J. frofozool.,37(4), 1990, pp. 17s-259 0 1990 by the Society of Protozoologists

Genes of Acanthamoeba: DNA, RNA and Protein Sequences (A Review)’ THOMAS J. BYERS, ERIC R. HUGO, and VALERIE J. STEWART Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210 ABSTRACT. This review summarizes knowledge about the structure of nuclear genes and mitochondria1 DNA in Acanthamoeba. The information about nuclear genes is derived from studies of DNA, RNA and protein sequences. The genes considered are those for 5S, 5.8s and 18s rRNA, actin I, profilins Ia/b and 11, myosins IB, IC and 11, and calmodulin. All of the sequences show strong similarities to comparable sequences from other organisms. Introns have been found in the actin and myosin genes. The location of the actin intron is unique, but many of the myosin introns occur at the same sites as introns in myosins of other organisms. Sequence comparisons, especially of 5s and 5.8s rRNA and actin, support previous evidence, based primarily on 18s rRNA, that Acanthamoeba genes are at least as closely related to those of higher plants and animals as they are to various other protistan genera. The functional organization of the promoter region for the nuclear rDNA transcription unit has been studied extensively, but there is a need for information about the functional organization of regulatory sequences for other genes. Restriction fragment length profile (RFLP) studies of mitochondria1 DNA reveal relatively high levels of overall sequence diversity, but information on the structure and function of individual genes is needed. The RFLP appear to have potential as tools for taxonomic studies of this genus. Key words. Actin, calmodulin, myosin, profilin, 5 s rRNA, 5.8s rRNA, 28s rRNA, mitochondria1 DNA.

A

CANTHAMOEBA has long been recognized as an appropriate model for studies on a variety of problems in cell biology. A relatively large size, rapid growth in axenic culture using broth or chemically defined media, active motility, readily induced phagocytosis and an easily induced synchronous cell differentiation are some of this organism’s attractive features. Molecular aspects of the cell cycle and encystment have been reviewed recently [ 151. Understanding the genetics of acanthamoeba is the most important challenge at the present time. Nuclear chromosomes are very small and may be very numerous [reviewed in 13, 141. Evidence suggesting that Acanthamoebus are polyploid is accumulating, but is not yet conclusive. Reproduction appears to be asexual because there is as yet no convincing evidence for genetic recombination. A number of drug resistant lines have been used to test for recombination, but the results so far are inconclusive (Akins, R. A. 1981, Ph.D. Dissertation, The Ohio State University; Gast, R. J. & Byers, T. J., unpubl. results). The most extensive recent progress in Acanthamoeba genetics has been in investigations of nuclear gene structure and in the regulation of transcription by RNA polymerase 1. In addition, the successful separation of nuclear chromosomes by pulse field electrophoresis has permitted preliminary investigations of genechromosome linkages. The availability of nucleic acid and protein sequence information has stimulated studies of phylogenetic relationships between Acanthamoeba and other organisms. This paper reviews data on Acanthamoeba nuclear gene structure that have been derived from sequencing of the genes themselves or from studies of RNA and protein sequences. This information has been obtained almost entirely from work on the Neff strain of A. castellanii. Genomic DNA sequences and deduced amino acid sequences have been published for actin I [55], myosin heavy chains IB, IC, and I1 [12, 28, 35, 371 and 18s rRNA [25]. Complete amino acid sequences have been determined for actin I [78], profilins Ia/b and I1 [2, 31, and a partial amino acid sequence has been obtained for calmodulin [85]. Nucleotide sequences for 5s and 5.8s rRNA have been determined directly from the RNA [49]. Our review emphasizes comparisons between the amoeba genes and comparable genes from other sources. At present, no published sequences are available for Acanthamoeba mitochondria1 genes or their products. However, information on overall interstrain mtDNA sequence diversity is available and considered here. I Portions of this review were included in a presentation to the Vth International Conference on the Biology and Pathogenicity of FreeLiving Amoebae, Brussels, Belgium, August 7-1 1, 1989.

