(updated August 6, 2015)
Among the various free-living amoebae, the member of the genus Naegleria may have acquired the most notorious reputation. They are “THE Brain-Eating Amoeba,” although that title is rightly applied specifically to Naegleria fowleri. N. fowleri is the causative agent of primary amoebic encephalitis (PAM), an infection with mortality rates >90%. Other species have lower pathogenicity. The genus is part of the family Vahlkampfiidae. Members of Naegleria are widely distributed in soil and freshwater habitats throughout the world. Almost all human infections by Naegleria occur though contact in warm water. Global climate change thus may have an important impact on the frequency with which infections will occur in the future.
The Ohio State University amoebae group has not been involved directly in research in Naegleria. Naegleria is included on this website primarily to place information about the levels of molecular variation in Acanthamoeba into a greater context of free-living amoebae. All data provided here were obtained from the DNA databases, or through interactions with colleagues elsewhere who have focused more frequently on Naegleria. Most prominent among our collaborators and friends with interest in Naegleria are Johan de Jonckheere, Francine Marciano-Cabral and Govinda Vishvesvara.
THE MOLECULAR TAXONOMY OF NAEGLERIA
Prior to the use of DNA for classification, six species were generally recognized, based on cyst morphology, pathogenicity, temperature tolerance, isozyme pattens and immunological criteria. These were N. fowleri, N. australiensis, N. lovianiensis, N. gruberi, N. jadini, and N. thorntoni. The last species, N. thorntoni, has since been identified as not being a member of Naegleria, but rather belonging within the genus Tetramitus. With the availability of data on DNA sequences beginning in the 1990’s, new attention could be placed on understanding the interrelationships of isolates of Naegleria. Substantial levels of genetic variation have been observed within the genus Naegleria. The patterns of variation have been interpreted as indicating the presence of a large number of differentiated species of the genus, although the criterion for describing a new species are arbitrary and based often on only a single gene segment. The number of named species has increased tremendously because of molecular information. There have been over 50 names applied to putative species within Naegleria. Some of these names predate molecular approaches to classification, and have not been subsequently applied to taxa identified in any molecular study. Regardless, the number of named forms is about double the number currently recognized in Acanthamoeba.
Molecular studies of protozoa have traditionally started with an analysis of the nuclear rRNA sequences. This was the case with Naegleria. Clark and Cross (1988) first determined a sequence of the 18S rRNA gene for N. gruberi. Further studies in the late 1990’s provided information on other recognized species, especially spurred by the discovery of group I self-splicing in the genes of some species (De Jonckheere 1994). More detailed information on the 18S rRNA gene sequences of Naegleria are presented in a section below. Although the 18S rRNA gene provided moderate levels of genetic differentiation for the initial study of the phylogenetics of species of Naegleria, levels of variation for the 18S rRNA gene within a “species” of Naegleria seem to be less than that seen in “species” of Acanthamoeba, primarily because of the absence of the hypervariable segments of the gene that exist in Acanthamoeba. As a result, the study of species differences in Naegleria has moved to the use of sequences from other gene segments to resolve most questions of species identification.
Molecular approaches driven by the work of Johan de Jonckheere (1998) have utilized the variable segment of the ribosomal RNA transcription region that includes the ITS1-5.8rRNA-ITS2 segment as the primary molecular identifier of species and isolates in Naegleria. Analysis of this ITS segment has resulted in the proposal over the last 15 years for the existence of a large number of newly identified species. An example of the proposed relationships between the newly described species of Naegleria as defined by the use of the ITS region is shown in the accompanying figure, taken from the Tree of Life page edited by de Jonckheere. Further information on the use of the ITS region for species identification in Naegleria can be found in the papers of Johan de Jonckheere (Protist, 2004, 155:89; Experimental Parasitology, 2014, 145:52).
Ultimately, any comparison of the degree of genetic variation between Naegleria and Acanthamoeba becomes more problematic. There have been a number of investigations of Naegleria that have utilized the 18S rRNA gene, and the phylogenetic relationships among isolates of Naegleria can be obtained (as shown below), but the use of the 18S rRNA gene sequence for species identification has been displaced by information from the ITS region, because of the greater level of variation for the latter region. However, the ITS region has been used only sparingly in Acanthamoeba, and has not been applied over the breadth of the genus. No ITS sequences have been obtained for either Balamuthia or Vermamoeba.
THE 18S rRNA GENE OF NAEGLERIA
The location of the ribosomal RNA genes of Naegleria is ususual, but not unique, compared to some other protists. The genes are encoded on circular plasmids, which each code for a single copy of the rRNA transcription unit. The complete sequence in the plasmid in N. gruberi is ~14,000 nucleotide pairs in size (Maruyama and Nozaki, 2007). N. gruberi maintains 3,000–5,000 extrachromosomal copies of rDNA plasmids per cell.
Since the first sequence of the N. gruberi 18S rRNA gene sequence was reported (Clark and Cross, 1988), the sequences for this gene have been determined in a number of isolates of various species of Naegleria. Details are provided in the page “NAEGLERIA 18S rRNA SEQUENCES IN THE DNA DATABASES“.
The references and accession numbers for the sequences deposited in the DNA databases are given in the accompanying REFERENCES page.
MITOCHONDRIAL 16S-LIKE GENE SEQUENCES OF NAEGLERIA
In addition to the sequences of the 18S rRNA gene, a set of nine sequences have been determined for the mitochondrial ribosomal small subunit rRNA gene. These include two sequences extracted from the complete mitochondrial DNA genome sequences of N. gruberi and N. fowleri. Details are provided on a separate page.
NAEGLERIA IDENTIFIED AS UNCULTURED EUKARYOTIC SEQUENCES
As with other free-living amoebae, members of Naegleria are sometimes found within environmental microbiome studies. In such studies they are identified as uncultured eukaryotic sequences. Examining the DNA databases, we find 17 sequences in the DNA databases of 18S rRNA genes from uncultured eukaryotic organisms that appear to be putative isolates of Naegleria. These sequences range in size from 515 to 1433 bp, with 14 exceeding 1200 bp. Eleven of the environmental sequences appear to be closely related to the N. fowleri/lovaniensis cluster of 18S rRNA gene sequences, while the remaining six are scattered. Three are closely linked to N. sp. 62K4, one to ATCC 30294, one to N. clarki, and one to N. sp. SumV3/I.