an intron. The careful analysis of the sequence revealed that, unexpectedly, two group I introns of subclass IC1 are present in the gene. These are 663 and 503 nt in lengths and are located at insertion sites S943 and S956, at the ends of stems 33 and 34 of the rRNA, respectively. Introns occurring at the same sites, but as unique elements were already described in A. lenticulata (SchroederDiedrich et al. 1998). Phylogenetic analyses based on full intron sequences (Fig. 4, right panel) showed that both, the S943 and the S956 introns of the dog D8 strain cluster with the homologous introns found in the other A. lenticulata strains, although being more divergent with similarities of only 52–57%. Higher values (>80%) are found when considering the conserved core pairs P3–P7. By comparing the intron and 18S rDNA phylogenies (Fig. 4), good correspondences can only be observed for the S943 of A. lenticulata and between acanthamoebae T3 and T4 with S516, indicating unique genetic invasions followed by vertical inheritances. Although probably derived from common ancestors, the remaining introns, S956 and S1389, exhibit a pattern of horizontal spread, since they have clearly invaded distinct subgroups of A. lenticulata (S956) or distant genotype hosts (S1389). The S943 intron of A. jacobsi appears to be a distinct lineage, perhaps related to the S1389 intron, but as only one sequence and one host are known, additional data are needed to better evaluate its history. The predicted secondary structures of the dog D8 strain introns are shown in Fig. 5 and compared with those of homologous introns found in Acanthamoeba. Overall, the size and structure of the S943 intron of strain D8 (Fig. 5a) are similar to those of the S943 introns found in the other A. lenticulata strains (e.g., PD2S strain, Fig. 5b). The dog S943 intron presents a second helix P2.1 and an additional helix P5.3. The P5 parts and their extensions in the two molecules are however very similar, with P5.2 comprising two stems (P5.2a, P5.2b). It should be noted that the structure of S943 occurring in the PD2S and close strains considered here, is
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slightly different from that proposed by Schroeder-Diedrich et al. (1998), because during our previous studies, we identified the P5 pair differently, resulting in a restructuring of the entire P5 extension. The S956 intron of the dog strain (Fig. 5c), on the other hand, is slightly shorter (∼170 nt) than the corresponding homologous introns occurring in the other strains (e.g., Jc-1, Fig. 5d). Its structure has only a P2 helix, as well as shorter P6 and simpler P5 extensions.
Nuclear intron phylogeny To better evaluate intron relationships, conserved corebased molecular phylogeny was performed on a larger number of introns from different hosts (Fig. 6). As expected, introns with the same insertion site, although present in distant hosts, often cluster together, indicating that they have common origins. The various intron types of Acanthamoeba each form a coherent group, however intermingled with the other groups, confirming that they are distinct lineages. The common origin of the two introns of the dog strain D8 with their respective homologous introns is also confirmed. However, except for the Acanthamoeba T3 and T4 S516 introns that belong to a lineage widespread across many organisms, the relationships of the other Acanthamoeba introns are not yet clear. Beyond the possible links with introns of green algae and chytrids already suggested in previous studies, this analysis also reveals relationships with the introns of Rhynchomonas, a marine/freshwater bodonid nanoflagellate (Scheckenbach et al. 2006), and Chlamydomyzium, an oomycete parasitizing on nematodes (Beakes et al. 2014). All these introns have however different insertion sites. In addition, the low values and/or inconsistency of the nodes supporting the relationships would suggest that these Acanthamoeba introns are actually unique and have no known close relatives.
Discussion The presence of multiple nuclear introns in the rDNA is relatively common in algae and fungi and even a rule in slime moulds, but only occasionally reported in amoebae without further investigation. However, except for Acanthamoeba (Corsaro and Venditti 2018b; Gast et al. 1994; SchroederDiedrich et al. 1998) and twintron-containing vahlkampfiid amoebae (Tang et al. 2014; Wikmark et al. 2006), the study of these elements in amoebae has attracted little interest, so that they are often neither characterized, nor correctly recorded, nor even identified in the sequences deposited at GenBank. We reported herein the first case of two group I introns co-
are shown at the nodes (1000 replicates for ML and MP; 2000 for NJ). Filled and open circles, nodes with 100 or >90% BV support with all methods, respectively; asterisk, node supported but BV<50%; hyphen, node not supported.
