Molecular analysis of Acanthamoeba and free-living amoebae



October – November 2023 – updating in progress of pages.  Most page updated

July 2023:   FLAM 2023 is completed.  A great success.  Thanks to all at Université de Poitiers for their hospitality.

For more information on the research that was presented at FLAM 2023 see ABSTRACTS FROM FLAM23 . Next FLAM meeting set for Mexico.

June 2023 – A paper reports the identification of a new subgroup within the T4 sequence group, designated T4H (Parasitology Research 122: 1445–1450). While much less frequent than other T4 subtypes, the T4H subgroup is already represented within genome sequences in the DNA databases through SRA sequences (isolate SNN, SRX14501634) described below from June 2022. 


If anyone sees a paper that contains major changes for our understanding of Acanthamoeba or other Free-living amoebae, please let us know.  If some important aspect of the subjects covered by this site seem to have been overlooked, please make suggestions.  Your comments are important to us.  Page maintained by Paul Fuerst


The genus Acanthamoeba represents one of the most ubiquitous protistan groups in nature. Members occupy a variety of ecological niches, ranging from soil to water as well as in dust and in the atmosphere, and can be isolated from the tropics to almost polar conditions.   The first standard strain of Acanthamoeba was isolated by Castellani (1930) as a contaminant of a yeast culture.   More than 20 species of Acanthamoeba have been described, based on morphological criteria (Visvesvara, 1991).  Various species of Acanthamoeba have been implicated as the cause of several different infections of vertebrates.  These diseases can range from infections of the skin, through more frequent infections of the eye (Acanthamoeba keratitis, AK), to very serious and potentially lethal infections of the brain (granulomatous amoebic encephalitis, GAE), or even to diffuse multiorgan infiltration.  Each of these manifestations except AK is very rare in immunocompetent individuals.  It is clear that Acanthamoeba is a growing threat to health around the world, even if we restrict our focus to only the role of these amoebae in the severe sight-threatening ocular infection AK (Lorenzo-Morales et al., 2013).

To better understand how Acanthamoeba is implicated in various diseases, molecular markers were developed beginning in the 1970’s (proteins) and 1980’s (nucleic acids) to classify and group specific isolates.  Many DNA sequences  have been collected and deposited in the international DNA databases by researchers working on Acanthamoeba.  At the  of 2017, more than 20,000 single gene nucleotides sequences, not including EST or genome sequences had been deposited in the DNA databases (DDBJ/EMBL/GenBank).  Most of this web site is devoted to better understanding the meaning of this DNA information.

The most widespread molecular marker used to study Acanthamoeba has been the DNA sequence of the nuclear 18S rRNA gene.   This has been a major focus of researchers at The Ohio State University (OSU).

Researchers at OSU have been involved in studying Acanthamoeba, and related groups of free-living amoebae (Balamuthia, Protacanthamoeba and Vermamoeba/Hartmanella) for more than 40 years (see the associated web page on this site).   Among their most important contributions to date has been the development of the genotype classification system, based on the sequences of the 18S rRNA gene.  This system of “sequence types” has been adopted by most researchers of Acanthamoeba and is now routinely applied to classify isolates of Acanthamoeba.



The genotype classification system was developed based on seminal papers in 1996 and 1998.   Based on DNA sequences of the small-subunit ribosomal RNA gene (18S rRNA), the typing system has seen the expansion of sequence types from the original four in 1996 to the present identification and analysis of more than twenty sequence types (T1-T22).   Work is continuing to determine the relationship between species names within the genus and the diversity of Acanthamoeba isolates and the relationship between species names and sequence types.   Questions also exist concerning the possible importance of variation within sequence types, including a possible allele designation system to further classify genetic variability within Acanthamoeba sequence types. 



This site will include summaries of various ongoing research, information about the metanalysis of the DNA sequences in the international DNA databases, and an ongoing update of the status of sequence types, sub-types and allele designations for Acanthamoeba.  The site also contains information about genetic variation in two genera closely related to Acanthamoeba: Balamuthia and Protacanthamoeba, and information about some other protist taxa, especially Vermamoeba and Naegleria.      



Information about the classification of sequence types is provided on the page “ACANTHAMOEBA SEQUENCE TYPES” .  Subpages provide information about the phylogenetic relationships of sequence types of Acanthamoeba, and on information about subtypes within the T4 and T2/T6 sequence types.

The page “ACANTHAMOEBA DNA DATABASES”  provides information about the number of 18S rRNA gene sequences that are deposited in the DNA databases.  Information on the genes that have been used for phylogenetic analysis are provided on separate major subpages.  