NUCLEAR GENOME Size and copy number. The total cellular DNA content of the Neff strain ranges between 1 and 2 pg for uninucleate amoebas during log phase [reviewed in 13, 141, but the number of genome copies (ploidy level) remains uncertain. Measurements of nuclear DNA content made in our laboratory by L. E. King [ 1980, Ph.D. Dissertation, The Ohio State University] are consistent with a total DNA content of lo4 bp. Measurements of kinetic complexity [ 11, 331 favor a haploid genome size of -4-5 x lo7 bp. More recent studies utilizing pulse field electrophoresis to size chromosomes suggest a genome as small as 2.3-3.5 x lo7 b p [63; Jantzen, H. & Schultze, I., abstr., Vth International Conference on the Biology and Pathogenicity of Free-Living Amoebae]. The pulse field data are likely to be underestimates, but still are consistent with the likelihood that Acanthamoeba is polygenomic. For comparison, the haploid genome sizes for the yeast Saccharomyces and the slime mold Dictyostelium are -2 x l o 7and -5 x lo7bp, respectively [46]. Ribosomal RNA repeat unit. The ribosomal gene (rDNA) repeat unit of eukaryotes typically includes one set of 18S, 5.8s and 28s rRNA genes plus spacer regions located between genes and between neighboring sets. (The generic designations used here for eukaryotic rRNA will be used throughout this review.) The repeat unit of Acanthamoeba is 12 kbp. It may be of a single kind since there is no evidence for sequence or size heterogeneity [ 181. The size range for repeat units in other organisms extends from 9 kbp for Saccharornyces and Neurospora to 44 kbp for mouse and human rDNA [46]. The number of repeat units can be up to several thousand copies i n other organisms [47], but is about 600 copies in Acanthamoeba (Paule, M. R., pers. commun.). The amoeba rRNA coding sequences are arranged 5’- 18S5.8s-28s-3’ as found in all prokaryotes and eukaryotes [47]. There is no evidence for introns in any of the Acanthamoeba rRNA genes. The 28s coding sequence is subdivided into two segments of 2,400 bp (28Sa) and 2,000 bp (28Sb) separated by an internal transcribed spacer (ITS) of about 200 bp [ 181. The ITS is excised from the primary transcript. The 28Sa and 28Sb rRNA molecules remain separate in the large ribosomal subunit (LSU) where they occur as a 28Sa:28Sb:5.8S hydrogen-bonded complex of unknown structure [ 18, 721. In most eukaryotes the LSU rRNA exists in the ribosome as a 28S:5.8S complex with the 5.8s molecule hydrogen-bonded to the 5’ end of the 28s molecule [26]. Presumably the same is true for Acanzhamoeba. Subdivision of the mature 28s molecule is not unique to Acanthamoeba, but also occurs in trypanosomatids [70] and

17s

18s

J. PROTOZOOL., VOL. 37, NO. 4, JULY-AUGUST 1990

other organisms. In the relatively extreme case of Crithidia fasciculata, the LSU rRNA complex includes seven separate species-six of which are derived from the 28s molecule [70]. One ITS that subdivides the 28s gene of Crithidia maps to a highly variable region designated D7c. This region is the location of the Acanthamoeba ITS [70]. 18s ribosomal RNA. The Acanthamoeba 18s rRNA gene has been sequenced in its entirety [25]; its coding sequence is 2,303 bp long. Protozoan 18s rRNA vary widely in length from 1,244 bp in the sporozoan Vairimorpha necatrix to 2,305 bp in Euglena gracilis, but most fall in the range of 1,800-2,300 [19]. Acanthamoeba has the 2nd largest 18s gene sequenced to date. The amoeba RNA has 10 regions that are not typical of other eukaryotes. However, a proposed secondary structure indicates that the molecule readily fits models proposed for other eukaryotes [26]. In the model, the majority of the extra bases are assigned to extended helical regions rather than to unpaired loops. The extra bases are present in the mature rRNA as well as the gene and, therefore, are not introns. Our laboratory has obtained nearly complete (>90%) nucleotide sequences for 18s rDNA from the Ma and Castellani strains of A. castellanii. Comparisons of these sequences with that of the Neff strain of the same species reveal sequence divergences of 3% or less (Gast & Byers, unpubl. results). Comparable studies based on 278 conserved sites in 18s rRNA from four species of Naegleria found 0.7-6.1% divergence among different species [9]. Phylogenetic studies based on 18s rRNA sequences arrive at the conclusion that Acanthamoeba is at least as closely related to higher animals and plants as to other protozoans [68]. One particularly interesting observation is the relatively low homology between the rRNA of Acanthamoeba and Naegleria, thus, suggesting a distant evolutionary relationship between the two amoebas [9, 16, 681. The promoter for the ribosomal transcription unit (RTU) is the only Acanthamoeba gene regulatory sequence for which detailed structural and functional analyses have been published. Expression of the typical eukaryotic RTU, and rRNA precursor that includes the sequences for 18S, 5.8s and 28s rRNA, is initiated from a promoter that overlaps the 5’ end of the RTU, but mostly lies in the adjacent flanking region [43]. In Acanthamoeba, the binding sites for RNA polymerase I (RNAPI) and a trans-acting transcription initiation factor (TIF) occur in the region from -47 to +8 relative to the transcription start site. Deletion mutation analysis [43], single base substitutions [42] and DNase I footprinting [8] have been used to identify Acanthamoeba promoter sites at which protein-DNA interactions essential for transcription occur. Paule and colleagues have concluded that binding of TIF is strongly influenced by the base sequence between - 3 1 and -28 (TAAA). In addition, TIF makes numerous other contacts with DNA over the region from - I7 to -45 or - 55 that affect transcription. The addition of RNAPI extends DNase protection from - 17 to $20, but binding of polymerase to the promoter depends on protein-protein interactions with TIF and is DNA sequence-independent [41]. The region between - 20 and +8 is required for initiation by RNAPI, but not for TIF binding [32]. Sollner-Webb & Tower [69] have suggested a general model for the eukaryotic rDNA promoter that is bipartite. The model proposes a “proximal promoter domain” that spans the region -40 to + 5 relative to transcription start sites, binds the necessary transcription factors, and is sufficient for effective initiation under most optimized conditions in vitro. It also proposes “upstream promoter domains” that extend an additional 120 base pairs in the 5’ direction, however, its effects can only be discerned under more stringent assay conditions. The Acanth-