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occurring in the same 18S rRNA gene of an Acanthamoeba strain, as well as their characterization and a comprehensive phylogenetic analysis of the nuclear introns present in these amoebae. Currently, at least five distinct types of introns are known to invade the 18S rDNA of Acanthamoeba by inserting into four different sites (Fig. 1). The S516 intron appears to be specific for the T3/T4 lineage (Corsaro and Venditti 2018b; Gast et al. 1994; Ledee et al. 1996), whereas the presence of the S943 or S956 introns characterizes many strains representing genotype T5 (Schroeder-Diedrich et al. 1998; this study). Most likely quite early these introns have invaded the respective host, in which they probably spread in different ways. On the other hand, the S1389 intron has clearly a separate history of horizontal transfer among distantly related T5 and T4 strains (Corsaro and Venditti 2018b), while the uniqueness of the S943 found in A. jacobsi (Corsaro et al. 2017) requires other data to be analysed. Only the S516 intron has obvious affinities with several homologous introns of other organisms, forming a robust clade. This intron has spread among different hosts, in which it has been inherited several times vertically, as also shown by intron-HEG coinheritance and intron-rDNA coevolution that can be observed especially for the case of heterolobosean amoebae and Acanthamoeba T4 (Corsaro and Venditti 2018b; Haugen et al. 2004; Wikmark et al. 2006). In addition, the data suggest that a more complex history is underlying in Acanthamoeba, consistent with the intron life cycle model, assuming an early invasion of a HEG-containing S516 intron in the T3/T4 lineage, followed by multiple and differential losses during its vertical inheritance (Corsaro and Venditti 2018b). The case of A. lenticulata (genotype T5) is of interest because, despite the relatively small number of strains analysed in their 18S rDNA full length (n = 16), particularly with respect to genotype T4 (∼300), this species has the largest (n = 12) and most diverse (n = 3) intron population (Table 1) (Schroeder-Diedrich et al. 1998; this study). Our analysis indicates that the S943 and S956 introns early invaded A. lenticulata. It is possible that the co-presence of these two introns in the dog D8 strain reported herein is only fortuitous, each intron having indeed its own independent history. Data available on the other basal strain, TUMSJ-341, suggest that it contains only the S956 intron, but the reliability of this part of the sequence is very uncertain (Corsaro and Venditti 2019). While the S943 intron was inherited vertically within the PD2S subgroup, showing similarity values of ≥97% among strains that are 100% identical at the rDNA level, the S956 intron spread horizontally from the basal lineage to strains Jc-1 and 25-1 belonging to distinct subgroups (Fig. 4). The existing distances with strain D8 introns nevertheless suggest that this picture might be uncomplete, although probably not very different. A higher diversity seems to exist within A. lenticulata, as indicated by the partial sequences (Fig. 3). Unfortunately, these mostly correspond to fragment GTSA.B1, and therefore they can
only confirm that the S516 intron is apparently never present in this species. Indeed, these data cannot tell us about the possible presence and distribution among these strains of the three introns specific for T5 (Fig. 1). The characterization of additional sequences of introns and complete rDNAs will certainly be very useful to improve our knowledge. It should be underlined that molecular strategies commonly used to characterize the strains do not allow the recognition of introns (Fig. 1). Strain identification is indeed mostly carried out using the primer set JDP1/JDP2, which amplify the fragment ASA.S1 containing the hypervariable region 29-1 useful for genotyping as diagnostic fragment 3 (DF3) (Booton et al. 2002; Schroeder et al. 2001), and none of the four intron insertion sites are included within. The amplimers, GEF which increases the discriminatory power by including the entire E23 region (Risler et al. 2013), and Nelson which targets the 45-1 region (Mathers et al. 2000), also do not cover any of the known sites. Even the longer amplimer GTSA.B1 covers only one insertion site (S516). Therefore, it would not be surprising that many introns remained undetected. Furthermore, except for two T4 strains, no sequence is available for the LSU rDNA, which could also contain introns, as it occurs in other organisms. It is thus very likely that our knowledge of the distribution and diversity of nuclear introns in Acanthamoeba is only at the beginning.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Author contributions M.B. Rott collected samples and isolated the strain. M. Köhsler & J. Walochnik performed DNA sequencing. D. Corsaro & D. Venditti analysed intron structures and built phylogenetic trees. D. Corsaro & M. Köhsler drafted the manuscript. All authors reviewed the manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/ j.ejop.2019.01.007.