The most important GENE subpage under ACANTHAMOEBA DNA DATABASES” provides information on the nuclear 18S rRNA gene, including information about the number of DNA sequences that have been identified as belonging to specific sequence types.   Further subpages of the 18S rRNA gene provide information on (1) “almost complete” 18S rRNA sequences and (2) partial 18S rRNA sequences.  Additional subpages provide information concerning (3) the difference between sequence types and alleles of the 18S rRNA gene in Acanthamoeba, (4) PCR primers and amplicons that have been used in the analysis of the sequences of the 18S rRNA gene and (5) the the secondary structure of the 18S rRNA sequence.  Finally, a subpage provides a detailed reference list of the publications that have contained information on those sequences deposited  into the Acanthamoeba 18S rRNA sequence database.  

A second major GENE subpage under ACANTHAMOEBA DNA DATABASES” provides information on the mitochondrial gene for the small ribosomal subunit (16S-like) rRNA.

As information on genome sequences has begun to proliferate, we have included information on the nuclear and mitochondrial genome sequences for 19 isolates of Acanthamoeba.

In the future, additional pages will provide details on other genes used to study the phylogenetic structure of Acanthamoeba.  These include additional mitochondrial genes for the cytochrome oxidase subunit I (COX-I or COI), and a tandem tRNA region.  Additional pages are also planned to include the nuclear protein coding genes for the proteins beta tubulin (β-tub),  elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (G3PD),  Glycogen Phosphorylase (Glyphos), and the Dictyostelium Ras subfamily protein, RasC.



The third major page (“BALAMUTHIA MANDRILLARIS” ) provides information concerning the DNA database sequences for Balamuthia mandrilarisB. mandrillaris has been a focus of the OSU research group.  This page also includes information about the nuclear and mitochondrial genome for an isolate of Balamuthia and the mitochondrial genome of 10 other isolates.



A page (“PROTACANTHAMOEBA“) has been added that details the molecular information that is available for the third genus that currently is recognized as a member of the Acanthamoebaidae.  Two species, P. caledonica and P. bohemica, have been studied using molecular methods.  The sequences of the 18S rRNA gene and the COXI gene of Protacanthamoeba caledonica have been studied by the OSU research group.



The next major page (“VERMAMOEBA“) details information about sequences that have been deposited in the DNA databases from isolates of the free-living limax amoeba Vermamoeba vermiformis (formerly Hartmannella vermiformis).  References for sequences and accession numbers are provided on a sub-page).

The levels of genetic variation in V. vermiformis and, levels compared to those seen in Acanthamoeba are included for sequences that are close to the full length of the 18S rRNA gene.  These comparisons will be of great use, since, among free-living amoebae, the number of sequences from V. vermiformis in the DNA databases is exceeded only by Acanthamoeba, and they provide natural comparisons with respect to life history and environmental niche.

Allele designations, as also recorded for the set of almost full length 18S rRNA gene sequences as a starting point for further analysis of variation in Vermamoeba.



Although the OSU FLA lab has not directly worked on any members of the genus Naegleria, the species of this important FLA group are of interest to us and the general FLA community.   We have included pages related to sequence variation in the ribosomal 18S RNA genes of Naegleria so that they can be available as an additional comparison group for the information being collected on Acanthamoeba and Balamuthia.  Additionally, information on mitochondrial 16S-like rRNA genes are provided.



In the future, additional taxa may be added to this page, possibly including other amebozoan groups, including unidentified eukaryotic sequences attributed to amebozoan related undescribed forms.

An additional page is (still) under construction which will provide information on the relationship between Acanthamoeba and its associated intracellular bacteria.



Additional pages provide information about the OSU Acanthamoeba group, including a list of OSU researchers and their publications on free-living amoebae.



We are working on providing a resource for other investigators to access DNA sequences from Acanthamoeba that have not been deposited in the international DNA databases.  Our experience in interacting with the Acanthamoeba research community suggests that many workers have collected DNA information on isolates that was never forwarded to DDBJ/EMBL/GenBank.  As we have collated the data described on this site, we have begun to see patterns that were not perceived without large collections of data.  We would be very grateful to hear from any member of the community who has such unpublished or undeposited DNA sequences.  We are very willing to assist in the preparation of an entry for that data which would be forwarded to the DNA databases.  The more data, the greater the possibility of gaining new insights into the biology of Acanthamoeba


This site maintained by Paul A. Fuerst, Academy Professor Emeritus, Department of Evolution, Ecology and Organismal Biology, The Ohio State University (