1

NOTE 1 2

Mitosomes in Trophozoites and Cysts of the Reptilian Parasite 3

Entamoeba invadens 4 5

Maria A. Siegesmund1, Adrian B. Hehl2 and Mark van der Giezen1* 6 7

1 Centre for Eukaryotic Evolutionary Microbiology, Biosciences, College of Life & 8 Environmental Sciences, University of Exeter, UK. 9

2 Institute of Parasitology, University of Zürich, Switzerland. 10 11 12 13 14 15 *Corresponding author: 16 Dr. Mark van der Giezen 17 Centre for Eukaryotic Evolutionary Microbiology 18

Biosciences 19 College of Life & Environmental Sciences 20 University of Exeter 21 Stocker Road 22 Exeter EX4 4QD 23 UK 24 Tel.: +44 1392 723483 25 e-mail: m.vandergiezen@exeter.ac.uk 26

27 28

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.05172-11 EC Accepts, published online ahead of print on 30 September 2011

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29 Heat shock protein genes led to the discovery of mitosomes in Entamoeba histolytica but 30

mitosomes have not been described for any other Entamoeba species nor have they been 31

identified in the cyst stage. Here, we show that the distantly related reptilian pathogen 32

E. invadens contains mitosomes, in both trophozoites and cysts, suggesting all 33

Entamoeba species contain these organelles. 34

35

Mitochondria have played a crucial role during eukaryotic evolution. They enabled and 36

facilitated the development of multi-cellular life by relaxing the energetic constraints facing 37

prokaryotes (11). The discovery of genes encoding proteins that are normally targeted to 38

mitochondria in other eukaryotes (3) in the genome of Entamoeba histolytica, an organism 39

previously thought to represent an earlier phase of eukaryotic life, raised doubts about the 40

presumed premitochondrial status of this intestinal parasite. Antibodies raised against one of 41

these, the mitochondrial chaperonin Hsp60, clearly showed the presence of an organelle, 42

which was called mitosome (21) or crypton (13). When the mitochondrial-like leader 43

sequence of Hsp60 was removed, the protein accumulated in the cytosol, a phenotype that 44

could be reversed by replacing the presequence with a genuine mitochondrial targeting 45

sequence from another species suggesting that the discovered organelle was indeed 46

mitochondrial in nature (21). 47

Although mitosomes have been discovered in other former Archezoa (22, 25) the 48

function of these organelles is not obvious. Analyses of the genomes of these human 49

pathogens only suggested a handful of genes whose products are targeted to mitosomes. Most 50

of these are ‘structural’ in nature and encode heat shock proteins and metabolite or protein 51

importers. Only a few ‘functional’ enzymes have been discovered and those involved in iron-52

sulfur cluster assembly seem to be a common denominator for all mitosomes. Unexpectedly, 53

these proteins of clear mitochondrial ancestry have been replaced by lateral gene transfer 54

with a much simpler system in E. histolytica (1, 24). Whether these proteins are genuinely 55

mitosomal in this organism remains a matter of dispute (14, 16). The most thorough attempt 56

to understand mitosomal function in E. histolytica employed mass spectroscopy on Percoll 57

purified mitosomes (16). Frustratingly, two thirds of the 95 identified proteins were 58

hypothetical proteins. However, this study indicated that E. histolytica mitosomes are 59

involved in sulfate activation (16), a thus far unique mitosomal trait of the Entamoeba 60

organelles. 61

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There are two stages in the E. histolytica life cycle, a motile trophozoite stage found 62

inside the human host and a resistant infectious cyst which is excreted by infected 63

individuals. The factors controlling encystation and excystation in E. histolytica are poorly 64

understood and our lack of knowledge is further hampered by our inability to induce cyst 65

formation in vitro. As a consequence, nothing is known about what happens to mitosomes in 66

cysts. The reptilian pathogen E. invadens acts as a proxy for the study of cyst formation as it 67

is relatively straightforward to induce encystation in this Entamoeba species (18). As 68

mitosomes have never been identified in any other member of the genus Entamoeba and there 69

is no knowledge regarding the fate of mitosomes in cysts, we have studied the distribution of 70

mitosomes in the reptilian pathogen Entamoeba invadens. 71

One of us previously demonstrated that the mitochondrial-type Hsp70 (mHsp70) 72

chaperone is enriched in mitosomal fractions (20). In order to validate that work, which was 73

performed using a heterologous mHsp70 antibody (23), we obtained a homologous antibody 74

raised against recombinant E. histolytica mitosomal Hsp70. To maximize recombinant 75

protein production, codon usage was converted from E. histolytica to Escherichia coli using 76

JCat (7). Subsequently, a synthetic gene was constructed, which included a C-terminal 77

histidine tag for protein purification and the restriction site BamHI at both termini to enable 78

cloning into the BamHI site of the pET-3c expression vector. The poly-His tagged 79

recombinant protein was produced in Escherichia coli BL21(DE3)pLysY cells and purified 80

under non-denaturing conditions by immobilized metal ion affinity chromatography using Ni-81