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Hewett, M.K., Robinson, B.S., Monis, P.T., Saint, C.P., 2003. Identification of a new Acanthamoeba 18S rRNA gene sequence type, corresponding to the species Acanthamoeba jacobsi Sawyer, Nerad and Visvesvara, 1992 (Lobosea: Acanthamoebidae). Acta Protozool. 42, 325–329. Jobb, G., von Haeseler, A., Strimmer, K., 2004. TREEFINDER: a powerful graphical analysis environment for molecular phylogenetics. BMC Evol. Biol. 4, 18, http://dx.doi.org/ 10.1186/1471-2148-4-18. Khan, N.A., 2009. Acanthamoeba: Biology and Pathogenesis. Caister Academic Press, Norfolk, Great Britain, 290 pp. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874, http://dx.doi.org/10.1093/molbev/msw054. Ledee, D.R., Hay, J., Byers, T.J., Seal, D.V., Kirkness, C.M., 1996. Acanthamoeba griffini. Molecular characterization of a new corneal pathogen. Invest. Ophthalmol. Vis. Sci. 37, 544–550. Li, Z., Zhang, Y., 2005. Predicting the secondary structures and tertiary interactions of 211 group I introns in IE subgroup. Nucleic Acids Res. 33, 2118–2128. Magnet, A., Henriques-Gil, N., Galván-Diaz, A.L., Izquiedo, F., Fenoy, S., Del Aguila, C., 2014. Novel Acanthamoeba 18S rRNA gene sequence type from an environmental isolate. Parasitol. Res. 113, 2845–2850, http://dx.doi.org/ 10.1007/s00436-014-3945-2. Mathers, W.D., Nelson, S.E., Lane, J.L., Wilson, M.E., Allen, R.C., Folberg, R., 2000. Confirmation of confocal microscopy diagnosis of Acanthamoeba keratitis using polymerase chain reaction analysis. Arch. Ophthalmol. 118, 178–183, http://dx.doi.org/10.1001/archopht.118.2.178. Michel, F., Westhof, E., 1990. Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J. Mol. Biol. 216, 585–610, http://dx.doi.org/10.1016/0022-2836(90)90386-Z. Molet, B., Ermolieff-Braun, G., 1976. Description d’une amibe d’eau douce: Acanthamoeba lenticulata sp. nov. (Amoebida). Protistologica 12, 571–576. Nuprasert, W., Putaporntip, C., Pariyakanok, L., Jongwutiwes, S., 2010. Identification of a novel T17 genotype of Acanthamoeba from environmental isolates and T10 genotype causing keratitis in Thailand. J. Clin. Microbiol. 48, 4636–4640, http://dx.doi.org/10.1128/JCM.01090-10. Page, F.C., 1988. A New Key to Freshwater and Soil Gymnamoebae. Freshwater Biological Association, Ambleside, pp. 92–97. Possamai, C.O., Loss, A.C., Costa, A.O., Falqueto, A., Furst, C., 2018. Acanthamoeba of three morphological groups and distinct genotypes exhibit variable and weakly inter-related physiological properties. Parasitol. Res. 117, 1389–1400, http://dx.doi.org/10.1007/s00436-018-5824-8. Qvarnstrom, Y., Nerad, T.A., Visvesvara, G.S., 2013. Characterization of a new pathogenic Acanthamoeba species, A. byersi n. sp., isolated from a human with fatal amoebic encephalitis. J. Eukaryot. Microbiol. 60, 626–633, http://dx.doi.org/10.1111/jeu.12069. Risler, A., Coupat-Goutaland, B., Pélandakis, M., 2013. Genotyping and phylogenetic analysis of Acanthamoeba isolates associated with keratitis. Parasitol. Res. 112, 3807–3816, http://dx.doi.org/10.1007/s00436-013-3572-3. Scheckenbach, F., Wylezich, C., Mylnikov, A.P., Weitere, M., Arndt, H., 2006. Molecular comparisons of freshwater
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ScienceDirect European Journal of Protistology 68 (2019) 88–98
Recovery of an Acanthamoeba strain with two group I introns in the nuclear 18S rRNA gene Daniele Corsaroa,∗ , Martina Köhslerb , Danielle Vendittia , Marilise B. Rottc , Julia Walochnikb a
CHLAREAS-12, Rue Du Maconnais, F-54500 Vandoeuvre-lès-Nancy, France Institute of Tropical Medicine and Specific Prophylaxis, Centre for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Kinderspitalgasse 15, 1090 Vienna, Austria c Departamento de Microbiologia, Imunologia e Parasitologia, Instituto de Ciências Básicas da Saúde, Setor de Parasitologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil b
Received 7 December 2018; received in revised form 16 January 2019; accepted 16 January 2019 Available online 28 January 2019
Abstract Nuclear group I introns are parasitic mobile genetic elements occurring in the ribosomal RNA genes of a large variety of microbial eukaryotes. In Acanthamoeba, group I introns were found occurring in the 18S rDNA at four distinct insertion sites. Introns are present as single elements in various strains belonging to four genotypes, T3 (A. griffini), T4 (A. castellanii complex), T5 (A. lenticulata) and T15 (A. jacobsi). While multiple introns can frequently be found in the rDNA of several algae, fungi and slime moulds, they are usually rare and present as single elements in amoebae. We reported herein the characterization of an A. lenticulata strain containing two introns in its 18S rDNA. They are located to already known sites and show basal relationships with respective homologous introns present in the other T5 strains. This is the first and unique reported case of multiple nuclear introns in Acanthamoeba. © 2019 Elsevier GmbH. All rights reserved. Keywords: Acanthamoeba; Acanthamoeba lenticulata; Group I intron; Nuclear IC1 introns
Introduction Free-living amoebae of the genus Acanthamoeba (Amoebozoa: Discoidea, Centramoebida) are major predators of bacteria and small eukaryotes, ubiquitous in terrestrial and aquatic environments. They are medically important as potential pathogens for humans and other vertebrates, and furthermore they can host a wide range of microbial pathogens playing a role in their transmission (Khan 2009). Molecular phylogenetic analyses based on the nuclear gene for the small subunit (SSU) ribosomal RNA (18S rDNA) have identified ∗ Corresponding
author. E-mail address: [email protected] (D. Corsaro).
