Thomas J. Byers

Use of Subgenic 18S Ribosomal DNA PCR and Sequencing for Genus and Genotype Identification of Acanthamoebae from Humans with Keratitis and from Sewage Sludge

ABSTRACT This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bpAcanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.

The Evolutionary History of the Genus Acanthamoeba and the Identification of Eight New 18S rRNA Gene Sequence Types

ABSTRACT The 18S rRNA gene (Rns) phylogeny of Acanthamoeba is being investigated as a basis for improvements in the nomenclature and taxonomy of the genus. We previously analyzed Rns sequences from 18 isolates from morphological groups 2 and 3 and found that they fell into four distinct evolutionary lineages we called sequence types T1‐T4. Here, we analyzed sequences from 53 isolates representing 16 species and including 35 new strains. Eight additional lineages (sequence types T5‐T12) were identified. Four of the 12 sequence types included strains from more than one nominal species. Thus, sequence types could be equated with species in some cases or with complexes of closely related species in others. The largest complex, sequence type T4, which contained six closely related nominal species, included 24 of 25 keratitis isolates. Rns sequence variation was insufficient for full phylogenetic resolution of branching orders within this complex, but the mixing of species observed at terminal nodes confirmed that traditional classification of isolates has been inconsistent. One solution to this problem would be to equate sequence types and single species. Alternatively, additional molecular information will be required to reliably differentiate species within the complexes. Three sequence types of morphological group 1 species represented the earliest divergence in the history of the genus and, based on their genetic distinctiveness, are candidates for reclassification as one or more novel genera.

Identification and Distribution of Acanthamoeba Species Genotypes Associated with Nonkeratitis Infections

ABSTRACT Acanthamoeba is a free-living protozoan genus found in a wide variety of natural habitats, including water, soil, and air. Pathogenic isolates of Acanthamoeba are medically relevant as the causative agent of sight- threatening Acanthamoeba keratitis (AK), serious infections of other organs, and fatal granulomatous amebic encephalitis. Previous work employing DNA sequences of nuclear and mitochondrial small-subunit rRNA genes (SSU rRNA genes) determined the genotypic diversity of Acanthamoeba and found that many named species of Acanthamoeba are associated with particular genotypes. These studies also concluded that nearly all AK infections result from a single molecular genotype: T4. Here, we asked whether Acanthamoeba clinical isolates from non-AK infections are also associated with particular genotypes. DNA sequence determination of nuclear SSU rRNA genes was employed for genotypic identification of 29 isolates of Acanthamoeba from non-AK infections. Sequence analysis demonstrates that T4 is the predominant genotype in non-AK infections, including those in brain, cerebrospinal fluid, nasal passages, skin, and lung. Rare genotypes (T1, T10, and T12) have been isolated from brain infections. We conclude that genotype T4 is the primary genotype in non-AK Acanthamoeba infections, as was the case in AK infections. However, the genotypes that were isolated from brains have not been observed in environmental isolates of Acanthamoeba, and their natural ecological niche is unknown.

Subgenus systematics of Acanthamoeba : Four nuclear 18S rDNA sequence types

ABSTRACT Classification of Acanthamoeba at the subgenus level has been problematic, but increasing reports of Acanthamoeba as an opportunistic human pathogen have generated an interest in finding a more consistent basis for classification. Thus, we are developing a classification scheme based on RNA gene sequences. This first report is based on analysis of complete sequences of nuclear small ribosomal subunit RNA genes (Rns) from 18 strains. Sequence variation was localized in 12 highly variable regions. Four distinct sequence types were identified based on parsimony and distance analyses. Three were obtained from single strains: Type T1 from Acanthamoeba castellanii V006, T2 from Acanthamoeba palestinensis Reich, and T3 from Acanthamoeba griffini S‐7. T4, the fourth sequence type, included 15 isolates classified as A. castellanii, Acanthamoeba polyphaga, Acanthamoeba rhysodes, or Acanthamoeba sp., and included all 10 Acanthamoeba keratitis isolates. Interstrain sequence differences within T4 were 0%–4.3%, whereas differences among sequence types were 6%–12%. Branching orders obtained by parsimony and distance analyses were inconsistent with the current classification of T4 strains and provided further evidence of a need to reevaluate criteria for classification in this genus. Based on this report and others in preparation, we propose that Rns sequence types provide the consistent quantititive basis for classification that is needed.

Identification of Balamuthia mandrillaris by PCR Assay Using the Mitochondrial 16S rRNA Gene as a Target

ABSTRACT Balamuthia mandrillaris is an opportunistic pathogen that causes granulomatous amebic meningoencephalitis in animals, including humans. Based on sequence analysis of mitochondrial small-subunit-rRNA genes, we developed primers that amplify a Balamuthia-specific PCR product. These primers will be useful for retrospective analyses of fixed tissues and possible identification of Balamuthia in vivo.

Growth, Reproduction, and Differentiation in Acanthamoeba

Publisher Summary Acanthamebas are attractive models for the study of several major problems in eukaryotic cell and developmental biology. The ease with which a variety of strains can be grown in axenic or chemically defined media and the simplicity of the single-cell differentiation are very useful attributes. Acanthamebas grow either as monolayer or suspension cultures. Individual amebas migrate extensively when A . castellanii is seeded in axenic medium on glass, plastic, or bacteria-free agar surfaces and, consequently, fail to form colonies. Colony formation is observed when A . castellanii is grown in or on agar with yeast or bacteria. The occurrence of lipophosphonoglycan instead of glycoprotein in the plasma membrane, the prominent role of pinocytosis in soluble nutrient uptake, the branched mitochondrial electron transport chain, the phenomenon of amitosis, and the induction of differentiation by inhibitors of mitochondrial macromolecule synthesis are all unusual features. Moreover, acanthamebas have are beneficial in the study of the effect of visible light on cell multiplication and the influence of cell concentration on nuclear gene expression.