-

-

amoeba equivalent of the proximal promoter domain is the region from -47 to + 8 that contains the binding sites for RNA PI and TIF. Transcription of rDNA tends to be species-specific with little cross-reactivity between promoters and trans-acting factors from different eukaryotes [69]. This imay be partially explained by the observation that the primary sequences of RNAPI promoters from various eukaryotes show little homology, except for closely related species [69]. The species specificity of transcription and the lack of sequence homology have caused current research on RNAPI promoters to focus on sequence motifs with common functions rather than common primary sequences [69]. In this regard, it might be especially worthwhile to examine interstrain variability in the promoter sequences of Acanthamoeba. Studies of mitochondria1 DNA sequence variability (see below) reveal a broad range of sequence divergence within a relatively small sample of the large number of available strains. If the amoeba nuclear gene promoter sequences also show interstrain divergence, then this organism shciuld be an excellent subject for studies on the evolution of sequence motifs. Studies on the RTU and its transcription in Acanthamoeba are among the most complete for this gene family for any organism. Although gene function is not the emphasis of this review, we wish to mention the existence of excellent studies on the mechanism of initiation by the amoeba RNAPI [ 6 ] , promoter occlusion in response to the passage of polymerases from an upstream promoter [7:/,and regulation of transcription during differentiation by modification of RNAPI [ 5 , 601. 5.8s ribosomal RNA. The’5.8S rRNA nucleotide sequence has been determined directly from the RNA [49]. The molecule is 162 bases long, whereas, the lengths of 5.8s molecules from an assortment of other eukayotes range from 154 in Tetrahymenu to 17 1 in Crithidia (Table I). Most interspecific variation occurs in a region of the molecule designated the “G C-rich hairpin.” As in the case of tht: 5s species, the 5.8s rRNA of Acanthamoeba is at least as homologous to RNA from other eukaryotic kingdoms as it is to IRNA of other protozoans (Table 1) [see also 641. (Homology is used throughout this review to indicate nucleotide or amino a’cid identity.) MacKay & Doolittle proposed a secondary structure for the 5.8s rRNA of Acanthamoeba [49]. But, as they pointed out, it might only exist in solution since the molecule normally is hydrogen-bonded to the 5’ end of‘28S rRNA in ribosomes. More recently, Gutell & Fox [26] have proposed secondary structures for 5.8s rRNA in complex with 28s rRNA for various eukaryotes. We observe that the amoeba 5.85 molecule would easily fold into a structure similar to those illustrated by Gutell & Fox, but a complete model for the ribosomal form awaits sequencing of the 5’ end of the 28s rRNA. 5s ribosomal RNA. In prokaryotes and a few lower eukaryotes such as Saccharomyces and Dictyostelium [47], the 5 s rRNA gene is an integral part of the ribosomal repeat unit. The Acanthamoeba 5 s molecule is 1 19 nucleotides long, thus, being similar to 5 s molecules from other organisms where the typical size is about 120 nucleotides [Table I]. A secondary structure has been proposed for Acanthamoeba 55; rRNA [49] that differs slightly from the recent minimal structure proposed for eukaryotes by Wolters & Erdmann [82], but we observe that the amoeba sequence is compatible with the latter structure as well. In most eukaryotes, including Acanthamoeba, 5 s genes are unlinked to the repeat unit [47]. The 5 s genes often occur in hundreds or thousands of copies [47]. In Neurospora crassa, seven different isoforms of the 5 s rRNA have been described [65]. In Acanthamoeba, only one 5 s rRNA sequence has been obtained to date [49]. Whether different isoforms exist remains to be seen. Sequence comparisons between Acanthamoeba 5 s rRNA and

+

19s

EYERS ET AL.-GENES OF ACANTHAMOEBA Table 1. Ribosomal RNA nucleotide sequence homologies.a Total 5s rRNA

Acanthamoeba castellanii Tetrahymena thermophila Crithidia fascrculata Physarum polycephalum Dictyosielium discoideurn Saccharomyces cerevisiae Xenopus laevis Homo sapiens Oriza sativa

Total

Coreb

5.8s rRNA

5.8s rRNA

bp

% Homol.

bp

Yo Homol.

bp

Yu Homol.

1 I9

I00 70 69 81 65 66 63 68 70

162 154 171 155 162 158 159 I58 164

100

135

100

65 58 57 36 77 67 67 75

137 132 133 136 136 135 140

64 60 65 81 68 69 78

120 120 120 119 121 I20 121 1 I9

The 5s sequences are from [82]; the 5.8s sequences are from [22, 26, 49, 58, 59, 741. Homologies were calculated using GENEPRO (Riverside Scientific Enterprises, Seattle, WA) and are comparisons with the corresponding rRNA from Acanthamoeba. The core is the sequence remaining after deleting the G+C-rich hairpin based on the secondary structures of Spencer et al. [lo] and Gutell & Fox [26].

