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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Parasitol Int. Author manuscript; available in PMC Jun 7, 2011.
Published in final edited form as:
PMCID: PMC3109654

Mitochondrial-type hsp70 genes of the amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and two microsporidians[star]


Genes encoding putative mitochondrial-type heat shock protein 70 (mit-hsp70) were isolated and sequenced from amitochondriate protists, Giardia intestinalis, Entamoeba histolytica, and two microsporidians, Encephalitozoon hellem and Glugea plecoglossi. The deduced mit-hsp70 sequences were analyzed by sequence alignments and phylogenetic reconstructions. The mit-hsp70 sequence of these four amitochondriate protists were divergent from other mit-hsp70 sequences of mitochondriate eukaryotes. However, all of these sequences were clearly located within a eukaryotic mitochondrial clade in the tree including various type hsp70 sequences, supporting the emerging notion that none of these amitochondriate lineages are primitively amitochodrial, but lost their mitochondria secondarily in their evolutionary past.

Keywords: Hsp70, Amitochondriate protists, Phylogeny, Giardia, Entamoeba, Microsporidia

1. Introduction

Evidence for secondary loss of mitochondrial function in four major amitochondriate protist lineages, diplomonads, parabasalids, entamoebidae and microsporidia has been obtained in recent years by the detection of mitochondrion-related chaperon genes that are coded in the nuclear DNA (for reviews see refs. [15]). These include chaperonin (cpn60) in Entamoeba histolytica (entamoebidae), Trichomonas vaginalis (parabasalids), and Giardia intestinalis (diplomonads) and mitochondrial-type heat shock protein 70 (mit-hsp70) in T. vaginalis and three microsporidians, Nosema locustae, Vairimorpha necatrix, and Encephalitozoon cunuculi. In addition to these chaperon genes, two other mitochondrion-related genes have also been reported recently. One is encoding ADP–ATP carrier proteins in T. vaginalis [6] and the other is iscS in T. vaginalis and G. intestinalis [7].

Phylogenetic reconstruction of each of these proteins demonstrated without exception that sequences from amitochondriate lineages are clearly located within the corresponding eukaryotic mitochondrial clades. Although the functions of mitochondrial-type chaperons in amitochondriate protists still have to be established, cpn60 and mit-hsp70 were shown to be localized to the hydrogenosome in T. vaginalis [8], while in E. histolytica, cpn60 is localized in a recently identified double membrane bounded organelle, called mitosome [9] or crypton [10]. This evidence suggests a common evolutionary origin of these organelles with the mitochondrion [1113], although this view is not universally accepted [14,15].

Since until very recently mit-hsp70 sequences were reported only from two amitochondriate lineages, parabasalids and microsporidia, we isolated mit-hsp70 genes from the other amitochondriate lineages, diplomonads and entamoebidae, in order to analyze their evolutionary relationships. Here we report complete sequences of the genes putatively encoding mit-hsp70 from G. intestinalis, E. histolytica, and a microsporidian, Encephalitozoon hellem, and a partial one containing most of the mit-hsp70 open reading frame (ORF) from an another microsporidian, Glugea plecoglossi. While this work was under way, a mit-hsp70 sequence became available from E. histolytica [16]. Our phylogenetic analysis demonstrated that all mit-hsp70 sequences from these organisms are located within a eukaryotic mitochondrial clade, supporting the emerging notion that none of these amitochondriate lineages are primitively amitochodrial, but lost their mitochondria secondarily in their evolutionary past.