https://doi.org/10.1016/j.ejop.2019.01.007 0932-4739/© 2019 Elsevier GmbH. All rights reserved.
at least 21 lineages in Acanthamoeba (genotypes T1–T21) (Corsaro and Venditti 2010, 2018a; Corsaro et al. 2015, 2017; Gast et al. 1996; Hewett et al. 2003; Magnet et al. 2014; Nuprasert et al. 2010; Qvarnstrom et al. 2013; Stothard et al. 1998), which correspond only in part to the morphologically described species (Page 1988). Sequencing of the complete 18S rRNA gene from different strains also revealed the presence of nuclear introns in Acanthamoeba. These were first identified in several strains of A. griffini and A. lenticulata (genotypes T3 and T5, respectively) (Gast et al. 1994; Ledee et al. 1996; Schroeder-Diedrich et al. 1998), subsequently in a strain of A. jacobsi (genotype T15) (Corsaro et al. 2017), and more recently in different strains of the A. castellanii complex (genotype T4), which are the sole to have remnants
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of homing endonuclease genes (HEG) (Corsaro and Venditti 2018b). Group I introns are mobile genetic elements that spread by reverse splicing (RNA level) or via an encoded endonuclease (DNA level). They are found in the viral, bacterial and nuclear and organelle genomes of a wide variety of organisms, where they disrupt the genes encoding for RNAs and proteins, while leaving the final products functional because they are spliced during transcription. Group I introns are catalytic RNAs (ribozymes) that once folded, present all a conserved series of nine base-paired regions (P1–P9). These are organized into three helical stacks referred to as the scaffold domain (P4–P6), the catalytic domain (P3, P7 and proximal P8 and P9) that hosts the binding site for the guanosine cofactor for splicing, and the substrate domain (P1 and P2) made up of both intron and exon sequences (Hedberg and Johansen 2013). Based on the overall structural motifs and sequence variations in the core pairs (P3–P7), group I introns are classified into five major classes IA, IB, IC, ID and IE, and fourteen subclasses (IA1–IA3, IB1–IB4, IC1–IC3, ID, IE1–IE3) (Li and Zhang 2005; Michel and Westhof 1990; Suh et al. 1999). Nuclear group I introns are IC1 and IE introns found only in the nuclear rRNA genes of various microbial eukaryotes. They are especially frequent in Fungi, green and red algae (Plantae), and myxogastrid slime moulds (syn. myxomycetes) (Amoebozoa: Mycetozoa), and often the same rDNA operon may contain multiple introns at different insertion sites that are not related to each other and may also belong to distinct subclasses (Hedberg and Johansen 2013; Haugen et al. 2005). Algae and fungi usually contain two to three introns in their SSU rDNA, while myxogastrids may contain five or more introns and up to 20 introns considering also the LSU rDNA. In other organisms such as amoebae, nuclear group I introns are rarely reported, usually as single elements. In Acanthamoeba, nuclear introns occur at one of four sites in the 18S rDNA (Corsaro and Venditti 2018b; Corsaro et al. 2017; Gast et al. 1994; SchroederDiedrich et al. 1998) (Fig. 1), and recent phylogenetic analyses indicated that they form distinct lineages within subclass IC1 (Corsaro and Venditti 2018b; Corsaro et al. 2017). Our study reports on an unusual strain of A. lenticulata carrying two introns in its 18S rDNA. These occur at previously recorded positions and have low similarities with respective homologous introns found in other Acanthamoeba strains although sharing common origins. We present herein the characterization of this Acanthamoeba strain and its two nuclear introns as well as the first comprehensive phylogenetic analysis including all known nuclear introns in the genus (Table 1).
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Material and Methods Sample origin and amoeba culture The Acanthamoeba strain dog-T5/D8 was isolated from nasal mucosa of an injured dog, in Brazil. The strain was previously genotyped as T5 by partial sequencing (ASA.S1 fragment) and characterized for temperature growth, mannitol osmo-tolerance and ability to induce cytopathogenic effect (CPE) on mammalian culture cells (Carlesso et al. 2014). Strain D8 was cultivated on non-nutrient agar (NNA) plates with Escherichia coli as food bacteria, and plates were evaluated daily by microscopy. Morphology of trophozoites and cysts was studied by light microscopy, according to Page (1988).