Table 2. Profilin, actin and myosin amino acid sequence similarities.a No. amino acids

Similarity”

Reference‘

O/o

Profilin: A . castellanii, pII pIa/b S. cerevisiae Human Mouse Bovine

125 125 126 139 140 139

100 87-89(94) 59(64) 36(52) 31 (52) 36(55)

2 3 57 I , 44 71 I , 56

Actin: A. castellanii, actin I Entamoeba histolytica Plasmodium falciparum P. polycephalum, Ppa35 S. cerevisiae Human, nonmuscle y-actin skeletal muscle a-actin Mouse, nonmuscle &actin

374 375 375 375 374 374 375 374

100

55 20 81 27 23 21 29 75

Myosin, type I1 heavy chain:“ A . castellanii. MI1 D. discoideum, LMHC Caenorhabditis elegans unc 54 muscle Rat, embryonic skeletal muscle Myosin, type I heavy chain? A . castellanii, MIB M IC D. discoideum Bovine, BMIHC

846 819

89 75 97 87 95 92 95 100

-60

28 80, 40

850

55

38.28

838

57

73,28

670 670 760 691

100

37 35,37 36, 36 31, 37

72 80 54

We calculated similarity values for profilin and actin using GENEPRO. Similarity includes amino acid identities and conserved replacements using the following replacement groups: AG, DE, FY, ILV, KR, NQ, ST. Estimates for myosins were from the literature and were higher than ours due to use of a larger set ofconserved amino acid replacements. For each protein type, the n/n similarity is a comparison with the 1st protein in the set. Values in parentheses are for N-terminal -34 amino acids only. References for profilin and actin are for the sequence source. For myosin, the lefthand reference is the sequence source and the righthand reference is the source of the similarity estimate. We calculated the similarity for D. discoideum. Analysis limited to amino acids in globular head region.

5s rRNA from other organisms (Table 1) [see also 491 support previous claims that sequence homologies among the protozoans are less than among animals, but no greater than between protozoans and animals, or protozoans and plants [68, 701. The homology between Acanthamoeba and Physarum is an apparent exception since it falls within the range of homologies found among 5s rRNA of higher animals [49]. Actin. Acanthamoeba has at least three actin genes [34, 551. Sequence information is available for one, the actin I gene [55]. It has not been demonstrated rigorously that this is an expressed gene, but its nucleotide sequence is totally consistent with the complete amino acid sequence determined for the major actin isoform present in the amoeba [78]. Actins from many sources are highly conserved molecules of 374-375 amino acids (Table 2). The Acanthamoeba genomic sequence encodes a protein of 375 amino acids including an N-terminal Ac-Met-Gly-Asp sequence. The major isoform of actin in the amoeba lacks the N-terminal methionine and begins with Ac-Gly-Asp [62]. Posttranslational removal of the N-terminal methionine and sometimes additional amino acids are characteristic of all actins [6 I]. The presence of glycine at the N-terminal position is unusual, but not unprecedented, since it also occurs in an actin from Entamoeba histolytica [20]. The Acanthamoeba actin I gene contains one intron of 129 base pairs that is located just after the codon for amino acid 105. Introns have been found in at least 14 different positions in actin genes [62], but the site of insertion in actin I is unique. Vandekerckhove & Weber [79] have subdivided the actins of higher animals into nonmuscle cytoplasmic actins and four classes of muscle actin. They propose that the nonmuscle actins ((3- and y-actins) are most closely related to the actins of invertebrates and some amoebas (Dictyostelium and Physarum). Plant actins, which also are cytoplasmic, are relatively highly variable [30, 661 and are thought to have evolved independently [61]. The actin I amino acid sequence of Acanthamoeba. with 95% similarity to human nonmuscle y-actin, 95% to mouse nonmuscle p-actin, and 92% to human skeletal a-actin, is slightly more closely related to cytoplasmic and muscle actins of mammals than it is to most other protistans where, for example, the similarity is 97% t o Physarum, 89% to Entamoeba, 87% to Saccharomyces and 75% to Plasmodium (Table 2). This is another indication of the relatively high molecular diversity among protistans. Profilin. At least 20 cytoskeletal proteins have been isolated from Acanthamoeba [40, 48, 50, 611. The majority are actinbinding proteins other than myosin and have not been se-

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J. PROTOZOOL., VOL. 37, NO. 4, JULY-AUGUST 1990

quenced. However, several isoforms of the small actin-binding protein profilin have been sequenced [2, 31. Profilin prevents actin polymerization and may be involved in sequestering actin monomers in a variety of organisms [6 I]. Profilins have three primary domains, a weakly charged N-terminal region that is relatively conserved and may be an actin-binding domain, an alanine-rich middle region, and a COOH-terminal region that is rich in basic amino acids and may interact with phosphatidylinositol 4,5-biphosphate in the regulation of actin binding [61]. The profilins of Acanthamoeba have an actin capping activity not typically seen in other profilins [61]. Only one isoform of profilin has been observed in humans, but there are three or more isoforms in Acanthamoeba-profilin I1 and two or more forms of profilin I (thus, the designation Ia/ b). Amino acid sequences have been determined directly, or deduced from cDNAs for human [ 1,441, bovine [ 1, 561, mouse [71], yeast [57] and acanthamoeba [2, 31 profilins. The mammalian proteins have 139-140 amino acids, yeast has 126 and Acanthamoeba has 125 (Table 2). The isoforms of acanthamoeba profilin I have not been separated, but are recognized by the presence of two different amino acids at five different positions ~31. Similarity of sequences among the various profilins is highest in the N-terminal region corresponding to the 1st 34 amino acids of the Acanthamoeba protein (Table 2). In this region, the amoeba profilins Ia/b and I1 are 94% similar to each other, whereas, profilin I1 is 64% similar to yeast profilin and 5 1-55% similar to mammalian profilin. In contrast, homology among the three mammalian proteins is -97% in the N-terminal region. It has been proposed that the N-terminal region may be the binding site involved in sequestration of actin and that this may account for the relatively high degree of similarity [3]. The COOH-terminal region of the acanthamoeba protein has a 16 amino acid sequence that is very similar to internal sequences found in two actin capping proteins, human gelsolin and Physarum fragmin [77]. A lysine residue at position 115 within this sequence can be cross-linked to a glutamic acid at position 364 in the COOH-terminal end ofAcanthamoeba actin and, thus, probably is involved in actin binding [77]. The profilin COOH-terminal sequence is not present in human profilin which does not have capping activity, and, therefore, it has been suggested that the sequence may be associated with capping activity V71. Myosin. Korn & Hammer [40] define myosin as ". . . any protein that binds F-actin and has ATPase activity that is activated by F-actin." Most muscle and nonmuscle myosins that have been studied are classified myosin 11; they are hexameric polypeptides including two heavy chains presumed to be identical and two pairs of light chains. A smaller, but growing group of myosins are classified myosin I; they consist of one heavy chain and, possibly, one light chain. The heavy chains include two major domains, an N-terminal region that forms a globular head and has the ATPase activity and a C-terminal tail region. Myosins I and I1 from various sources tend to be similar in the head domain, but extensive variability is found in the tail. Myosin I1 polypeptides associate through their tail regions to form myosin filaments, whereas, myosin I tails do not form filaments. One Acanthamoeba myosin I1 heavy chain gene (MII) has been sequenced. The gene codes for a protein of 1,509 amino acids and has three small introns located at the 5' end [28]. Myosin I1 is the amoeba myosin most similar to muscie myosins. It has a globular head domain of 846 amino acids and a tail domain of 663 amino acids. The head domain is 64% similar (similarity is explained in Table 2) to the 1st 8 19 amino acids of a myosin I1 heavy chain from Dictyostelium, 55% similar to the 1st 850 amino acids of the unc 54 muscle myosin of Cae-