2. Materials and methods

2.1. Cloning and sequence analysis

Giardia intestinalis, strain WB, clone 6 (ATCC 30957), Entamoeba histolytica, strain HM-1:IMSS (ATCC 30459), Encephalitozoon hellem (ATCC 50451) isolated from the urine of an adult patient with AIDS (gift of Dr E.S. Didier, LA, NY), and Glugea plecoglossi collected from infected Ayu fishes in the Biwako Lake in Japan were used. Genomic DNA was extracted from G. intestinalis and E. histolytica with the use of a blood and culture DNA kit (QIAGEN, Chatsworth, CA, USA) according to the manufacturer's protocol. Extraction of the two microsporidian genomic DNA was described previously; E. hellem [17] and G. plecoglossi [18]. Using degenerate primers synthesized based on the conserved amino acid residues in the alignment of hsp70 sequences and unique primers synthesized based on the partial sequencing results, major parts of the mit-hsp70 genes were amplified by polymerase chain reaction (PCR), and the corresponding products were cloned, and sequenced. The amino acid and the corresponding nucleotide sequences used for synthesizing the degenerate primers are: G. intestinalis, GIDLGTTN (forward) (5′-GGNATHGAYYTNGGNACNACNAA-3′), and F(D/E)(I/V)DANG (reverse) (5′-CCRTTNGCRTCNAYNTCRAA-3′); E. histolytica, GIGLGTT (forward) (5′-GGN ATHGGNYTNGGNACNAC-3′), V(F/Y)DLGGGT (forward) (5′-GTNTWYGAYYTN GGNGGNGGNAC-3′), MTRMPLVQ (reverse) (5′-TGNACNARNGGCATNCKNGTCAT-3′), and EVTFDIDA (reverse) (5′-GCRTCDATRTCRAANGTNACYTC-3′); E. hellem, V(F/Y)DLGGGT (forward) and MTRMPLVQ (reverse); G. plecoglossi, GIGLGTT (forward), V(F/Y)DLGGGT (forward), MTRMPLVQ (reverse), and FEA(A/S)DANG(M/I) (reverse) (5′-ATNCCRTTNGCRTCNSHNGCYTCRAA-3′). An uneven PCR method was used to obtain N- or C-terminal portion of each gene [19]. Specific primers used for the uneven PCR were based on the sequence information already obtained, while arbitrary primers were synthesized based on the sequences described in Chen and Wu [19]. Plasmid vectors, pT7 (Novergen, Madison, WI, USA) and pCR2.1 (Invitrogen, Carlsbad, CA, USA), were used for cloning the PCR amplified bands. A ZAP express genomic DNA library of E. hellem [17] was also screened with a PCR based fragment as a probe to isolate the C-terminal portion of the gene. Nucleotide sequences were determined on both strands with the use of an automated DNA sequencer (ABI model 310 Genetic Analyzer, Perkin-Elmer Cetus, Norwalk, CT, USA).

In addition, G. intestinalis cDNA encoding mit-hsp70 was cloned and sequenced. Total RNA of G. intestinalis was extracted with the use of Tri Reagent LS-RNA/DNA/Protein isolation reagent (Molecular Research Center, Cincinnati, OH, USA), according to the manufacturer's instructions. Total RNA was treated with Super-Script™ II Rnase HReverse Transcriptase (RT) (GIBCO BRL, Life Technologies, Rockville, MD), and the resultant RNA:cDNA duplex was used as template for RT-PCR analysis. A set of primers that covers an entire part of the ORF was synthesized based on the sequence information of the genomic DNA. The amplified 2.0 kb fragment was cloned and sequenced as described above.

2.2. Sequence alignment and phylogenetic reconstruction

The deduced mit-hsp70 sequences of the four amitochondriate protists were aligned with all hsp70 homologs currently available in the database by manual editing. Based on the alignment, 375 unambiguously aligned positions were used for phylogenetic analyses. Selected positions are (G. intestinalis numbering): 17–22, 26–27, 29–47, 57–58, 68–83, 117–146, 150–189, 208–222, 226–254, 277–280, 282–286, 322–352, 356–408, 413–418, 420–525 and 527–537. The maximum likelihood method of protein phylogeny was applied for the data set by using PROTML program in MOLPHY version 2.3 [20], since the method was shown to be the most robust against the violation of rate constancy [21,22], and since evolutionary rates for different lineages in the present data set showed extreme variation. The JTT-F model was used for amino acid replacement process. Since the number of operational taxonomic units to be analyzed was large, topology search for obtaining the best tree was performed by the use of the local rearrangement (−R) and the quick stepwise OTU addition procedure (−q −n2000) options of the PROTML program. A neighbor-joining tree was used as an initial tree for the −R option analysis. Alternative tree topologies produced by the search step were used for the RELL bootstrap analysis [23].