18S rDNA molecular analysis Amoebae were harvested from agar plates, washed and resuspended in sterile saline solution. Whole cell DNA was extracted with the QIAamp DNA mini kit (Qiagen GmbH, Germany), according to the manufacturer’s recommendations. The amoebal 18S rDNA was amplified employing the SSU1 and SSU2 primers complementary to the 5 and the 3 ends of the 18S rRNA gene and a series of internal primers p1fw–p3rev (Walochnik et al. 2004) and additionally primers 892/1262 (Wilcox et al. 1992). Fragments were amplified with a standard PCR program (1 min at 95 ◦ C, 1 min at 54 ◦ C, and 2 min at 72 ◦ C) for 35 cycles. PCR products were ® sequenced in both directions using the AB BigDye sequenc® ing kit and an ABI PRISM automated sequencer (AB Life Technologies, Vienna, Austria). The final sequence was carefully assembled and edited using BioEdit, and then was deposited in GenBank (ID. MK248592). 18S rDNA molecular analyses were carried out as described previously (Corsaro et al. 2015). Briefly, sequences were aligned using MAFFT, thus ambiguous sites were excluded manually using BioEdit. Maximum Likelihood (ML) tree (GTR + + I:4 model, 1000 replicates) was built with TREEFINDER (Jobb et al. 2004). Pair-wise similarity values were calculated with BioEdit.
Intron characterization The secondary structures of introns were inferred by manually identifying the conserved core paired helices P3–P7, using as guide the reference intron sequence alignment (Michel and Westhof 1990) and previous works on Acanthamoeba introns (Corsaro and Venditti 2018b; Corsaro et al. 2017; Gast et al. 1994; Schroeder-Diedrich et al. 1998). The remaining portions of the molecules were determined using Mfold.
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Fig. 1. Schematic draw of the Acanthamoeba 18S rDNA showing (upper) the insertion sites of the introns (black triangles) and their distribution among genotypes, and (down) the relative positions of some PCR primers (arrows) and amplimers (solid lines). Numbers on the 18S rDNA refer to A. castellanii (Genbank ID U07413) positions. Hypervariable regions are indicated by gray boxes. Note that there is no relationship between the S943 introns of A. lenticulata and A. jacobsi, which although located at the same site, form two distinct types. Table 1. Summary of Acanthamoeba strains harboring nuclear 18S rDNA group I introns. GT
T3
T4
T5
T15 a Partial
Species/strain
18S rDNA
Intron
bp
bp
2812 2781 1976a 1974a 2812
518 494 523 523 518b
Acanthamoeba castellanii complexc AcaVN14 Salt cave, Slovakia KA/E4 Human keratitis, Korea KA/E5 Human keratitis, Korea KA/E21 Human keratitis, Korea KA/MSS2 Ocean sediment, Korea KA/MSS6 Ocean sediment, Korea KA/MSS7 Ocean sediment, Korea KA/MSG23 Ocean sediment, Korea
2395a 3242 3236 3262 3275 3256 3275 3583
933 957 959 982 969 959 967 1275
Acanthamoeba lenticulata PD2ST ATCC 30841 NJSP-3-2 ATCC 50429 E18-2 ATCC 50690 118 ATCC 50706 53-2 ATCC 50691 Egy3 25-1 ATCC 50707 Jc-1 ATCC 50428 TUMSJ-341
Swimming pool, France Sewage plant, New Jersey, USA Sewage dump site, Atlantic Ocean, USA Human nasal mucosa, Germany Sewage dump site, New York Bight, USA Human corneal scraping, Egypt Human nasal mucosa, Germany Freshwater stream, New York, USA Eutrophic lake sediment, Malaysia
2950 2936 2935 2934 2930 2950 2973 2992 2990d
Dog D8
Injured dog, Brazil
3449
407-3a ATCC 50692
Waste dump site, Atlantic Ocean, USA
3012
656 642 641 640 636 656b 679 699 639 663 503 721
Acanthamoeba jacobsi Pool-4-37
Thermal pond, Italy
2891
558
Acanthamoeba griffini S7T ATCC 30731 H37 ATCC 50702 AcaVN06 AcaVNAK05 Egy4
Origin
Beach bottom, Connecticut, USA Human keratitis, Scotland, UK Air conditioner, Slovakia Human keratitis, Czech Republic Human corneal scraping, Egypt
sequences (GTSA.B1 fragment). identical to type strains S7 and PD2S. c All introns in T4 strains contain endonuclease pseudogenes. d 18S rDNA likely chimeric. b Sequences
GenBank ID Site
S516
S516
S1389
S943
S956 S943 S956 S1389 S943
U07412 S81337 GQ397468 GQ905499 MF350347 GQ397476 AF349045 AY148954 EF140633 AY173013 AY173014 AY173015 AY176047 U94741 U94738 U94735 U94736 U94737 MF350346 U94740 U94739 AF352391 MK248592 U94734 KY513796
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Fig. 2. LM pictures of strain D8; a: trophozoite; b: cysts. Scale bar = 10 m.
