(updated October 2023)
Alleles within sequence type T3 had been described in only one publication (to our knowledge) until our recent review (Fuerst & Booton, 2020). Alleles within sequence type T11 had never been described.
We defined alleles within T3 during our study of isolates from water samples in Hong Kong (Booton et al. J. Clin. Micro. 2002). As with alleles of sequence type T4, the alleles represent variability within the ASA.S1 subsequence of the 18S rRNA, specifically the primary nucleotide sequence of the variable region of segment DF3 (stem 29-1 of the Acanthamoeba 18S rRNA).
In our original study, we observed 5 different alleles within sequence type T3. As mentioned elsewhere in our discussion of alleles within T4, the results of our Hong Kong study were not an attempt to use the allele designations as a phylogenetic tool. Rather, they are used simply as a means of categorizing the isolates observed in the study.
ALLELES WITHIN SEQUENCE TYPE T3
There were (as of October 2023) 400 isolates whose 18S rRNA gene sequences have been categorized into sequence type T3. The data set of T3 sequences has been examined to identify alleles that occurred more than once in this collection of sequences. By January 2020, there were ten alleles that were identified in more than a single T3 isolate. Since that time, 3 additional alleles have been seen in at least two isolates. In addition, two of the alleles in our original study (alleles T3/02 and allele T3/05) have been observed only within the initial single isolates that we reported in the study of Hong Kong isolates of Acanthamoeba. One additional allele (T3/06 ) occurred in the “almost complete” sequences from isolates of A. pearcei (Sawyer 205-1). While two sequences of this isolate are being reported, they actually represent the same allele seen in the non-axenic (ATCC 50435) and axenic (ATCC 50436), versions of the isolate.
The 58 “almost complete” sequences identified as T3 (including the two duplicate sequences from A. pearcei strain 205-1), were examined to see if any of the allele sequence within the DF3 region of the gene used for allelic identification in any of these longer sequences had a unique variant sequence. Only the almost complete sequence found in the two versions of A. pearcei (Sawyer 205-1) were “unique”, carrying the T3/06 allele. Ten of the “almost complete sequences carried the T3/01 allele (including A. polyphaga Panola Mountain, ATCC 30487). Two of the “most complete isolates carried allele T3/03, while long sequences from 42 isolates carried allele T3/04. This latter group included A. griffini H37 (ATCC 50702). Allele T3/11 was found in two “almost complete” sequences, including that from A. griffini S-7 (ATCC 30731).
The sequences of the 13 identified allele classes are given in the following:
Of the 18S rRNA sequences from 400 isolates of T3, 282 could be categorized into an allele class. The remaining isolate sequences were not classified because they either: (1) did not completely overlap the region of the 18S rRNA sequence used to identify alleles [n=69 isolates], (2) had ambiguous nucleotide sites within the region [n=3], (3) sequences that appear to be mixed samples [n=3], or (4) were partial 18S rRNA sequences that were classified as singletons in the allelic region [n=40 isolates].
Four alleles (T3/01, T3/03, T3/04 and T3/12) are found frequently in the DNA databases. The two isolates found carrying allele T3/11 were both “almost complete” sequences. The frequencies of the 14 allele classes (in October 2023) were:
ALLELES WITHIN SEQUENCE TYPE T11
Isolates within sequence type T11 were not reported as part of studies described in previous reports until our 2020 paper. This page represented the first description of alleles of the DF3 region within the T11 sequence type, while we reported results through 2019 in our paper (Fuerst & Booton, 2020).
As of October 2023, 219 isolates had been categorized into sequence type T11. Examination of the dataset of T11 sequences identified 12 alleles that occurred more than once. In addition, among the eleven “almost complete” sequences identified as T11, two isolates (A. stevensoni [AF019069, allele T11/05], and A. sp. E13 [GU808311, allele T11/07]) each possessed a unique variant sequence that we have also designated as an allele. Thus, 14 alleles are currently listed for T11. The sequences of the alleles are given below:
Of the 219 isolates of T11, only 132 could be categorized into an allele class. As previously noted, the sequences of the remaining isolates were not classified by allele because they either: (1) did not completely overlap the region of the 18S rRNA sequence used to identify alleles [n=38], (2) had ambiguous nucleotides in the region [n=14], or (3) were partial 18S rRNA sequences that were classified as singletons in the allelic region [n=35].
Twelve T11 alleles occur more than once in the DNA databases. However, unlike the distribution of alleles in sequence types T4 or T3, alleles in sequence type T11 show only slight evidence of a dominant allele or alleles, with six alleles occurring in 10-23 isolates, and only one allele exceeding 20 occurrences.
Allele T11/01 occurs in two “almost complete” sequences, including A. hatchetti BH-2 (ATCC 30730,[AF019068] ), Allele T11/02 includes five of the “almost complete” sequences (including one “almost complete” sequences that was classified as belonging to the species A. hatchetii, isolate 4RE [AF251937]. Allele T11/06 occurs in five long sequences. Allele T11/11 occurs in 19 long sequences, while allele T11/12 occurs in 4 long sequences and allele T11/14 occur in 3 long isolates. Single “almost complete” isolates are included in the counts for alleles T11/03, T11/05 (in ATCC 50388), T11/07, T11/09 and T11/13.
The frequencies of isolates within the various allele classes (in October 2023) were:
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