3. Results and discussion

3.1. Mit-hsp70 sequences of four amitochondriate protists

Putative mit-hsp70 ORFs were 1923 bp (640 aa), 1797 bp (598 aa., and 1782 bp (593 aa), respectively for G. intestinalis, E. histolytica, and E. hellem. A truncated reading frame of 1542 bp (513 aa) probably lacking only a few N-terminal residues, was obtained for G. plecoglossi. All of these sequences were not interrupted by intronlike sequences. A cDNA sequence encoding G. intestinalis mit-hsp70 ORF was the same as the genomic DNA sequence with the exception of a synonymous substitution on the third codon position. No introns were seen in the ORF, being congruent with the finding that none of the reported G. intestinalis sequences have introns [24] A BLAST search [25] revealed high similarity of the deduced amino acid sequences to mit-hsp70 and bacterial-type hsp70 (dnaK) sequences, identifying these as mit-hsp70 homologs.

The sequence of the putative ORF of the E. histolytica (strain HM-1:IMSS. mit-hsp70 was 98.5% identical to a mit-hsp70 ORF of the same strain, recently reported [16]. The two sequences differed at 10 substitutions and three insertions, resulting in the putative translations in eight replacements and an insertion. The marked difference indicated the presence of different copies. A BLAST search of the genome sequencing project database of the same E. histolytica isolate (http://www.tigr.org/tdb/edb2/enta/htmls/. demonstrated that at least two different mit-hsp70 sequences exist in the database and that both the above two ORFs are likely to be present in the database. However, these two sequences may represent allelic heterogeneity of the same locus, as already mentioned by Bakatselou et al.[16], because 5′- and 3′-non-coding regions of these two sequences were very similar with each other, and the level of conservation seems to be unusual for different copy genes. Approximately 80 bp upstream of the initiation codon of the E. hellem mit-hsp70 gene, a putative tryptophanyl-tRNA synthetase was coded on the opposite strand. A comparable gene organization is seen in another microsporidian, Encephalitozoon cuniculi [26].

3.2. Comparison of hsp70 sequences

Deduced mit-hsp70 amino acid sequences obtained in this study were aligned with other mithsp70 sequences and bacterial-type, chloroplast-type, cytosolic-type and endoplasmic reticulum (ER)-type hsp70 sequences (data not shown, the alignment is available from T.H. upon request). On the N-terminal portion, short extensions of 9, 12 and 16 residues, were found in G. intestinalis, E. histolytica, and E. hellem sequences, respectively, just 5′ of the highly conserved motif GIDLGTT. No similarity was detected among these three extension sequences. However, the E. hellem extension was very similar to that seen in E. cuniculi [26], and the E. histolytica extension was similar to the N-terminal sequences of some T. aginalis hydrogenosomal proteins, as also noted by Bakatselou et al. [16]. Two mit-hsp70 signatures recognized at first by Germot et al. [27] were present in all the sequences reported here (Fig. 1). The first motif (GDAWV) is shared by mitochondrial and proteobacterial hsp70s, and the second motif (YSPSQI) is generally conserved in mit- and α-proteobacterial-hsp70s. Several conservative amino acid replacements were found in the corresponding motifs of the sequences reported here, and the first motif of G. plecoglossi and both motifs of G. intestinalis were quite divergent from the consensus.

Fig. 1
Two signature motifs found in mit-hsp70 sequences of eukaryotes including amitochondriate protists and in homologous proteobacterial sequences. Amino acid residues within the originally defined motifs[27], GDAWV and YSPSQI, are shown in bold face characters. ...

The mit-hsp70 sequences of G. intestinalis and G. plecoglossi are most divergent of all the hsp70 homologs currently available. In addition, the G. intestinalis sequence contained several short insertions that are not found in other mit-hsp70s and other type hsp70 sequences, while the G. plecoglossi sequence had a long deletion on the C-terminal portion. The E. histolytica mit-hsp70 sequence was also divergent, but no large insertion/deletion was found in the sequence. The E. hellem mit-hsp70 sequence showed high similarity with the E. cuniculi homolog, and neither the Encephalitozoon homolog was as divergent as other mit-hsp70 sequences of microsporidia.

