Strategies for Maximizing ATP Supply in The

Hacker, Christian, Howell, Matthew, Bhella, David, and Lucocq, John (2014) Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC. Cellular Microbiology, 16 (4). pp. 565-579. ISSN 1462-5814 Copyright © 2013 The Authors http://eprints.gla.ac.uk/93829/ Deposited on: 22 May 2014 Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk Cellular Microbiology (2014) 16(4), 565–579 doi:10.1111/cmi.12240 First published online 6 December 2013 Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC Christian Hacker,1 Matthew Howell,1 David Bhella2 the mitochondria-vacuole interaction site. Thus and John Lucocq1* E. cuniculi appears to maximize ATP supply by 1School of Medicine, University of St Andrews, North direct binding of mitochondria to the para- Haugh, St Andrews, Fife KF16 9TF, UK. sitophorous vacuole bringing this organelle within 2MRC – University of Glasgow Centre for Virus 0.020 microns of the growing vegetative form of Research, 8 Church Street, Glasgow G11 5JR, UK. the parasite. ATP-delivery is further enhanced by clustering of ATP transporting porins in those Summary regions of the outer mitochondrial membrane lying closest to the parasite. Microsporidia are obligate intracellular parasites with extremely reduced genomes and a depend- ence on host-derived ATP. The microsporidium Introduction Encephalitozoon cuniculi proliferates within a Microsporidia are intracellular eukaryotic parasites known membranous vacuole and we investigated how the to cause economically important disease in several ATP supply is optimized at the vacuole–host inter- animals, including fish, honey bees and silk worms face. Using spatial EM quantification (stereology), (Desportes-Livage, 2000; Lom and Nielsen, 2003; we found a single layer of mitochondria coating Cornman et al., 2009). They also induce clinically signifi- substantial proportions of the parasitophorous cant disease in immune-suppressed humans, including vacuole. Mitochondrial binding occurred preferen- individuals suffering from AIDS (Didier and Weiss, 2011). tially over the vegetative ‘meront’ stages of the Effective and specifically targeted drugs are still scarce parasite, which bulged into the cytoplasm, thereby (Costa and Weiss, 2000; Didier et al., 2005). increasing the membrane surface available for More than a thousand species of microsporidia have mitochondrial interaction. In a broken cell system been described with many more likely to be discovered mitochondrial binding was maintained and was (Williams and Keeling, 2011). The typical life cycle of typified by electron dense structures (< 10 nm microsporidia involves environmental dissemination in the long) bridging between outer mitochondrial and form of a resistant spore, which is able to infect suitable vacuole membranes. In broken cells mitochondrial host cells using a unique apparatus termed the polar tube. binding was sensitive to a range of protease treat- The polar tube is preassembled in the spore as a coiled ments. The function of directly bound mitochon- organelle that unfurls and acts as a nanosyringe to enter dria, as measured by the membrane potential the host cell cytoplasm. The extended tube then functions sensitive dye JC-1, was indistinguishable from as a conduit for guiding the sporoplasm into the host cell other mitochondria in the cell although there environment (Wittner and Weiss, 1999; Franzen, 2005). was a generalized depression of the membrane Once in place the parasite proliferates through vegetative potential in infected cells. Finally, quantitative (or meront) cell stages and differentiates into sporont, immuno-EM revealed that the ATP-delivering sporoblasts and finally the spore. In many microporidians mitochondrial porin, VDAC, was concentrated at growth and differentiation take place inside a vacuole which, depending on the species, can be either derived from the host cell or secreted by the microsporidium itself (Hollister et al., 1996; Rönnebäumer et al., 2008). Received 9 September, 2013; revised 11 October, 2013; accepted 11 November, 2013. *For correspondence. E-mail jml7@st-andrews. Originally thought to be early offshoots in evolution, it ac.uk; Tel. (+44) 1334 463547; Fax (+44) 1334 462570. became clear that microsporidia have undergone extreme © 2013 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. cellular microbiology 566 C. Hacker, M. Howell, D. Bhella and J. Lucocq genomic reduction, divesting themselves of metabolic membrane via electron dense bridging-structures pathways (Weidner et al., 1999; Williams and