ACANTHAMOEBA STRAINS ISOLATED FROM ORGANS OF FRESHWATER FISHES

Contrary to data on Acanthamoeba infections in humans, little is known about infections in fishes. The present study combines the description of strains isolated from fishes with presentation of an improved method for subgeneric classification. Acanthamoeba spp. were isolated aseptically from tissues of 14 (1.7%) of 833 asymptomatic fishes collected in rivers and streams in the Czech Republic. Acanthamoebae successfully cloned from 10 of the 14 isolated strains were examined here. Morphology of these isolates was evaluated using light optics plus scanning and transmission electron microscopy. Cyst morphology, which varied extensively within and among clones, was most like morphological group II, but species-level classification was considered impossible. A distance analysis based on 442 bases in an 18S rDNA polymerase chain reaction fragment of about 460 bp placed the isolates in a clade composed of sequence types T3, T4, and T11, the 3 subdivisions of morphological group II. Fluorescent in situ hybridization (FISH) using oligonucleotide probes indicated that all isolates belong to a single subdivision of group II, the T4 sequence type. It has been concluded that the fish isolates are most closely related to strains commonly isolated from human infections, especially Acanthamoeba keratitis. The shorter diagnostic fragment sequences have proved nearly as useful as complete 18S rDNA sequences for identification of Acanthamoeba isolates.

Molecular biology of DNA in Acanthamoeba, Amoeba, Entamoeba, and Naegleria

Publisher Summary This chapter discusses the molecular biology of DNA in acanthameba, ameba, entameba, and naegleria. The chapter reviews a reasonably current review about gene structure and the overall organization of the DNA and its metabolism. The four genera of amebas that have been studied most extensively are described. The genus ameba includes large free-living organisms. The cell cycle is simple; replication is by binary fission and differentiation is unknown. These amebas are large enough to manipulate by various surgical procedures, and this attribute has been exploited extensively. Cultures typically are grown using the ciliated protozoan tetrahymena as the major food organism. The genus acanthameba includes small amebas that are ordinarily free-living, but can be opportunistic pathogens of animals and humans. Such organisms are considered amphizoic. In a few cases, infection has been lethal, but these organisms are ubiquitous in nature and most humans appear to have good resistance to infections. The taxonomy of acanthameba is confusing, and it is unclear whether pathogenicity is restricted to certain species or whether all strains are capable of it. acanthameba replicate by binary fission. They differentiate into dormant cysts (that is, they encyst) during adverse environmental conditions. The genus naegleria includes small amebas that can differentiate either into a cyst or a flagellated stage. In naegleria, it is clear that certain species are pathogenic and that other species are not. The pathogenic Naegleria fowleri is highly virulent and fatal to humans. The genus entameba is distinct from the rest because it includes anaerobes and parasites. The life cycle includes a phase of replication by binary fission and encystment, which occurs in response to environmental factors. Nuclear division can continue in the cyst in the absence of cytoplasmic division. Species are typically identified according to their hosts. Entameba histolytica, the human parasite, and Entameba invadens, a parasite of reptiles, are the two most commonly studied species. Both can be grown axenically under anaerobic conditions. Entameba moshkovskii is interesting because it appears to be free-living.

Interstrain mitochondrial DNA polymorphism detected in Acanthamoeba by restriction endonuclease analysis.

The genus Acanthamoeba includes pathogenic and nonpathogenic strains of amebas with unclear taxonomic and evolutionary relationships. To explore these relationships further, we have examined mitochondrial DNA fragment patterns obtained for 15 Acanthamoeba strains by use of five restriction endonucleases. The mitochondrial DNA molecules were circular, averaging 41.6 +/- 1.5 kilobase pairs. Fragments resulting from endonuclease digestion of the DNA were separated by agarose gel electrophoresis. Ten distinct families of electrophoretic patterns (digestion phenotypes) were observed. Seven phenotypes were found for seven strains considered nonpathogenic or of unknown pathogenicity. Three phenotypes were associated with pathogenic strains. One of these phenotypes included a single pathogenic strain, a second included one pathogen and one strain of unknown pathogenicity, and the third included five pathogenic strains. The latter five were of widespread geographic origin and previously were assigned to two different species. The results suggest that extensive nucleotide sequence diversity occurs among strains from a single species of Acanthamoeba, but that subgroups of strains with similar sequences also occur. Thus, restriction enzyme analysis can identify clusters of strains and may be a useful approach to classification in the genus. Improvements in classification should help clarify relationships among pathogenic and non-pathogenic strains.

Group-I introns with unusual sequences occur at three sites in nuclear 18S rRNA genes of Acanthamoeba lenticulata.

Abstract Seven of eleven isolates of Acanthamoeba lenticulata were found to have group-I introns located at one of three positions within the 18S rRNA gene. The introns are 636–721-bp long and are absent from mature rRNA. They lack open reading frames that could encode any known endonucleases. Sequences of introns from the same site in different isolates are 86.0–98.9% identical, while from different sites they are 24.2–29.8% identical. The most closely related introns from other organisms are in the 18S rRNA genes of several green algae where the 17.0–23.6% identity is mostly limited to a highly conserved core of base pairs including P, Q, R and S. Because the A. lenticulata introns only occur in one Acanthamoeba lineage, they were probably acquired after the divergence of this species.