Microsporidia Interact with Host Cell Mitochondria via Voltage-Dependent Anion Channels Using Sporoplasm Surface Protein 1

Abstract

Microsporidia are opportunistic intracellular pathogens that can infect a wide variety of hosts ranging from invertebrates to vertebrates. During invasion, the microsporidian polar tube pushes into the host cell, creating a protective microenvironment, the invasion synapse, into which the sporoplasm extrudes. Within the synapse, the sporoplasm then invades the host cell, forming a parasitophorous vacuole (PV). Using a proteomic approach, we identified Encephalitozoon hellem sporoplasm surface protein 1 (EhSSP1), which localized to the surface of extruded sporoplasms. EhSSP1 was also found to interact with polar tube protein 4 (PTP4). Recombinant EhSSP1 (rEhSSP1) bound to human foreskin fibroblasts, and both anti-EhSSP1 and rEhSSP1 caused decreased levels of host cell invasion, suggesting that interaction of SSP1 with the host cell was involved in invasion. Coimmunoprecipitation (Co-IP) followed by proteomic analysis identified host cell voltage-dependent anion channels (VDACs) as EhSSP1 interacting proteins. Yeast two-hybrid assays demonstrated that EhSSP1 was able to interact with VDAC1, VDAC2, and VDAC3. rEhSSP1 colocalized with the host mitochondria which were associated with microsporidian PVs in infected cells. Transmission electron microscopy revealed that the outer mitochondrial membrane interacted with meronts and the PV membrane, mitochondria clustered around meronts, and the VDACs were concentrated at the interface of mitochondria and parasite. Knockdown of VDAC1, VDAC2, and VDAC3 in host cells resulted in significant decreases in the number and size of the PVs and a decrease in mitochondrial PV association. The interaction of EhSSP1 with VDAC probably plays an important part in energy acquisition by microsporidia via its role in the association of mitochondria with the PV. IMPORTANCE Microsporidia are important opportunistic human pathogens in immune-suppressed individuals, such as those with HIV/AIDS and recipients of organ transplants. The sporoplasm is critical for establishing microsporidian infection. Despite the biological importance of this structure for transmission, there is limited information about its structure and composition that could be targeted for therapeutic intervention. Here, we identified a novel E. hellem sporoplasm surface protein, EhSSP1, and demonstrated that it can bind to host cell mitochondria via host VDAC. Our data strongly suggest that the interaction between SSP1 and VDAC is important for the association of mitochondria with the parasitophorous vacuole during microsporidian infection. In addition, binding of SSP1 to the host cell is associated with the final steps of invasion in the invasion synapse.

Invasion of Host Cells by Microsporidia

“…As the sporoplasm forms a droplet at the tip of PT during germination we hypothesized that PTPs might be able to interact with sporoplasm proteins during the process of invasion. This concept is supported by the finding that a recently identified sporoplasm surface protein (SSP1) from E. hellem interacts with PTP4 in a yeast-two hybrid assay (Han et al, 2019).…”

“…In vitro method of PT germination References Amblyospora sp. 1.6M sucrose plus 0.2M KCl, pH 9 Undeen and Avery, 1984 Edhazardia aedis 0.1M KCl, pH 10.5 Frixione et al, 1992 Encephalitozoon hellem 140 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2, pH 9.5 or 7.5 with and without 5% H 2 O 2 Leitch et al, 1993He et al, 1996 Encephalitozoon hellem 140 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 5 mM KCl, pH 7.5 for 15min then 5% H 2 O 2 for another 15 min Han et al, 2019 Encephalitozoon intestinalis 140 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , pH 9.5 or 7.5 with and without 5% H 2 O 2 He et al, 1996 Encephalitozoon intestinalis Spores from urine resuspended in 0.025 N NaOH in phosphate buffered saline Beckers et al, 1996 Glugea fumiferanae Chlorides of alkali metal ions at pH 10.8: CsCl, RbCl, KCl, NaCl, or LiCl Ishihara, 1967 Glugea hertwigi Calcium ionophore A-23187 Weidner, 1982 pH shift from neutral (7.0) to alkaline (9.5) in 150 mM phosphate buffer 50 mM sodium citrate in 100 mM glycylglycine buffer pH 9.5 150 mM phosphate buffer in 100 mM glycylglycine buffer pH 9.5…”

“…Four additional PTPs (PTP2 through PTP5) have been identified and characterized using proteomic and antibody-based approaches, and proteomic data suggests that there are additional PTPs in the PT (Peuvel et al, 2002;Weiss and Becnel, 2014;Han et al, , 2019. PTP2 is found at the same genomic locus as PTP1.…”

“…Microsporidia can use glycolysis for energy generation, but this pathway, while active in spores, appears to not be active during the stage of intracellular growth and replication inside of the host cytosol (Dolgikh et al, 2011;Heinz et al, 2012;Williams et al, 2014). Thus, microsporidia depend on their host cells for energy and mitochondria accumulate around the microsporidia [this is clearly observable in Encephalitozoonidae residing in a parasitophorous vacuole within their host cells (Han et al, 2019)].…”

“…It is possible that the PT either pierces the host cell membrane in this synapse delivering the sporoplasm directly into the host cells, or that the sporoplasm itself may interact with the host cell membrane during invasion (Takvorian et al, 2013;. Based on observations on the Encephalitozoonidae (Han et al, , 2019, we hypothesize that that the sporoplasm interacts with the host cell membrane and an invasion vacuole is formed (Figure 3). Once the infectious sporoplasm enters the host cell it undergoes development into meronts (proliferative forms), sporonts, sporoblasts (developing spore) and finally mature spores (Visvesvara, 2002).…”