(March 2017: This section is still “under construction and revision”, but we wanted to let others see our thoughts. Note that it may always be “under construction”, because new ideas are likely to appear at any time). Please feel free to contact us about these, or feel free to use our thoughts as a starting point for new investigations. We also welcome any suggestions concerning your own unanswered questions that we would be happy to add to ours (and attribute to any of you that wish to contribute).
Much has currently (2016) been uncovered about how the gene and genome sequences obtained from natural isolates of Acanthamoeba provide insights into the evolution of the genus and of some related groups of free living amoebae. However, even as new details are revealed, the data provide clues suggesting that our understanding is very far from complete.
Below I will list a number of unanswered questions that emerged as I have conducted my analyses. My lab is no longer running at the level that it once was, and most of these questions are unlikely to ever be addressed by me or by my colleagues and students. I encourage any investigator who is interested by these questions to pursue answers. I also welcome additions to this list from others who have been puzzled by some aspect of the biology and genetics of Acanthamoeba and related forms, and who feel that the community of researchers working on free living amoebae may have ways to provide an answer. If answers to some of these questions appear, I will endeavor to report them and give due credit to those who pursued an answer.
QUESTION #1: [NOW ANSWERED!] Why is there no “nearly complete” sequence of the 18S rRNA gene from genotype T15 (Acanthamoeba jacobsi) ?
Context: Acanthamoeba jacobsi has been known for more than two decades (Sawyer, Nerad and Visvesvara. 1992. J. Helminthol. Soc. Wash. 59: 223-2260). Partial sequences ranging from 1443 – 1475 bases, potentially covering about 60% of the gene beginning at the 5′ end was reported by Hewett et al. in 2003 (Acta Protozool. 42, 325-329). The isolates analyzed included the isolate A. jacobsi 31-B (ATCC 30732). My own lab has also studied this isolate adding ~140 bases at the 3′ end of the reported sequence. This remains about 600+ bases short of a “nearly complete” sequence.
Several possible reasons could explain the lack of an almost-complete 18S rRNA gene sequence:
(1) It is possible that the gene in A. jacobsi includes an intron sequence near the spot where the partial sequences end. The intron might be long enough to interfere with efficient PCR amplification of the sequence for subsequent sequencing. Use of priming site within the terminal region of the partial sequences could be attempted to see whether a PCR product can be produced. [ONE OF THE SEQUENCES DOES CONTAIN AN INTRON]
(2) The PCR primer sequence at the 3′ end of the gene might have changed in A. jacobsi, no longer providing efficient amplification for the end of the sequence. Use of alternative conserved sequences within the 3′ ends of other Acanthamoeba taxa might provide successful amplification.
[SEE: Corsaro, D., Kohsler, M., Montalbano Di Filippo, M., Venditti, D., Monno, R., Di Cave, D., Berrilli, F. and Walochnik, J. 2017. Update on Acanthamoeba jacobsi genotype T15, including full-length 18S rDNA molecular phylogeny. Parasitol. Res. 116: 1273-1284. ]
QUESTION #2: What sequence type or “species” is represented by the genome sequence that has been labeled as A. royreba, genome project CDEZ.
The DNA that served as the source material for this genome sequence was originally labeled as having been obtained from a culture of A. royreba, ATCC 30884. Our lab studied ATCC 30884, and identified it as a T4 strain (placed in the sub-type T4-D of our analysis of T4 isolates). The genome sequence suggests that the isolate examined for the genome sequence had gene sequences that are not consistent with previous information on ATCC 30884, and, further, seem to represent no studied ATCC strain. Finally, the gene sequences are so different that they would represent a new sequence type (putatively T21). What is the source of this DNA ?
Context: The divergence of sequences of the nuclear 18S rRNA gene, and of the complete mitochondrial genome (especially the 16S-like small subunit rRNA gene and the COX-1 gene), and difficulty in identifying gene sequences for the nuclear genes for beta tubulin, Ef-1, G3pd, Glyphos, and rasC all suggest that the isolate from which DNA was obtained is not from the isolate previously identified as A. royreba, ATCC 30884, but does fall within the general patterns expected for an isolate that would be classified as Acanthamoeba. However, these sequences suggest that the isolate has never been previously encountered as any isolate deposited in the DNA databases.
What ATCC strain was actually cultured to provide the DNA sequenced in the genome study? The nuclear 18S rRNA gene and the mitochondrial genome sequence suggest that the isolate represents a new sequence type previously unknown. Since a number of ATCC isolates have never been studied genetically, the source and characteristics of this isolate remain a mystery.