NTA. Correct protein identity was verified by mass spectroscopy and this protein was 82

subsequently used for immunisation. E. histolytica HM-1:IMSS and E. invadens IP-1 were 83

grown using standard conditions. Cyst formation was induced according to established 84

protocols (18). For E. invadens trophozoite localization experiments, cells were washed in 85

PBS and fixed with 3 % formaldehyde in PBS for 45 minutes while mature cysts were fixed 86

using 3 % formaldehyde in PBS overnight at 4 °C. The dehydrated specimens were 87

rehydrated with PBS for 30 minutes, permeabilized with 0.2 % Triton X-100 in PBS for 20 88

minutes at room temperature and blocked for 2 hours with 2 % BSA in PBS. The cell 89

preparations were incubated with titrated E. histolytica Hsp60 (1:300) (a kind gift of Dr. C. 90

Graham Clark) and E. histolytica mHsp70 (1:100) (this study) antibodies in PBS with 2 % 91

BSA and 0.2 % Triton X-100 for 1 hour at room temperature in a humid chamber. Secondary 92

antibodies coupled to Alexa Fluor® 594 and 488 (Invitrogen), respectively, were used to 93

detect bound antibodies. Specimens were thoroughly washed in PBS with 0.5 % BSA and 94

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0.05 % Triton X-100 between incubations and finally embedded with Vectashield (Vector 95

Labs) or Glycergel (Dako) mounting medium. Nuclear DNA was detected with the 96

intercalating agent 4',6-diamidino-2-phenylindole (DAPI). Immunofluorescence image data 97

collection was performed on a Leica SP2 AOBS confocal laser-scanning microscope (Leica 98

Microsystems, Wetzlar, Germany) with an oil immersion objective (Leica, HCX PL APO 99

63x 1.4) and a pinhole setting of Airy 1 with twofold oversampling. Image stacks were 100

further processed using the Huygens deconvolution software package version 2.7 (Scientific 101

Volume Imaging, Hilversum, Netherlands). Three-dimensional reconstruction, volume 102

rendering and colocalization analysis were done with the Imaris software suite, version 7.2 103

(Bitplane, Zurich, Switzerland). 104

In order to understand the phylogenetic relationship of amoebozoan mHsp70, a 105

dataset of 39 protein sequences with 25 eukaryotic and 14 prokaryotic taxa was assembled. 106

Protein sequences were aligned using ClustalW in SeaView version 4.2.12. The dataset 107

contained 620 informative patterns from a total of 704 sites. Phylogenies were calculated 108

using the model-based maximum likelihood approach (ML) using PhyML (8) and the 109

Bayesian inference approach using MrBayes (17). For ML analyses, modelgenerator v.0.85 110

(10) suggested the model LG+G+F, with 8 rate categories and alpha shape parameter of 0.48 111

to fit the observed data best. Four Bayesian analyses were run using a mixed amino acid 112

model accommodating 4 rate+inv categories containing 4 chains each. One million 113

generations were calculated and trees sampled every 1,000 generations. The model stabilised 114

rapidly and 250 trees were discarded as burn-in. Mitochondrial targeting signals were 115

analysed using the localization prediction tools WoLF PSORT (9) and Mitoprot (4). 116

The presence of mitosomes in E. histolytica is well documented but there is no 117

information regarding the presence of these organelles for any other Entamoeba species. As 118

there is (partial) genome information available for several other Entamoeba species, we 119

decided to screen these genomes for the presence of Hsp60 and mHsp70. Putative Hsp60 and 120

mHsp70 sequences were identified in E. dispar and E. invadens but incomplete Hsp60 121

sequences lacking their N-termini could only be identified for E. terrapinae and E. 122

moshkovskii while no mHsp70 sequences could be identified with reasonable certainty in 123

these two species. As shown before (6), targeting signal prediction programmes have 124

difficulties identifying Entamoeba mitosomal presequences but alignment of the N-termini of 125

Hsp60 and mHsp70 clearly indicates the presence of presequences that are upstream of the 126

analogous prokaryotic N-terminus and which have been shown to be genuine targeting 127

signals in E. histolytica (Fig. 1). 128

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In order to confirm the mitochondrial nature of the identified putative mHsp70 we 129

conducted phylogenetic analyses including extended amoebozoan sampling (Fig. 2). Our 130

analyses clearly confirm the mitochondrial ancestry and monophyly of amoebozoan 131

mHsp70s. Although we correctly recovered the sister group relationship with the 132

opisthokonts, this node was only weakly supported. As shown previously (2), alpha-133

proteobacteria are basal to all eukaryotes in accordance with their supposed role as donor of 134

the mitochondrial endosymbiont. 135

To verify our in silico analyses, which clearly predict the Entamoeba mHsp70 to be 136

mitochondrial, we conducted laser-scanning confocal microscopy and three-dimensional 137

image rendering using the homologous Hsp60 and mHsp70 antisera on E. histolytica whole 138

cell preparations. The Hsp60 antibody localises in a discrete punctuate pattern and abundance 139

similar to previous reports (12, 16) and the homologous mHsp70 antiserum co-localises to the 140

same areas (Fig. 3A-D) confirming the earlier fractionation data (20). When these antisera 141

were used on the distantly related E. invadens, a similar localisation pattern was observed. 142