All available Acanthamoeba nuclear introns were already been retrieved from GenBank (Corsaro and Venditti 2018b). Sequences were thus aligned with a large set of nuclear IC1 introns based on secondary structure. The conserved core pairs (P3–P8) were selected to infer phylogeny as described previously (Corsaro et al. 2017), using maximum likelihood (GTR + model) (1000 replicates) with TREEFINDER (Jobb et al. 2004), and neighbour joining (NJ) (Jukes–Cantor model) (2000 replicates) and maximum parsimony (MP) (1000 replicates) with TBR (tree-bisectionreconnection, search level 1 with 10 random additions) with MEGA7 (Kumar et al. 2016). Nuclear IE introns were used as outgroup. In parallel, to retrace evolutionary history of introns within amoebae, UPGMA (Unweighted Pair-Group Method with Arithmetic Mean) and ML (GTR + + I:4) cladograms for introns and 18S rDNA, respectively, were build and compared. Trees were based on full-length sequences of the introns without HEG and intron-less 18S rDNA of Acanthamoeba, using MEGA7 and TREEFINDER, respectively, with 1000 replicates, as described previously (Corsaro and Venditti 2018b; Corsaro et al. 2015).
Fig. 3. Maximum likelihood tree of A. lenticulata. Strains for which complete 18S rDNA sequence is available are noted as “Alent”. For the remaining strains partial sequences are included, mostly the GTSA.B1 fragment. Lateral bars along the tree indicate possible subgroups (cf. Fig. 4). A. pyriformis (T21) was used as outgroup (not shown). Bootstraps values (>50%) after 1000 replicates are indicated at the nodes.
Results Morphology and physiological features The strain D8 shows characteristic morphological traits of Acanthamoeba, with trophozoites (approx. 25 m in length and 18 m in breadth) bearing multiple acanthopodia. Cysts (15 m in diameter) exhibit group III morphology, with an almost circular endocyst and a slightly undulate ectocyst (Fig. 2). The strain exhibits growth at 37 ◦ C and exhibited CPE on mammalian culture cells (Carlesso et al. 2014). Mor-
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Fig. 4. Distribution of group I introns in the Acanthamoeba tree. At left, maximum likelihood phylogeny of A. lenticulata (genotype T5) and representatives of other intron-bearing genotypes (T3, T4, and T15) and their close relatives, based on full 18S rDNA sequences without introns. A. pyriformis (T21) and A. comandoni (T9) were used as outgroup (not shown). At right, UPGMA cladogram based on complete sequences of the introns without HEG insertions. For both trees, bootstrap values >50% (1000 replicates) are shown at the nodes. Monophyletic groups/subgroups of sequences are highlighted by shaded areas. Solid and dotted lines connecting host and intron phylogenies indicate good correspondence or incongruence, respectively. The dog strain D8 investigated herein is in bold and correspondences with its introns are highlighted by a thick solid line.
phological and biological characters correspond well to those of A. lenticulata within the thermophilic acanthamoebae of group III (Molet and Ermolieff-Braun 1976; Page 1988), which are potentially pathogenic (De Jonckheere and Michel 1988).
18S rDNA analysis The full 18S rDNA of strain D8 is 3449 bp in length, being >1000 bp longer compared to the usual 2300-bp sequences of Acanthamoeba, and it shows similarity values of 97.1% (96.4–97.2%) with the other strains of A. lenticulata when complete sequences were compared after intron removal. The 18S rDNA sequence of the TUMSJ-341 strain was excluded because of its chimeric nature (Corsaro and Venditti 2019). In
phylogenetic reconstruction (Fig. 3), strain D8 clusters with a few environmental strains characterized by partial sequencing (GTSA.B1 fragment), isolated either from dust in Brazil (Possamai et al. 2018) or soil in Thailand (Chusattayanond et al. unpubl.), forming a basal subgroup of the species. Analyses performed by including the T5-type 5 -end sequence of TUMSJ-341 indicated that this strain also belongs to this subgroup (not shown). Most of the sequence variations within A. lenticulata are found in some regions of rDNA, notably the helices E23-1 and H10, and their distribution is congruent with the different subgroups and subtypes identified here (Supplementary Fig. 1). Phylogeny based on complete 18S rDNA sequences (Fig. 4, left panel) confirms that strain D8 branches early as sister to all other T5 strains, which are >99% similar each other and cluster into four tight subgroups, i.e.,
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Fig. 5. Predicted secondary structures of the S943 and S956 introns present in the nuclear 18S rDNA of A. lenticulata. The two intron types occurring together in the dog D8 strain (a, c) are compared with homologous introns found separately in distinct amoeba strains, e.g. PD2S (b) and Jc-1 (d). Sequences of the introns are in uppercase, those of the flanking exons are in lowercase. Paired elements (P1–P10) and the 5 and 3 splice sites (black arrowheads) are indicated. Structures of PD2S and Jc-1 introns modified from Schroeder-Diedrich et al. (1998).