Keeling, (< 10 nm length). The mitochondria bind preferentially to 2011) and reducing some organelles such as their mito- the early vegetative ‘meront’ cell stages that are them- chondria to tiny relict structures that carry out only selves only found at the vacuole periphery and bulge into a limited array of essential processes (Goldberg et al., the host cell cytoplasm. In a broken cell system we show 2008). Microsporidia are likely to be related to the fungal that this interaction is retained after removing soluble endoparasite phylum of the cryptomycota (James et al., cytosolic factors but in the same system is sensitive to 2013) and are now considered to have undergone a digestion by proteases. By using a fluorescent dye that reductive evolution which has resulted in simplified and reports on mitochondrial membrane potential, we find the streamlined adaptations to the intracellular parasitic life- function of adherent mitochondria is indistinguishable style (Wittner and Weiss, 1999). from mitochondria elsewhere in the cell. Interestingly, Most microsporidia appear to have lost the ability to we also find that VDAC molecules, the ATP exporting produce ATP in their relict mitochondria and they then rely voltage gated channels of the mitochondrial outer mem- on the uptake of nutrients and energy substrates from brane, become concentrated at the sites of mitochondrial their host (Williams and Keeling, 2003; van der Giezen binding. We conclude that E. cuniculi induces direct et al., 2005). Accordingly, emerging data have assigned protein-dependent binding of host mitochondria to nucleotide transporters to the plasma membrane of the the parasitophorous vacuole surrounding the vegetative parasite where they could ensure a continuous supply meront cell stages. This binding is combined with of ATP (Richards et al., 2003; Tsaousis et al., 2008) mitochondrial ATP-delivering channels at the vacuole which itself can pass through the membrane of the membrane to facilitate delivery of ATP from host cyto- parasitophorous vacuole (PV), known to be permeable to plasm to the growing and differentiating parasite. small molecules (< 10 kDa) (Rönnebäumer et al., 2008). Interestingly, as the parasite develops from its vegetative/ proliferative form into spores, these organisms appear Results/discussion increasingly independent of ATP delivery from the host as Quantification reveals a single layer of mitochondria they upregulate glycolysis (Heinz et al., 2012). at the PVM Another way in which microsporidia might influence delivery of nutrients and ATP is by re-organizing the host The highly simplified relict mitochondria of microsporidia cell cytoplasm by re-positioning and/or influencing the have lost the ability to produce ATP (Tsaousis et al., 2008; function of the host cell organelles. Previous studies have Williams et al., 2008), and so these organisms must suggested that a range of intracellular parasites can have evolved different strategies of sustaining their influence the organization of host cell cytoplasm in energy requirements. We tested the hypothesis that the close proximity to the infecting organism (Canning and microsporidian E. cuniculi can modulate its interaction Hollister, 1992; Sinai et al., 1997; Scanlon et al., 2004). In with the host to facilitate ATP delivery. Using conventional Toxoplasma gondii one rigorous, and high-resolution, electron microscopy we first investigated the nature and quantitative EM study clearly demonstrated close apposi- extent of the re-organization of the host cell cytoplasm tion of mitochondria to the vacuole surrounding the close to the PV. The membrane of the PV (PVM) can be parasites, although a subsequent study failed to show distinguished easily and careful study of E. cuniculi- such as relationship (Sinai et al., 1997; Magno et al., infected RK-13 cells revealed apparent clustering of 2005). Here we focused on the host–parasite interac- mitochondria around the PVM (Fig. 1) as described tion of Encephalitozoon cuniculi, which belongs to a in previous reports (Scanlon et al., 2004). A closer exami- microsporidian genus able to cause human infection nation of the host cell mitochondria in E. cuniculi-infected (Didier and Weiss, 2006) and harbours one of the smallest cells revealed that mitochondria appear to be closely eukaryotic genomes found to date (Katinka et al., 2001; applied to the PV (Fig. 1A–C). In order distinguish Keeling, 2001). Previous observations had indicated a between clustering/aggregation and direct

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