norhabditis and 57% similar to the 1st 838 amino acids of rat embryonic skeletal muscle myosin (Table 2). Regions of especially strong similarity in the head domain include the probable ATP-binding site and the active thiol regions, whereas, dissimilarity is greatest in the 1st 80 amino acids and in the locations corresponding to muscle myosin tryptic cleavage sites [28, 391. The MI1 of Dictyostelium, i.he only other protozoan MI1 sequenced to date, contains 2,116 amino acids, thus, making it 40% longer than Acanthamoeba MI1 [80]. The size difference is primarily in the tail domain. Even though the tails of these two nonmuscle myosins form a-helical coiled-coil rods typlcal of myosin 11, the primary sequences do not align with each other or with muscle myosin tail domains [28, 801. Clearly, the rodforming capability of these tails has been conserved rather than any specific amino acid sequence [28]. Two Acanthamoeba myosin I heavy chain genes (MIB, MIC) have been sequenced, but more are likely to be found because three isoforms ofmyosin I protein have been purified [48] and Southern blot analysis suggests that there may be up to six isoforms of myosin I genes [37]. Myosin IC, originally designated MIB, was the 1st myosin I gene from any organism to be completely sequenced [35]. It encodes a protein of 1,168 amino acids that contains 23 introns and corresponds to myosin heavy chain isoform IC [12]. Myosin IB, originally designated MIL, encodes a protein of 1,147 amino acids that contains 17 introns [37] and corresponds to myosin heavy chain isoform IB [l I]. At present, only partial amino acid sequences are available for MIA, the 3rd MI isoform. Both MIC and MIB code for conventional myosin-like globular heads and unconventional tails that do not form a-helical coiled coils or myosin filaments. The globular head domain amino acid sequences deduced from the genes for MIC and MIB (-670 N-terminal amino acids) are 72% similar to each other (Table 2). The 1st -240 amino acids of the tail domains are 56% similar and, thus, the tails are much less alike than the heads. The MIC and MIB diverge even more extensively from each other in the C-terminal 240 amino acids of the tail domain. This region begins in MIB with a unique stretch of 187 amino acids that is rich in glycine, proline and alanine (GPA-rich) and then terminates in a 53amino acid sequence that also is found in MIC, but at a different location. The C-terminal 240-amino acid portion of the MIC tail begins with a unique 56-,amino acid GPA-rich sequence, followed with the 53-amino acid conserved sequence found at the MIB C-terminus, and terminates with a unique 135-amino acid GPA-rich sequence [37]. The amino acid sequences of the MIC and MIB head region also are -55% similar to residues 15-705 of a bovine myosin 1 heavy chain [37] and -55% similar to residues 100-770 of rat and nematode muscle myosins [40]. A 185-amino acid subset of the 1st 240 amino acids of tail sequence from Acanthamoeba MIB and MIC is 46% similar to 185 amino acids near the C-terminal end of the bovine IMI heavy chain, but there are no other similarities between the bovine and amoeba tail sequences. A recently sequenced myosin I heavy chain gene from Dictyostelium [36] codes for a protem remarkably similar to Acanthamoeba MIB and MIC. The closest similarity is to MIB; the globular heads are 80% similar and the unconventional tail domains (excluding the GPA-rich domains) are 70% similar. The tail of the Dictyostelium myosin, like MIB, contains a single GPA-rich region. This region !contains 131 amino acids and is similar in several respects to the 186-amino acid GPA-rich region of MIB, but the two sequences could not be aligned in any single, unique way [36]. Jung et al. [36] suggest that the unusual amino acid composition and the net positive charge in the GPArich regions may be conserved in the MI heavy chain tails of

-

-

-

21s

EYERS ET AL-GENES OF ACANTHAMOEBA

Table 3. Mitochondria1genome size in Acantharnoeba calculated by summation of restriction fragments.*

I

2 3 4 5 6 7 8 9 10 11 I2 13 14 IS 16 17

No. enSpecies

1. A. astronyxis 2. A. castellanii 3. 4. 5. 6. 7. 8. A. polyphaga 9. 10.

11. 12. 13. 14. 15. 16. 17.