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Fig. 6. Phylogenetic relationships between IC1 subclass introns located in the nuclear rDNA operon of eukaryotes, with emphasis on those found in the Acanthamoeba 18S rDNA. The alignment is based on the conserved core P3 to P8. The two introns carried by the strain of A. lenticulata characterized in this study are in bold. Nuclear introns of the IE class are used as outgroup. Bootstrap values (BV) for ML/NJ/MP
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Jc-1, 45, PD2S, and 25-1. As expected, the complete 18S rDNA-based tree provides better resolution and higher bootstrap values, recovering the main subgroups as defined by variations in the E23-1, H10 and 45-1 helices. The slight difference with the partial tree in the internal cluster is probably due to the few variations in helices E10-1, E23-5 and 29-1, which could differentiate subtypes within the main groups.
Identification of introns The extraordinary length of the 18S rDNA of strain D8, even exceeding the sequences of the A. lenticulata strains known to contain introns by ∼500 bp also, strongly indicated the presence of an intron. The careful analysis of the sequence revealed that, unexpectedly, two group I introns of subclass IC1 are present in the gene. These are 663 and 503 nt in lengths and are located at insertion sites S943 and S956, at the ends of stems 33 and 34 of the rRNA, respectively. Introns occurring at the same sites, but as unique elements were already described in A. lenticulata (SchroederDiedrich et al. 1998). Phylogenetic analyses based on full intron sequences (Fig. 4, right panel) showed that both, the S943 and the S956 introns of the dog D8 strain cluster with the homologous introns found in the other A. lenticulata strains, although being more divergent with similarities of only 52–57%. Higher values (>80%) are found when considering the conserved core pairs P3–P7. By comparing the intron and 18S rDNA phylogenies (Fig. 4), good correspondences can only be observed for the S943 of A. lenticulata and between acanthamoebae T3 and T4 with S516, indicating unique genetic invasions followed by vertical inheritances. Although probably derived from common ancestors, the remaining introns, S956 and S1389, exhibit a pattern of horizontal spread, since they have clearly invaded distinct subgroups of A. lenticulata (S956) or distant genotype hosts (S1389). The S943 intron of A. jacobsi appears to be a distinct lineage, perhaps related to the S1389 intron, but as only one sequence and one host are known, additional data are needed to better evaluate its history. The predicted secondary structures of the dog D8 strain introns are shown in Fig. 5 and compared with those of homologous introns found in Acanthamoeba. Overall, the size and structure of the S943 intron of strain D8 (Fig. 5a) are similar to those of the S943 introns found in the other A. lenticulata strains (e.g., PD2S strain, Fig. 5b). The dog S943 intron presents a second helix P2.1 and an additional helix P5.3. The P5 parts and their extensions in the two molecules are however very similar, with P5.2 comprising two stems (P5.2a, P5.2b). It should be noted that the structure of S943 occurring in the PD2S and close strains considered here, is
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slightly different from that proposed by Schroeder-Diedrich et al. (1998), because during our previous studies, we identified the P5 pair differently, resulting in a restructuring of the entire P5 extension. The S956 intron of the dog strain (Fig. 5c), on the other hand, is slightly shorter (∼170 nt) than the corresponding homologous introns occurring in the other strains (e.g., Jc-1, Fig. 5d). Its structure has only a P2 helix, as well as shorter P6 and simpler P5 extensions.
Nuclear intron phylogeny To better evaluate intron relationships, conserved corebased molecular phylogeny was performed on a larger number of introns from different hosts (Fig. 6). As expected, introns with the same insertion site, although present in distant hosts, often cluster together, indicating that they have common origins. The various intron types of Acanthamoeba each form a coherent group, however intermingled with the other groups, confirming that they are distinct lineages. The common origin of the two introns of the dog strain D8 with their respective homologous introns is also confirmed. However, except for the Acanthamoeba T3 and T4 S516 introns that belong to a lineage widespread across many organisms, the relationships of the other Acanthamoeba introns are not yet clear. Beyond the possible links with introns of green algae and chytrids already suggested in previous studies, this analysis also reveals relationships with the introns of Rhynchomonas, a marine/freshwater bodonid nanoflagellate (Scheckenbach et al. 2006), and Chlamydomyzium, an oomycete parasitizing on nematodes (Beakes et al. 2014). All these introns have however different insertion sites. In addition, the low values and/or inconsistency of the nodes supporting the relationships would suggest that these Acanthamoeba introns are actually unique and have no known close relatives.
