Evidence for loss of mitochondria in Microsporidia from a mitochondrial-type HSP70 in Nosema locustae1

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Abstract

In molecular phylogenies based on ribosomal RNA, three amitochondriate protist lineages, Microsporidia, Metamonada (including diplomonads) and Parabasala (including trichomonads), are the earliest offshoots of the eukaryotic tree. As an explanation for the lack of mitochondria in these organisms, the hypothesis that they have diverged before the mitochondrial endosymbiosis is preferred to the less parsimonious hypothesis of several independent losses of the organelle. Nevertheless, if they had descended from mitochondrion-containing ancestors, it may be possible to find in their nuclear DNA genes that derive from the endosymbiont which gave rise to mitochondria. Based on similar evidence, secondary losses of mitochondria have recently been suggested for Entamoeba histolytica and for Trichomonas vaginalis. In this study, we have isolated a gene encoding a chaperone protein (HSP70, 70 kDa heat shock protein) from the microsporidian Nosema locustae. In phylogenetic trees, this HSP70 was located within a group of sequences that in other lineages is targetted to the mitochondrial compartment, itself included in the proteobacterial clade. In addition, the N. locustae protein contained the GDAW(V) motif shared by mitochondrial and proteobacterial sequences, with only one conservative substitution. Moreover, microsporidia, a phylum which was assumed to emerge close to the base of the eukaryotic tree, appears as the sister-group of fungi in the HSP70 phylogeny, in agreement with some ultrastructural characters and phylogenies based on α- and β-tubulins. Loss of mitochondria, now demonstrated for several amitochondriate groups, indicates that the common ancestor of all the extant eukaryotic species could have been a mitochondriate eukaryote.

Introduction

Microsporidia (phylum Microspora) [1]are obligate intracellular parasites which infect a wide diversity of hosts (900 known species), including fish, insects, mammals and some protozoa such as Apicomplexa and Ciliophora 2, 3. They are well-known for damage they cause to beekeeping (Nosema apis), fish farming (Glugea atherinae) and silkworm breeding (Nosema bombycis). They have been used as agents for biological control of insect pests, especially Nosema locustae against tropical grasshoppers [4]. They gained particular interest recently because they represent one of the most important groups of opportunistic parasites in AIDS patients [5]. Transmission of the parasite occurs by spores. Spores germinate in response to certain stimuli [6]and the sporoplasm contained in the spore is inoculated into a host cell where cycles of rapid divisions (merogony) occur and spores are formed (sporogony). Microsporidia do not possess mitochondria, peroxisomes or a typical Golgi apparatus, although an atypical Golgi apparatus has been described [7]and Golgi enzyme activities have been demonstrated [8]. Microsporidian genome sizes vary from 2.9 Mb in Encephalitozoon cuniculi [9]to approximately 19.5 Mb in Glugea atherinae [10]. They possess ribosomes with sedimentation coefficients identical to values characteristic of prokaryotes: 70S for the monosome, 50S and 30S for its subunits 11, 12. They also lack 5.8S rRNA; as in prokaryotes, they have a large subunit rRNA whose 5′ region corresponds to a part of the 5.8S rRNA [13]. These typical prokaryotic features have led Cavalier–Smith [14]to propose that Microsporidia were one of the earliest offshoots within the eukaryotic tree.

Eukaryotic trees based on small subunit rRNA [15]show microsporidia emerging very early in eukaryotic evolution together with diplomonads and trichomonads. The reliability of this result has been questioned, especially due to bias in G+C content [16]. Indeed, the relative order of emergence of the three taxa is sensitive to this bias [17]. However, phylogenies that are based only on transversions are less sensitive to bias in G+C content [18]and also support the early emergence of these three groups (Philippe, unpublished). Moreover, several phylogenies based on protein comparisons, such as isoleucyl tRNA synthetase [19], elongation factors EF-1α [20]and EF2 [21], also support this result. In contrast, phylogenies based on β-tubulin [22]and α-tubulin [23]provide a rather strong signal which locates microsporidia within the fungi.

The three earliest emerging groups in the eukaryotic phylogeny all lack mitochondria. This absence could be attributed to a primitive absence, to a secondary loss or to a conversion into another organelle. Since endosymbiotic events are followed by a massive transfer of genes from the symbiont genome to the host nucleus, relic genes could be retained even after the disappearance of the mitochondria. Such genes have been found in Entamoeba histolytica [24]and in Trichomonas vaginalis 25, 26, 27, 28providing strong evidence for loss of a typical mitochondrion or its conversion into an hydrogenosome, respectively.

Seventy kiloDaltons heat shock proteins (HSP70s) are good markers for putative endosymbioses. They are highly conserved and present in all organisms, Archaebacteria, Eubacteria and Eukaryota [29], since they participate in numerous cellular processes [30]. They stabilize newly made proteins and protect them from aggregation. They support the correct folding of mature proteins and are implicated in their translocation across membranes. In eukaryotes, they are present in most cellular compartments. They constitute a multigenic family, with HSP70s located in the cytosol, the endoplasmic reticulum and in organelles (mitochondria and chloroplasts), the last two being the result of a transfer from endosymbiont genes to the nucleus [31]. We have now found a mitochondrial-type HSP70 in N. locustae, which phylogenetic reconstructions place among the mitochondrial sequences, thus suggesting that mitochondrial endosymbiosis occurred before the emergence of Microsporidia.

Section snippets

DNA isolation

DNA was extracted from spores of a Nosema locustae strain (ATCC 30860), as described by Vossbrinck et al. [32]. Spores were isolated from cysts of infected grasshoppers, Melanoplus sanguinipes.

Cloning of the N. locustae HSP70 gene

We amplified two fragments of 400 and 1000 bp with degenerate primers corresponding to the HSP70 gene. These primers were based on highly conserved regions of the protein. PCR was carried out with sense primers A and M (5′-CCCGGGIATHGAYYTIGGNAC-3′ and 5′-CTCACCATCACAGATAGCTG-3′) corresponding to the amino

Isolation of the HSP70 gene

We obtained the entire sequence of an HSP70 gene from Nosema locustae encoding a protein of 622 residues. The HSP70 sequence is somewhat A+T rich (54.8%) as it has been shown for other Nosema genes 15, 19. The codon usage is of the same pattern as noted for isoleucyl tRNA synthetase of N. locustae [19]. We could exclude the possibility of contamination from host material because PCR amplification with universal primers did not amplify more than one other HSP70 (data not shown). Moreover,

Discussion

We have demonstrated, in the microsporidian N. locustae, the existence of a typical HSP70 gene that is homologous to genes encoding in other eukaryotes a mitochondrial protein. Phylogenetic analysis unambiguously placed N. locustae inside the mitochondrial group. In addition, this HSP70 displayed mitochondrial signatures only shared by α-Proteobacteria or by the whole group of Proteobacteria. Therefore, this HSP70 sequence probably had a mitochondrial origin in spite of the fact that N. locustae

Acknowledgements

This work was supported by a grant from the GREG (Groupement de Recherches et d'Études sur les Génomes, décision d'aide no. 94/125) and from the Réseau National de Biosystématique. AG was supported by a fellowship from the Ministère de l'Éducation Nationale, de l'Enseignement Supérieur, de la Recherche et de l'Insertion Professionnelle. We acknowledge the constructive criticisms of Dr Miklós Müller. We thank Dr Jacqueline Laurent for valuable suggestions and Dr Andrew Roger for reading the

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    Note: Nucleotide sequence data reported in this paper has been submitted to the GenBank™ data base under the accession number U97520.

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