Acanthamoeba sequence types

(Updated JULY  2021)

The amoebae now placed in the genus Acanthamoeba were first definitively discovered by Castellani and described by Volkonsky in 1930.  Morphological characters (primarily based on cyst structure), and cytological characteristics of nuclear division (such as those proposed by Pussard and Pons, Protistologica 13:557-598, 1977) were used to describe different species prior to application of biochemical or molecular approaches to systematic classification. Descriptions have been proposed for more than 20 putative species, based on such criteria.

Beginning in the 1970’s biochemical approaches (such as the use of isozyme patterns) were employed, followed in the early 1980’s by DNA based approaches such as RFLP analysis of mitochondrial DNA.  The 1990’s saw the introduction of DNA sequencing to the study of Acanthamoeba.  DNA sequence information from the nuclear small subunit ribosomal RNA gene (Rns) began to accumulate and a pattern of phylogenetic relationships began to be inferred.

Our lab contributed significantly to this effort with seminal papers by Gast et al. (J. Eukaryot. Microbiol. 43: 498-504. 1994 [Gast JEM96] and Stothard et al. (J. Eukaryot. Microbiol. 45:  45-54, 1998 [Stothard_JEM98]).  These papers set the framework for the current use of Rns genotype groups (sequence types).   By 2000, studies in our lab and elsewhere had identified at least 12 sequence types within Acanthamoeba, a number that has grown to 22 by 2018We initially proposed that different sequence types existed if two sequences showed >5% pairwise divergence between Rns sequences.  These criteria are being re-evaluated, given the expansion of available sequences for comparison, with the likelihood that the criterion for the identification of new types using Rns sequences will be reduced to 4% pairwise sequence differences.

Questions about diversity within sub-types have also arisen.  For sequence type T4, we have described the existence of significant sub-types (Fuerst, Booton and Crary, 2015).  Further discussion of sequence sub-types of T4 is given on another page.  Additional analyses continue to support these sub-types as significant evolutionary units.  In the same paper, sub-types were also described for the T2/T6 supertype.  Information is provided on a separate page. Significant sub-types also appear to exist within T3, T5 and T11.  More information will be provided in the future.

An alternative method of examining subtype diversity was the designation of “allele” type, originally raised in a paper from our lab (Booton et al, 2002; J. Clinical Microbiology 40: 1621-1625).   In the literature, allele types have currently been classified only within the T3  T4 sequence types.   Examination of other sequence types suggests that allele types can be defined at least within T2, T5, T6, T11, T13 and T15 sequence types, each of which have sufficient samples to allow reasonable comparisons that can rule out simple sequencing error.   Allele types are described in an associated page of this site for sequence types T4, T3 and T11, T5 and T15.  Analyses (details to be presented in the future) suggest that the designation of sub-types within T4 are supported strongly by examination of alleles.  Different alleles of the 18S rRNA gene seem to be sub-type specific, and do not seem to be shared between sub-types of T4.

Some details concerning the history of  the description of each sequence type is provided on a separate page.

A phylogenetic tree indicating the relationships among sequence types is given on an attached page.  (This tree does not yet include new sequence type T23, observed in 2021. T23 has a group III cyst ultrastructure, and would appear in the tree as a sister group to the clade that consists of sequence types T10, T12 and T14).


Sequence types

with arbitrary isolates designated as type isolate for each sequence type (as of June 1, 2014 [updated 2017]): 

Sequence Type: [representative isolate with Genbank (accession #)]

T1 – A. castellanii V006 [ATCC 50494] (U07400)

T2(*) – A. palestinensis Reich [ATCC 30870] (U07411)

T3 – A. griffini S-7 [ATCC 30731] (U07412)

T4(*) – A. castellanii [ATCC 30011] (U07413)

T5 – A. lenticulata Jc-1 [ATCC 50428] (U94739)

T6(*) – A. palestinensis 2802  [ATCC 50708] (AF019063)

T7 – A. astronyxis (Ray & Hayes) [ATCC 30137] (AF019064)

T8 – A. tubiashi OC-15C [ATCC 30867] (AF019065)