After elimination of the positions that could not be aligned reliably, 375 positions were used for the phylogenetic analysis. A difference matrix of these positions for 11 sequences is shown in Table 1. The pairwise differences revealed that mit-hsp70 sequence of amitochondriate protists, G. intestinalis, E. histolytica and microsporidia are highly divergent, in agreement with the alignment features described above. The differences between Homo sapiens and amitochondriate protists excluding T. vaginalis ranged from 34.1 to 50.7%. These are larger than the differences between H. sapiens and proteobacteria.

Table 1
Percentage pairwise amino acid difference between mitochondrial-type and proteobacterial-type hsp70s of various species (375 shared residues)

3.3. Phylogenetic reconstruction analysis of mithsp70 sequences

Preliminary phylogenetic analysis including 92 hsp70 homologs of various type clearly demonstrated that the mit-hsp70 sequences of amitochondriate protists reported here are located within the clade comprising of mit-hsp70 sequences of other eukaryotes and that the sister group of the mit-hsp70 clade is shared by α-proteobacteria (data not shown). Based on the preliminary analysis, we selected 24 hsp70 sequences for a more detailed phylogenetic analysis. This data set included most of the mit-hsp70 sequences except for very similar ones and six proteobacterial hsp70s.

Fig. 2 demonstrates the best tree of the maximum likelihood analysis using the PROTML program [20]. The tree clearly demonstrates that all the mit-hsp70 sequences from amitochondriate protists are monophyletic with other eukaryotic mit-hsp70 sequences with a high bootstrap proportion value (97%) confirming the mitochondrial-type features of these sequences. Within the mit-hsp70 clade, monophyly of animalia, fungi, microsporidia, plantae, and of euglenozoa are clearly reconstructed. The monophyletic origin of and the branching order within microsporidia are also clearly resolved, but the close relationship of N. locustae with G. plecoglossi is incongruent with the small subunit (SSU) rRNA phylogeny, in which Nosema and Varimorpha are the closest relatives [28,29]. However, since no SSU rRNA data is available from N. locustae and since monophyly of the genus Nosema has yet been established in the SSU rRNA phylogeny [28,29], we cannot simply compare the two trees.

Fig. 2
Phylogenetic tree of mit-hsp70 homologs using proteobacterial hsp70s as outgroups. The best tree obtained by the maximum likelihood analysis of protein phylogeny using the PROTML program is shown. The horizontal length of each branch is proportional to ...

The monophyly of three amitochondriate protists lineages, G. intestinalis, E. histolytica and microsporidia, is clearly supported by the high bootstrap proportion value (96%) in disagreement with the accepted eukaryotic phylogeny [30]. Since branch lengths of the tree leading to these amitochondriate protists are very long compared to those leading to the other eukaryotes, the positions of these three lineages are possibly affected by a long branch attraction of the phylogenetic reconstruction analysis [31]. Especially for G. plecoglossi and G. intestinalis, the lengths are extremely long, being comparable with the highly divergent sequence features of these organisms. The long branch lengths of these amitochondriate lineages suggest that the functional constraints have been relaxed. The function of mit-hsp70 might have been changed during the evolution of these organisms.


We thank Dr H.S. Zhang for extracting genomic DNA of Encephalitozoon hellem and for preparing its genomic DNA library, Dr G. Wu, Dr M. Hasegawa and Dr T. Yano for helpful discussions, and A. Deguchi and S. Kikuchi for technical assistance. This work was carried out under the ISM Co-operative Research Program (00ISM• CRP-2042, and 01ISM•CRP-2038) and was supported by grants from the Japanese Society for the Promotion of Science (10044219 and 13640709) to T.H. Work carried out at the Rockefeller University in New York was supported by the USPHS National Institutes of Health Grant (AI11942) to M.M. Work carried out at the Albert Einstein College of Medicine in New York was supported by the USPHS National Institutes of Health Grant (AI31788) to L.M.W. Visits of T.H. to the New York laboratories and of L.B.S. to the Hayama laboratory were supported by the US-Japan Co-operative Research Project by National Science Foundation and Japanese Society for the Promotion of Science (INT-9726707). Preliminary sequence data for E. histolytica is deposited regularly into the GSS division of Gen-Bank. The sequencing effort is part of the International Entamoeba Genome Sequencing Project and is supported by an award from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.


mitochondrial-type heat shock protein 70
open reading frame
polymerase chain reaction
reverse transcriptase
small subunit


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