QUESTION #3: what unknown types are out in nature that have been seen as only partial 18S rRNA gene sequences, and do they represent taxa close to known types, or are they quite unique ?
Context: A) The T20 situation. The most recent sequence type to be unequivocally identified was T20. The first T20 sequences submitted to the DNA databases was that from a study in which the OSU collaborated, the analysis of amoebae from a keel-billed toucan who apparently died following an infection. An organism was cultured from the liver of the bird, and a partial 18S rRNA sequence was obtained. At the time (analyses performed in 2006) the DNA database of 18S rRNA sequences from Acanthamoeba was much smaller (~600 sequences) than at the end of 2016 (~3700 sequences). When comparisons were made of the partial sequences with other sequences in the database, the sequence was unlike any other in the database at that time, but similar enough to the most frequent class (T4) that we assumed that it represented a divergent T4 sequence. Subsequently, a series of slightly longer sequences appeared that suggested the presence of a new sequence type. We went back to our sample and obtained the almost complete sequence of the sample. This indicated that the isolate was not, in fact a divergent T4 sample, but represented an entirely new sequence type, which was designated T20 (Fuerst, Booton and Crary, 2015: J. Eukaryotic Microbiology 62: 69-84.) Are there other seemingly divergent members of a sequence type or sub-type for which only partial sequence information has been obtained that actually represent additional new forms ?
B) (THE T99 CLASSIFICATION HAS NOW BEEN ANSWERED!): Information from environmental studies; the T99 question (among others). On this site, we list a sequence type with the classification T99. This designation is given to a number of sequences that were observed in an environmental study of the soil microbiome associated with trembling aspen. No equivalent organism has been cultured. The 18S rRNA gene sequences from these uncultured organisms show higher sequence similarity to sequences of Acanthamoeba than to those of any other taxa. However, they lack the expanded hypervariable sequence regions that characterize the Acanthamoeba 18S rRNA gene. Some related sequences have been retrieved in other environmental surveys of uncultured microbiomes. What are these organisms, and are they truly members of the Acanthamoebidae ?
QUESTION #4: Why are there so many ATCC strains for which there is no genetic sequence information.
Context: more than one-third of the “standard” strains available from either ATCC or CCAP have never been studied. [under construction].
QUESTION #5a: Is there a significant correlation between the specific sequence type or sub-type or allelic type of an Acanthamoeba isolate and the ability of that isolate to “host” intracellular bacterial forms (either obligate intracellular or facultative intracellular ?
Context: Not all Acanthamoeba that are isolated from nature are found to carry intracellular bacteria. Is this just random, or is it related to genetic background of the amoeba ? Are there specific genetic backgrounds (of the Acanthamoeba) that provide more hospitable environments for some bacteria ? Are some Acanthamoeba isolates (because of the specific genes or alleles that they carry in their genome) more likely to act as potential vectors of bacteria ?
QUESTION 5b: Is there a correlation between the specific sequence type or sub-type or allelic type of an Acanthamoeba isolate and the specific identification of intracellular bacteria that are harbored within that Acanthamoeba isolate ?
Context: Do some Acanthamoeba provide long-term environments for specific bacteria such that descendant amoebae will share both amoebic genetic identities and bacterial co-decendants ? There is a very small set of evidence that Acanthamoeba strains isolated by different laboratories, and which are classified as closely related based on sequence sub-types or allelic types also carry very closely related bacterial types.
QUESTION 6: How diverse is the sequence subtype T4E?
Context: This sequence subtype is characterized by a significant number of ATCC isolates (ATCC 30871, Page-23; ATCC 50711, Rodriguez; ATCC 50727, A. hatchetti 2AX1, ATCC PRA-113, hatchetti 2HH and the isolates in the series ATCC 50676 through ATCC 50684), as well as 7 other isolates for which almost complete 18S rRNA sequence have been obtained. However, almost 100 other isolates have been reported whose partial sequences indicate that they are related to the almost complete 18S rRNA sequences of subtype T4E, but with substantial (2-3% or more) sequence divergence. Over 15 different alleles of the 18S rRNA gene are represented within T4E, with 11 of these represented only by isolates for which no almost complete sequence has been obtained. How diverse are these incompletely studied isolates within a subtype that may have been among the first to diverge from with sequence type T4 ?