View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library Published in Parasitology Research, 107, issue 1, 233-238, 2010 1 which should be used for any reference to this work Phylogenetic evidence for a new genotype of Acanthamoeba (Amoebozoa, Acanthamoebida) Daniele Corsaro & Danielle Venditti Abstract Acanthamoeba are widespread free-living amoe- Introduction bae, able to cause infection in animals, with keratitis and granulomatous encephalitis as major diseases in humans. The genus Acanthamoeba comprises several species of Recent developments in the subgenus classification are free-living amoebae, widespread in many types of habitats, based on the determination of the nucleotide sequence of playing a role of major microbial predators. Many strains the 18S rDNA. By this mean, Acanthamoeba have been naturally harbour endosymbionts or may act as vector/ clustered into 15 sequence types or genotypes, called T1 reservoir for various microbial pathogens. Furthermore, to T15. In this study, we analysed near full 18S rDNA of trophozoites can be used as host cells to isolate an Acanthamoeba recovered from an environmental intracellular organisms (Horn and Wagner 2004;Corsaro sample and various unidentified Acanthamoeba sequences and Greub 2006). retrieved from GenBank. We provided phylogenetic Acanthamoeba spp. may cause diseases in animals, evidence for a new genotype, which we proposed to name including humans, like keratitis, encephalitis and dissemi- T16. nated tissue infections; thus, great interest is brought to identification of pathogenic species or strains (Marciano- Cabral and Cabral 2003; Visvesvara et al 2007). : Acanthamoeba species have been traditionally clustered D. Corsaro (*) D. Venditti into three morphological groups, on the basis of cyst CHLAREAS, Chlamydia Research Association, features (Pussard and Pons 1977). However, successive 12 rue du Maconnais, 54500 Vandoeuvre-lès-Nancy, France studies based on biochemical and molecular methods have e-mail: [email protected] pointed out in many cases incoherences in strain/species clustering, proving also that some species, e.g. Acantha- D. Corsaro moeba polyphaga, are polyphyletic. Gast et al. (1996) and Swiss Federal Research Institute WSL, Wetlands Research Group, Station 2, Stothard et al. (1998) proposed to classify Acanthamoeba 1015 Lausanne, Switzerland strains on the basis of 18S ribosomal RNA gene (18S rDNA) sequence similarities. By analysing more than 50 D. Corsaro strains from the three morphogroups, they identified 12 École Polytechnique Fédérale de Lausanne (EPFL), Laboratory of Ecological Systems, Station 2, sequence types or genotypes, named T1 to T12, where each 1015 Lausanne, Switzerland genotype should correspond to natural species or species complex (Gast et al. 1996; Stothard et al. 1998). This D. Corsaro approach has been largely adopted, and three new geno- Laboratory of Soil Biology, University of Neuchâtel, rue Emile Argand 11, types have been established, called T13 to T15 (Horn et al. Neuchâtel 2009, Switzerland 1999; Gast 2001; Hewett et al. 2003). 2 This study focuses on the phylogenetic analysis of an pairwise comparison, under optimal global alignment, using Acanthamoeba strain, isolated from a freshwater pond, and all sites and indels but excluding introns, and by removing of other Acanthamoeba sequences, identified as candidates common and terminal gaps. for novel genotypes. Results and discussion Materials and methods Heavy fungal overgrowth prevented subculture and isola- Sample origin and DNA extraction tion of the strain cvX. Cysts onto agar appeared wrinkled, with a polygonal endocyst; therefore, we considered this Amoebae were recovered from a freshwater pond contain- strain as belonging to group II. DNA analyses were then ing decaying vegetable matter (South Italy) onto bacterised performed on extracts of amoebae scraped directly from a 1.5% non-nutritive agar during a study focusing on the contaminated agar plate. Amoebae resulted negative for search for chlamydiae in the environment (Corsaro and chlamydiae. Venditti, 2009). Fungal overgrowth persisted in subcultures Amplicons obtained with CAT1/1137R (∼1,400 bp) and even after 24-h HCl treatment of cysts; thus, agar plates 892cF/Br (∼1,120 bp), starting from both DNA extracts and were eliminated. Prior to discharge plates, a small quantity after PCR with CAT1/Br (∼2,200 bp), give identical of amoebae was recovered from the least contaminated agar sequences. Thus, a unique Acanthamoeba intronless se- and centrifuged in Page’s amoeba saline buffer (three times quence of 2,157 bp was obtained. At BLAST, our sequence at 200×g, 5 min). Total DNA was extracted by repeated revealed almost identical (99.2) to the strain U/HC1, passages through a 24-gauge needle after freezing–thawing. isolated from a human keratitis in Brazil, of undefined PCR for the amoeba 18S rDNA was performed by using genotype (Alves et al. 2000). Other closest relatives were several primer sets: CAT1 (5′-CAT GCA TGT CTA AGT strains of genotype T13, showing sequence identity of 94%, ATA AGC-3′) with GSPr (5′-TTC AC <G/A> GTA AAC while following entries in the BLAST all showed sequence <G/A> ATC TGG GC-3′) (modified from Stothard et al. identity values <93%. 