A. comandoni A. culbertsoni

A. grifini A. palestinensis A. rhysodes A . species

Overall average

Strainh

Ray & Hayes Boyce Chang Ma Castellani Neff Lewin Nagington Jones (Garcia) Wang Page 23 Comandon A- 1 Griffin Reich Sin& HOV-6

kbp

44.3 45.1 44.9 41.8 42.4 41.6 43.2 43.6 41.5 39.9 43.3 40.7 44.3 39.4 39.2 39.8 48.8 42.5

zymes Std. dev. used

0.5 1.1 0.3 1.1 0.8 1.0 0.7 0.9 0.8 1.2 1.5 1.4 0.9 0.4 0.6 1.2 0.7 2.4

7 6 5 5 7 10 6 4 7 4 4 5 4 4 4 10 4

1 2 3 4

5 6 7 8 9 10 I1 12 I3 14 IS 16 I7

J

Sizes are based on measurements of five gels for each enzyme. Strains are identified in [lo]. Strains 15 and 16 also have been classified as A. castellanii [lo].

Acanthamoeba and Dictyostelium rather than any specific amino acid sequence. It is significant that the coding sequences of amoeba MIB and slime mold myosin I are more alike than the coding sequences for the two amoeba myosin I isoforms. It will be interesting to see whether the functional roles of the three proteins sort out in a similar way. The evolutionary affinities of Acanthamoeba myosins with those of higher forms also are evident in the location of introns. For example, nine of the 16 introns that are found in the globular head region of MIC occur at the same locations as in rat skeletal myosin. Introns found in the unc 54 muscle myosin gene of the nematode and in MI myosin of Dictyostelium also occur at intron locations comparable to those in one or the other of the Acanthamoeba myosin I genes [35]. Calmodulin. The calcium-binding regulatory protein calmodulin has a relatively conserved primary structure of 148 amino acids. Acanthamoeba calmodulin has been partially sequenced [85]. A putative sequence positioning 130 amino acids has been obtained by direct determination and by deduction from comparisons of amino acid compositions of its tryptic peptides with the corresponding peptides of the bovine polypeptide. The amoeba and bovine amino acid sequences are -85% homologous [85]. In comparison, the calmodulin sequences of Tetrahymena pyriformis [83], Dictyostelium discoideum [5 I], and Trypanosoma brucei gambiense [76] all are 89% homologous with the bovine sequence.

-

MITOCHONDRIAL GENOME Size and copy number. The mitochondria1 DNA (mtDNA) of Acanthamoeba consists of circular molecules. Estimates of genome size based on the summation of restriction fragment sizes (Table 3) and measurements of circular molecules by electron microscopy [ 101give an average of 42.5 kbp for 17 strains representing seven to nine different species. The mitochondria1 DNA of Acanthamoeba tends to be in the midrange of sizes for other protozoans as indicated by the following examples: 5058 kbp for 7 strains of vahlkampfid amoebas [52]; 20-39 kbp for kinetoplast maxicircles of various trypanosomatid species [67]; 45-64 kbp for 6 species of Tetrahymena [53]; and 43 kbp

1 1

Fig. 1. Agarose gel electrophoreticrestriction fragment patterns for mtDNA from 17 strains of Acantharnoeba. (a) Cla I digests. (b) EcoR I digests. Fragment sizes represented are average values for five replicas. Conditions for electrophoresis are described in [lo]. Numbers above lanes refer to the strains listed in Table 3. The Chang strain used here was ATCC 30898 and the Boyce strain was obtained from G. S. Visvesvara. Both differ from those previously used [lo].

for Paramecium aurelia [2412. The mtDNA copy number has been estimated at -3,300 for a log phase amoeba of the Neff strain [ 141. Similar calculations for Naegleria estimate -400 per amoeba, but the total DNA content ofthis organism is much lower than for Acanthamoeba [ 141. DNA sequence diversity. At this time, very little is known about the gene complement or details of mtDNA nucleotide sequences in Acanthamoeba. However, restriction enzyme digests reveal extensive interstrain mtDNA sequence diversity. This variability was originally explored as a possible basis for measuring genetic distances within the genus and, especially, for helping to determine whether pathogenic isolates would group into clusters of closely related strains. The rationale for this approach is based on the successful use of mtDNA variability to study phylogenetic relationships in a variety of other organisms including Tetrahymena [4, 45, 531. A relatively high frequency of mtDNA restiction fragment Kbp determined from length: pm

x

3.08

=

kbp.