However, as the representative images in Figs. 3 and 4 show, we regularly detect at least 143

tenfold fewer organelles in E. invadens. This clearly suggests that this distant Entamoeba 144

species (19) contains mitosomes, as well allowing us to suggest all species in the genus do 145

contain this organelle. Although perhaps discounted by most, some still entertain the 146

possibility of genuine extant Archezoa without mitochondria (5, 15). Systematic 147

demonstration of mitochondria (or mitosomes/hydrogenosomes) in all branches of eukaryotes 148

therefore has its merits. 149

Finally, as no information about mitosomes in cysts exists for any species we decided 150

to address this issue using E. invadens where encystation can easily be induced in vitro (18). 151

The distribution and abundance of mitosomes resembles that of E. invadens trophozoites 152

(Fig. 4). No mitosome-organising center can be identified in cysts suggesting that the 153

inheritance of these organelles in Entamoeba is stochastic. 154

In conclusion, our work clearly demonstrates the presence of mitosomes in the 155

reptilian parasite E. invadens, which is distantly related to E. histolytica. The presence of 156

these organelles in both E. histolytica and E. invadens suggests that all Entamoeba spp. 157

contain this organelle. We also show that mitosomes are abundant in the infectious cysts 158

suggesting that these enigmatic organelles may play a role in this important life-cycle stage as 159

well. 160

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We thank Dr. C. Graham Clark (London School of Hygiene and Tropical Medicine) for 162

kindly providing the Entamoeba strains and the Hsp60 antibody and for critical reading of the 163

manuscript. This work was supported by a University of Exeter PhD-studentship to MvdG for 164

MAS and the work in the laboratory of ABH is supported by the Swiss National Science 165

Foundation (Grant # 31003A-125389). 166

167

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Figure legends. 249

250

FIG. 1. Analyses of the amino-terminal regions of the mitochondrial chaperones Hsp60 (A) 251

and mHsp70 (B) from the Amoebozoa. The bacterial homologue from Rickettsia prowazekii 252

is shown for comparison. A. castellanii Hsp60 is not shown due to being incomplete at the N-253

terminus. Predicted or confirmed cleavage sites are indicated by a dash while the asterisk 254

denotes an incomplete N-terminus. The underlined residues for E. histolytica Hsp60 have 255

been shown to be required for mitosomal targeting (21). 256

257

FIG. 2. Phylogenetic relationships of eukaryotic mHsp70 and prokaryotic DnaK homologues. 258

An unrooted maximum likelihood tree produced by PhyML is shown. Circles at the nodes 259

represent bootstrap values as determined using PhyML and posterior probabilities (pp) as 260

determined by MrBayes (black circle: bootstrap > 60 % and pp > 0.9; half circle black on he 261

left: bootstrap > 60 % and pp < 0.9; half circle black on the right: bootstrap < 60 % and pp > 262

0.9). The long branch leading to the Entamoeba spp. has been shortened by 50 %. The 263

Acanthamoeba castellanii mHsp70 was removed from the analyses due to its erratic 264

placement in the analyses in addition to it failing the compositional χ2-test in PUZZLE. 265

Accession numbers of sequences used are available on request. 266

267

FIG. 3. Localization of Entamoeba histolytica and E. invadens mitosomes using Hsp60 and 268

mHsp70 antibodies. Orthogonal projection views of the volume images reconstructed from 269

image stacks showing (A-D) E. histolytica and (E-H) E. invadens trophozoites with DNA 270

labelling using DAPI. (A, E) Hsp60 labelling, inset: corresponding bright-field image. (B, F) 271

mHsp70 labelling. (C, G) Merged image. (D, H) Projection images of the colocalization 272

channel generated with the Imaris colocalization module (grey) depicting voxels in which 273

significant overlap of both labels was detected. Inset: scatterplots indicating the extent of 274

colocalisation within the volume image. Bars equal 3 μm. 275

276

FIG. 4. Orthogonal projection views of the volume images reconstructed from image stacks 277

showing the localization of Entamoeba invadens mitosomes in mature cysts using Hsp60 and 278

mHsp70 antibodies. (A) Hsp60 labelling, inset: corresponding bright-field image. (B) 279

mHsp70 labelling. (C) Merged image. (D) Depiction of voxels with signal overlap as in Fig. 280

3 D and H. Bars equal 2 μm. 281

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