1999), or 1137R (5′-GTG CCC TTC CGT TCA AT-3′), and Figure 1 shows a phylogenetic tree based on a partial 892cF (5′-GTC AGA GGT GAA ATT CTT GG-3′) with Br ∼1,450-bp portion of the 18S rDNA, in order to include (5′-GAT CCT TCT GCA GGT TCA C-3′). Primers 1137R also the genotype T15, corresponding to the species and 892cF were from Schroeder et al. (2001), and Br was Acanthamoeba jacobsi (Hewett et al. 2003), for which only modified from Gast (2001). Reaction conditions were 5 min this part of the gene is available. However, this gene portion at 94°C, followed by 40 cycles of 94°C 45 s, 56°C 30 s, is a diagnostic region comprising eight variable sequence 72°C 1 min, with final extension of 72°C 5 min. PCR regions able to differentiate among all the 15 genotypes products were purified and sequenced by using the same (Schroeder et al. 2001). In this partial tree, our sequence PCR primers, and screened at BLAST for their identity. emerged in a highly supported holophyletic clade with the strains of genotype T13 and clustered unambiguously with Phylogenetic analysis the strain U/HC1. The genotype T15, presented as the sister group of the genotype T13 in the original report of Hewett The obtained sequence was aligned with closest relatives, et al. (2003), emerged as an independent clade in the retrieved after BLAST (www.ncbi.nlm.nih.gov), and with present analysis. representatives of each Acanthamoeba genotype. Multiple To identify genotypes, pairwise similarity values were alignments were obtained with clustalX and edited with determined by considering available full sequences, i.e. BioEdit. Introns were excluded from the analysis. Phylo- >2,100 bp. Pairwise similarity value between the strains genetic reconstructions were made on a partial ∼1,450-bp U/HC1 and cvX was of 99.17% (dissimilarity 0.8%). diagnostic region of the 18S rDNA, in order to include also Sequence from cvX strain showed 16 point mutations from the genotype T15 (Hewett et al. 2003; Schroeder et al. that of U/HC1, 10 of which are in the diagnostic region, 2001), and on full sequences (T15 excluded). Treeing and two nt insertions. T13 genotype was represented by methods used were neighbour-joining, minimum evolution three strains, showing values of 96.0–97.7% (dissimilarity (NJ, ME; p-distance) and maximum parsimony (MP), with 2.3–4%): UWC9 isolated from contact lens case (Fritsche et MEGA3 (Kumar et al. 2004), and maximum likelihood al. 1993), and TUMK-23 and UWET39 from soils (Horn et (ML, GTR+gamma+I:4 model), with TREEFINDER (Jobb al. 1999; Hewett et al. 2003). Pairwise similarity value et al. 2004), with bootstrap test of 1,000. Following between the group U/HC1 and cvX and the T13 strains was Stothard et al. (1998), genetic similarity between full 93.6–94.6% (dissimilarity 5.4–6.4%). Following the crite- sequences (∼2,200 bp) was calculated with BioEdit by rion of a 5% of dissimilarity (Stothard et al. 1998), the 3 78/55 Acanthamoeba sp. E-5C (AB425955) 77/92 Acanthamoeba sp. OB3b-3A (AB425945) 92/82 polATCC30872 91/92 Acanthamoeba sp. KA/MSG27 (AY173011) group 37/- 95/97 Acanthamoeba sp. SE2-6F (AB425949) -/- 99/99 A. ‘polyphaga’ ATCC30872 (AY026244) 61/- Acanthamoeba sp. EI4 (AM408799) 69/79 OX1 group 67/- A. ‘polyphaga’ OX1 (AF019051) 25/- A. palestinensis Reich (U07411) 81/- 95/- Acanthamoeba sp. EFW15 (DQ992189) Acanthamoeba sp. EFW7 (DQ992192) T2 91/73 Acanthamoeba sp. EFW9 (DQ992193) 98/63 86/88 Acanthamoeba sp. EI5 (AM408800) Reich group 86/84 61/- A. ‘pustulosa’ GE3a (AF019050) -/- 58/- -/41 ‘Comandonia operculata’ ATCC50243 (AY033896) 55/- -/- A. ‘palestinensis’ 2802 (AF019063) 94/81 Acanthamoeba sp. KA/MSS1 (AY172999) T6 92/94 A. ‘hatchetti’ 11DS (AF251939) 82/43 T 81/82 A. castellanii Castellani (U07413) A. lugdunensis L3a (AF005995) T4 83/65 A. royreba Oak Ridge (U07417)T 84/92 A. polyphaga Page-23 (AF019061) 79/- -/- A. triangularis SH621 (AF316547) 64/- T -/- 99/96 A. griffini S-7 (U07412) T3 99/99 A. griffini H37 (S81337) 48/20 51/66 A. hatchetti BH-2 (AF019068) T11 83/70 A. stevensoni RB-F1 (AF019069) 96/96 100/99 A. lenticulata ATCC50706 (U94736) T5 100/100 A. lenticulata PD2S (U94741) 99/99 Acanthamoeba sp. PN13 (AF333609) 100/100 T14 84/76 Acanthamoeba sp. PN15 (AF333607) 93/93 A. culbertsoni Lilly A1 (AF019067) T12 93/71 99/92 64/59 97/98 A. healyi V013 (AF019070) T10 Acanthamoeba sp. VOO6 (U07400) T1 59/- 87/68 94/98 -/- 95/95 A. jacobsi ATCC30732 (AY262360) 100/100 94/- A. jacobsi AC080 (AY262361) T15 -/- 100/99 A. jacobsi AC194 (AY262362) 100/100 AcaVNAK01 (GQ905495) 86/91 Acanthamoeba sp.