22s

J. PROTOZOOL., VOL. 37, NO. 4, JULY-AUGUST ECORI 0

BclI

0

BglII

0

1990

htI

I

Boyce

P

TPPTT TPPTtPP P

P P P ? T 'A PPWP

Chang

PPI T P V T TPPTtPP P Fm i

P P P ? T W PPWP

Neff

Kilobase pairs Fig. 2. Restriction site maps for the Boyce, Chang, Neff, and Ray & Hayes (formerly Ray [lo]) strains. Two restriction sites in Chang that are not in Boyce are indicated by a shaded bar. The 3rd polymorphic site mentioned in the text is revealed by Sul I (not illustrated). A map for Neff has been obtained previously by Bohnert and Heermann (see [14]). length polymorphisms [RFLP] has been observed in both intraand interspecific acanthamoeba comparisons [ 10, 171. We found that interstrain nucleotide sequence differences ranged from 413% and, thus, were comparable to differences among sibling species of ciliates [lo]. The interspecific variation in Acanthamoeba sequences was as large as the intraspecies variation [ 101. With the addition of new data collected since our initial observations, RFLP for 17 strains of Acantharnoebu representing seven to nine species are available (Fig. 1). In this sample, we have observed 16 different fragment length phenotypes. Where available, restriction site maps reflect the variation in RFLP patterns (Fig. 2). The mtDNAs of two nonpathogenic strains that have been classified as different species, Lewin (#7, A . castellanii) and Nagington (#8, A . polyphaga), are identical when examined with five different enzymes (Fig. 1). Two other strains, one isolated from a human bone infection (#2, Boyce) and one pathogenic to mice (#3, Chang), differ at only 3 restnction sites out of 44 6-base sites examined (Fig. 2). (These two strains originally appeared to be identical to each other and to three additional pathogenic strains [ 101, but this apparently was an error based on cross-contaminated cultures.) Two strains, one pathogenic (#4, Ma) and one thought to be nonpathogenic ( # 5 , Castellani), have very similar restriction fragment length profiles with several enzymes (e.g. Eco R1) and very different profiles with other enzymes (e.g. Cla I) (Fig. 2). Recently, T. Endo and associates (pers. commun. and abstr. of the Vth International Conference on the Biology and Pathogenicity of FreeLiving Amoebae) have found mtDNA RFLP corresponding to both of these strains in eye infections or contact lens containers in Japan. Milligan & Band [52] have used restriction digests of mtDNA

and an analytical approach similar to ours to investigate the relatedness of Nueglerzu and other vahlkampfid amoebas. They have found interstrain divergence of 3-14%, very similar to our results, but the intraspecific variation of 3-7% is significantly less than the interspecific variation of 12-149'0. This seems to indicate that species can be identified more reliably in Naegleria than in Acunthamoeba. The availability of a larger sample of mitochondria1 RFLP should help significantly, however, with the problem of classification of' Acanthamoeba. CONCLUSIONS The primary sequences of nuclear DNA, RNA and proteins that are available for Acunthamoeba all indicate that the genes of this amoeba are similar to those found in other organisms. Actin has the most highly conserved amino acid sequence among the acanthamoeba proteins that have been studied. Calmodulin also is highly conserved, but the amino acid sequence is not complete. The overall profilin arid myosin amino acid sequences have diverged significantly more from comparable sequences in other organisms, but in both cases sequences are more conserved in the N-terminal halves of the molecules. Data available to date support previous evidence, based on rRNA sequences, that Acanthamoeba genes are at leaijt as closely related to those of higher organisms as they are to those of other protozoans [68]. Relatively little information is available, however, about intraand interspecific variation in gene nucleotide sequences in most genera of protozoans. Acunthamoeba should be an excellent genus in which to explore this variation. The actin and three myosin genes all have introns. Although there is no evidence yet for conservation of overall intron sequences, the introdexon splice junction sequences conform to

EYERS ET AL-GENES OF ACANTHAMOEBA

the [GT . . . AG] consensus patterns for other organisms and many of the amoeba myosin introns are positioned at sites where introns occur in other eukaryotes [28, 35, 37, 541. T h e single intron in acanthamoeba actin I occurs at a unique position. A great deal is known about the promoter region and regulation of ribosomal R N A transcription by R N A polymerase I [5-8, 32, 41-43]. T h e challenge now is to obtain comparable information about transcription mediated by R N A polymerases I1 and 111. Sequences bearing some resemblance to T A T A or CCAAT boxes have been found i n the 5’ flanking regions of the actin and two myosin genes. Possible polyadenylation sites also have been found. A t present, the very small set of genes that have been sequenced makes it difficult to identify the important regulatory sites. A much larger set ofgenes needs to be examined. In addition, this approach needs t o be complemented by development of in vitro transcription systems for study of interactions between genes and regulatory factors. Further study of Acanthamoeba genes would be enhanced considerably by the development of vectors that could be used for transformation. If this organism is polyploid, it will be necessary to devise special strategies for in vivo studies of gene modifications. O n e promising approach has recently been used for studies on the effects o f mutated genes in the polygenomic macronucleus of Tetrahymena [84]. Alterations of telomere sequences and the biological consequences of these changes were studied following microinjection of mutated telomerase genes into the macronucleus. T h e mutant genes were carried on a plasmid vector that replicated at > lo3copies per cell and, thus, enabled them to compete successfully with the resident wildtype telomerase genes. With an appropriate vector, a similar strategy might be used for studying selected genes in Acanthamoeba. T h e use o f antisense oligonucleotides to inactivate specific gene products is another strategy that may prove useful and should be explored. The mitochondrial genome ofAcanthamoeba deserves further study. Recent comparisons of mitochondrial nucleotide sequences in Tetrahymena illustrate their promise for protozoan phylogenetic studies [53]. This is especially true for the most closely related species. T h e R F L P data available for Acanthamoeba may be useful both for clinical identification of amoebas and for phylogenetic comparisons. Nucleotide sequences of selected genes would be more informative, however, in both cases. A t present, the organization of genes within the mitochondrial genome and the sequences of individual genes are almost totally unknown. These need to be examined. Variations in nuclear gene D N A sequences among different strains of Acanthamoeba may be very useful for the development of oligonucleotide probes that can be used in clinical situations to identify isolates. W e have obtained very promising results from studies o n variation in 18s r R N A sequences among six strains of Acanthamoeba (Gast & Byers, unpubl. results). Although rRNA genes are especially suited for clinical applications, variation in other conserved genes such as actin might also be useful in this regard and should be investigated. ACKNOWLEDGMENTS The authors wish to thank Paul A. Fuerst for extensive help with sequence comparisons a n d the manuscript reviewers for a number of important suggestions. Some of the unpublished work from the authors’ laboratory was supported by grant R03EY08237 awarded to T.J.B. by the National Eye Institute. LITERATURE CITED 1. Ampe, C., Markey, F., Lindberg, U. & Vandekerckhove, J. 1988.