Discussion The presence of multiple nuclear introns in the rDNA is relatively common in algae and fungi and even a rule in slime moulds, but only occasionally reported in amoebae without further investigation. However, except for Acanthamoeba (Corsaro and Venditti 2018b; Gast et al. 1994; SchroederDiedrich et al. 1998) and twintron-containing vahlkampfiid amoebae (Tang et al. 2014; Wikmark et al. 2006), the study of these elements in amoebae has attracted little interest, so that they are often neither characterized, nor correctly recorded, nor even identified in the sequences deposited at GenBank. We reported herein the first case of two group I introns co-
are shown at the nodes (1000 replicates for ML and MP; 2000 for NJ). Filled and open circles, nodes with 100 or >90% BV support with all methods, respectively; asterisk, node supported but BV<50%; hyphen, node not supported.
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occurring in the same 18S rRNA gene of an Acanthamoeba strain, as well as their characterization and a comprehensive phylogenetic analysis of the nuclear introns present in these amoebae. Currently, at least five distinct types of introns are known to invade the 18S rDNA of Acanthamoeba by inserting into four different sites (Fig. 1). The S516 intron appears to be specific for the T3/T4 lineage (Corsaro and Venditti 2018b; Gast et al. 1994; Ledee et al. 1996), whereas the presence of the S943 or S956 introns characterizes many strains representing genotype T5 (Schroeder-Diedrich et al. 1998; this study). Most likely quite early these introns have invaded the respective host, in which they probably spread in different ways. On the other hand, the S1389 intron has clearly a separate history of horizontal transfer among distantly related T5 and T4 strains (Corsaro and Venditti 2018b), while the uniqueness of the S943 found in A. jacobsi (Corsaro et al. 2017) requires other data to be analysed. Only the S516 intron has obvious affinities with several homologous introns of other organisms, forming a robust clade. This intron has spread among different hosts, in which it has been inherited several times vertically, as also shown by intron-HEG coinheritance and intron-rDNA coevolution that can be observed especially for the case of heterolobosean amoebae and Acanthamoeba T4 (Corsaro and Venditti 2018b; Haugen et al. 2004; Wikmark et al. 2006). In addition, the data suggest that a more complex history is underlying in Acanthamoeba, consistent with the intron life cycle model, assuming an early invasion of a HEG-containing S516 intron in the T3/T4 lineage, followed by multiple and differential losses during its vertical inheritance (Corsaro and Venditti 2018b). The case of A. lenticulata (genotype T5) is of interest because, despite the relatively small number of strains analysed in their 18S rDNA full length (n = 16), particularly with respect to genotype T4 (∼300), this species has the largest (n = 12) and most diverse (n = 3) intron population (Table 1) (Schroeder-Diedrich et al. 1998; this study). Our analysis indicates that the S943 and S956 introns early invaded A. lenticulata. It is possible that the co-presence of these two introns in the dog D8 strain reported herein is only fortuitous, each intron having indeed its own independent history. Data available on the other basal strain, TUMSJ-341, suggest that it contains only the S956 intron, but the reliability of this part of the sequence is very uncertain (Corsaro and Venditti 2019). While the S943 intron was inherited vertically within the PD2S subgroup, showing similarity values of ≥97% among strains that are 100% identical at the rDNA level, the S956 intron spread horizontally from the basal lineage to strains Jc-1 and 25-1 belonging to distinct subgroups (Fig. 4). The existing distances with strain D8 introns nevertheless suggest that this picture might be uncomplete, although probably not very different. A higher diversity seems to exist within A. lenticulata, as indicated by the partial sequences (Fig. 3). Unfortunately, these mostly correspond to fragment GTSA.B1, and therefore they can
only confirm that the S516 intron is apparently never present in this species. Indeed, these data cannot tell us about the possible presence and distribution among these strains of the three introns specific for T5 (Fig. 1). The characterization of additional sequences of introns and complete rDNAs will certainly be very useful to improve our knowledge. It should be underlined that molecular strategies commonly used to characterize the strains do not allow the recognition of introns (Fig. 1). Strain identification is indeed mostly carried out using the primer set JDP1/JDP2, which amplify the fragment ASA.S1 containing the hypervariable region 29-1 useful for genotyping as diagnostic fragment 3 (DF3) (Booton et al. 2002; Schroeder et al. 2001), and none of the four intron insertion sites are included within. The amplimers, GEF which increases the discriminatory power by including the entire E23 region (Risler et al. 2013), and Nelson which targets the 45-1 region (Mathers et al. 2000), also do not cover any of the known sites. Even the longer amplimer GTSA.B1 covers only one insertion site (S516). Therefore, it would not be surprising that many introns remained undetected. Furthermore, except for two T4 strains, no sequence is available for the LSU rDNA, which could also contain introns, as it occurs in other organisms. It is thus very likely that our knowledge of the distribution and diversity of nuclear introns in Acanthamoeba is only at the beginning.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Author contributions M.B. Rott collected samples and isolated the strain. M. Köhsler & J. Walochnik performed DNA sequencing. D. Corsaro & D. Venditti analysed intron structures and built phylogenetic trees. D. Corsaro & M. Köhsler drafted the manuscript. All authors reviewed the manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/ j.ejop.2019.01.007.
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