T9 – A. comandoni [ATCC 30135] (AF019066)

T10 – A. culbertsoni Lilly A-1 [ATCC 30171] (AF019067)

T11 – A. hatchetti BH-2 [ATCC 30730] (AF019068)

T12 – A. healyi V013 OC-3A/AC-020 [ATCC 30866] (AF019070)

T13 – UWC9 [ATCC PRA-3] (AF132134)

T14 – PN13 (AF333609)

T15 – A. jacobsi 31-B [ATCC 30732] (AY262360)

T16(#) – U/HC1 (AY026245)

T17 – Ac E1a (GU808277)

T18 – CDC:V621 (KC822461)

T19 – USP-AWW-A68 (KJ413084)

T20** – OSU 04-020 (DQ451161)

                                          T21**** – A. pyriformis (KX840327)

T22*** – “A. royreba” genome project (CDEZ01000000)

T23**** – “A. bangkokensis sp. nov.”

(MZ272148 and MZ272149)

T99($) – NO LONGER VALID Acanthamoeba environmental clone Amb_18S_1223 (EF023782)


(*) – Note that T2, T4 and T6 have significant subtypes, some of which approach the level of significant sequence differences (see subtypes on attached pages).  The phylogenetic relationships among T4 subtypes and among subtypes in the T2-T6 supertype are shown on an attached pages.  

(#) – Note that several different sequence variants have been designated as T16.  U/HC1 receives priority.  Type T20 represents another group of sequences originally designated as T16.

(**) – Type T20 was previously designated T19 on this page.  The T19 sequence type was reported in a recent paper (Magnet et al. 2014, Parasitol Res 113:2845–2850).  T20 is described in a report  [P.A. Fuerst, G.C. Booton & M. Crary (2015) Phylogenetic Analysis and the Evolution of the 18S rRNA Gene Typing System of Acanthamoeba. Journal of Eukaryotic Microbiology 62: 69-84].

**** – T21 – Acanthamoeba pyriformis – reported by Tice et al, 2017.

(***) – Type T22 is represented by the 18S rRNA gene sequence and the mitochondrial 16S-like rRNA gene sequence extracted from the WGS archive of the genome project for “A. royreba” genome project (CDEZ01000000).  These sequences are not consistent with the placement of A. royreba based on the sequence previously obtained from the strain ATCC 30884.  The ATCC 30884 strain is the putative source of material for the genome project.  The sequences are not consistent with any previously reported sequences from Acanthamoeba, and place the unidentified ATCC isolate into a new sequence type because of differences exceeding 6% from all previous almost-complete 18S rRNA sequences and a similar unique and divergent identification for the mt-DNA 16S-like rRNA gene sequence.  The two rRNA sequences for this genome project are listed on the page for Acanthamoeba genomes.   

($) – The T99 sequence type is no longer valid.  It was originally identified as encompasses a set of environmental samples, some of which include almost complete 18S rRNA gene sequences.  These sequences showed closer similarity to Acanthamoeba than to any other taxa.  In a paper in 2017 (Corsaro and Venditti, Parsit Res) these sequences were shown to be chimeras between 18S rRNA sequences from Acanthamoeba T13 and portions of sequences from nematodes and cercozoans.   Partial sequences that seemed to represent “T99” samples were reported from several different locations throughout the world, but must be considered sequencing artifacts from environmental surveys.

(****) – Type T23 was observed in water samples from Thailand (Putaporntip et al., 2021).

NOTE: re-numbering of Sequence type T19. 

In mid June 2014 a sequence was deposited in the DNA databases that may be a new sequence type.  It is now designated T19 by priority in the literature: 

T19 – Acanthamoeba sp. USP-AWW-A68 (KJ413084). 

Information about the environmental and geographical source of this isolate has now been published by Magnet et al. 2104 (see footnote above).

New and additional information will be added when available in the databases.

Additional subtypes may be present in T3, T5 and others, especially in the Group I Acanthamoeba (T7, T8, T9, T17 and T18), which are relatively rare in the DNA databases. 

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