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Received 1-30-90; accepted 4- 12-90

J. Protosod., 37(4), 1990, pp. 294-333 0 1990 by the Society of Protozoologists

Epidemiology of Free-Living Ameba Infections I GOVINDA S. VISVESVARA and JEANETTE K. STEHR-GREEN Division of Parasitic Diseases and Division of HIV/AlDS. Centerfor Infectious Diseases, Centersfor Disease Control, Public Health Service, U S . Department of Health and Human Services, 1600 Ctifton Road, Atlanta, Georgia 30333 ABSTRACT. Small free-living amebas belonging to the genera Acanthamoeba and Naegleria occur world-wide. They have been isolated from a variety of habitats including fresh water, thermal discharges of power plants, soil, sewage and also from the nose and throats of patients with respiratory illness as well as healthy persons. Although the true incidence of human infections with these amebas is not known, it is believed that as many as 200 cases of central nervous system infections due to these amebas have occurred worldwide. A majority (144) of these cases have been due to Naegleriufowleri which causes an acute, fulminating disease, primary amebic meningoencephalitis. The remaining 56 cases have been reported as due either to Acanthamoeba or some other free-living ameba which causes a subacute and/or chronic infection called granulomatous amebic encephalitis (CAE). Acanthamoeba, in addition to causing CAE, also causes nonfatal, but nevertheless painful, vision-threatening infections of the human cornea, Acantharnoeba keratitis. Infections due to Acanthamoeba have also been reported in a variety of animals. These observations, together with the fact that Acanthamoeba spp., Naegleria fowleri, and Hartmannella sp. can harbor pathogenic microorganisms such as Legionella and or mycohacteria indicate the public health importance of these amebas. Key words. Acanthamoeba, Acanthamoeba keratitis, acquired immunodeficiency syndrome, contact lens, granulomatous amebic encephalitis, Naegleria, primary amebic meningoencephalitis.

S

MALL free-living amebas belonging to the genera Acanthamoeba and Naegleria occur world-wide. They have been isolated from a variety of habitats. Naegleria spp., e g , have been isolated from fresh water ponds and lakes, domestic water supply, thermal discharges of power plants, hot springs and spas, swimming pools, hydrotherapy pools, remedial pools, aquaria, soil, sewage and even nasal passages of healthy children [22, 301. Acanthamoeba spp. also have been isolated from soil, fresh water, bottled mineral water, mushrooms and vegetables, brackish and sea water as well as ocean sediments, cooling towers of electric and nuclear power plants, physiotherapy pools and medicinal pools, swimming pools, heating, ventillating and air conditioning units, dialysis units, gastrointestinal washings, dental units, dust in air, sewage, and bacterial, fungal, and mammalian cell cultures. In humans they have been found in the nose and throat of patients with respiratory illness as well as from healthy persons, and in bronchial secretions, ear discharge, and stool I Based in part on a keynote address presented by G. S. Visvesvara at the Vth International Conference on the Biology and Pathogenicity of Free-Living Amoebae, Brussells, Belgium, August 7-1 1, 1989.

samples of patients with diarrhea. More recently they have been isolated from hot tubs, contact lens-care solutions, and intrauterine contraceptive devices [2, 8, 12, 22, 45, 58, 64, 65, 751. These amebas have also been implicated in humidifier fever, and an allergic hypersensitivity pneumonitis illness. Although the true incidence of human infections with these amebas is not known, it is believed that as many as 200 cases of central nervous system (CNS) infections due to these amebas have occurred world-wide. A majority of these cases (144) have been due t o Naegleria fowleri. The remaining 56 cases have been reported as due either to Acanthamoeba or some other free-living ameba (Table 1). Acanthamoeba, in addition to causing CNS disease, is also known to cause nonfatal, but nevertheless painful, infections of the human cornea, Acanthamoeba keratitis. Infections due to N. fowleri. At this time six species of Naegleria have been described in the literature. They are N . andersoni, N. austratiensis, N . fowleri, N. gruberi, N. jadini, and N . lovaniensis. Although both N . australiensis and N . fowleri are known to be thermophilic and pathogenic to mice [22, 30, 451, only N . fowleri is thought to cause disease in humans. Most of the human isolates have been shown to be N. fowleri by various