Evolution of an Iron-Detoxifying Protein: Eukaryotic and Rickettsia Frataxins Contain a Conserved Site Which Is Not Present in Their Bacterial Homologues.

Friedreich's ataxia is a neurodegenerative disease caused by mutations in the frataxin gene. Frataxin homologues, including bacterial CyaY proteins, can be found in most species and play a fundamental role in mitochondrial iron homeostasis, either promoting iron assembly into metaloproteins or contributing to iron detoxification. While several lines of evidence suggest that eukaryotic frataxins are more effective than bacterial ones in iron detoxification, the residues involved in this gain of function are unknown. In this work, we analyze conservation of amino acid sequence and protein structure among frataxins and CyaY proteins to identify four highly conserved residue clusters and group them into potential functional clusters. Clusters 1, 2, and 4 are present in eukaryotic frataxins and bacterial CyaY proteins. Cluster 3, containing two serines, a tyrosine, and a glutamate, is only present in eukaryotic frataxins and on CyaY proteins from the Rickettsia genus. Residues from cluster 3 are blocking a small cavity of about 40 Å present in E. coli's CyaY. The function of this cluster is unknown, but we hypothesize that its tyrosine may contribute to prevent formation of reactive oxygen species during iron detoxification. This cluster provides an example of gain of function during evolution in a protein involved in iron homeostasis, as our results suggests that Cluster 3 was present in the endosymbiont ancestor of mitochondria and was conserved in eukaryotic frataxins.

Fusarium graminearum Infection Strategy in Wheat Involves a Highly Conserved Genetic Program That Controls the Expression of a Core Effectome.

Fusarium graminearum, the main causal agent of Fusarium Head Blight (FHB), is one of the most damaging pathogens in wheat. Because of the complex organization of wheat resistance to FHB, this pathosystem represents a relevant model to elucidate the molecular mechanisms underlying plant susceptibility and to identify their main drivers, the pathogen's effectors. Although the F. graminearum catalog of effectors has been well characterized at the genome scale, in planta studies are needed to confirm their effective accumulation in host tissues and to identify their role during the infection process. Taking advantage of the genetic variability from both species, a RNAseq-based profiling of gene expression was performed during an infection time course using an aggressive F. graminearum strain facing five wheat cultivars of contrasting susceptibility as well as using three strains of contrasting aggressiveness infecting a single susceptible host. Genes coding for secreted proteins and exhibiting significant expression changes along infection progress were selected to identify the effector gene candidates. During its interaction with the five wheat cultivars, 476 effector genes were expressed by the aggressive strain, among which 91% were found in all the infected hosts. Considering three different strains infecting a single susceptible host, 761 effector genes were identified, among which 90% were systematically expressed in the three strains. We revealed a robust F. graminearum core effectome of 357 genes expressed in all the hosts and by all the strains that exhibited conserved expression patterns over time. Several wheat compartments were predicted to be targeted by these putative effectors including apoplast, nucleus, chloroplast and mitochondria. Taken together, our results shed light on a highly conserved parasite strategy. They led to the identification of reliable key fungal genes putatively involved in wheat susceptibility to F. graminearum, and provided valuable information about their putative targets.

Nuclear hormone receptors promote gut and glia detoxifying enzyme induction and protect C. elegans from the mold P. brevicompactum.

Animals encounter microorganisms in their habitats, adapting physiology and behavior accordingly. The nematode Caenorhabditis elegans is found in microbe-rich environments; however, its responses to fungi are not extensively studied. Here, we describe interactions of C. elegans and Penicillium brevicompactum, an ecologically relevant mold. Transcriptome studies reveal that co-culture upregulates stress response genes, including xenobiotic-metabolizing enzymes (XMEs), in C. elegans intestine and AMsh glial cells. The nuclear hormone receptors (NHRs) NHR-45 and NHR-156 are induction regulators, and mutants that cannot induce XMEs in the intestine when exposed to P. brevicompactum experience mitochondrial stress and exhibit developmental defects. Different C. elegans wild isolates harbor sequence polymorphisms in nhr-156, resulting in phenotypic diversity in AMsh glia responses to microbe exposure. We propose that P. brevicompactum mitochondria-targeting mycotoxins are deactivated by intestinal detoxification, allowing tolerance to moldy environments. Our studies support the idea that C. elegans NHRs may be regulated by environmental cues.

Mitochondria as a Cellular Hub in Infection and Inflammation.

Mitochondria are the energy center of the cell. They are found in the cell cytoplasm as dynamic networks where they adapt energy production based on the cell's needs. They are also at the center of the proinflammatory response and have essential roles in the response against pathogenic infections. Mitochondria are a major site for production of Reactive Oxygen Species (ROS; or free radicals), which are essential to fight infection. However, excessive and uncontrolled production can become deleterious to the cell, leading to mitochondrial and tissue damage. Pathogens exploit the role of mitochondria during infection by affecting the oxidative phosphorylation mechanism (OXPHOS), mitochondrial network and disrupting the communication between the nucleus and the mitochondria. The role of mitochondria in these biological processes makes these organelle good targets for the development of therapeutic strategies. In this review, we presented a summary of the endosymbiotic origin of mitochondria and their involvement in the pathogen response, as well as the potential promising mitochondrial targets for the fight against infectious diseases and chronic inflammatory diseases.

Modeling the Life Cycle of the Intramitochondrial Bacterium "Candidatus Midichloria mitochondrii" Using Electron Microscopy Data.

"Candidatus Midichloria mitochondrii" is a Gram-negative bacterium that lives in strict intracellular symbiosis with the hard tick Ixodes ricinus, forming one of the most intriguing endosymbiosis described to date. The bacterium is capable of durably colonizing the host mitochondria, a peculiar tropism that makes "Ca. Midichloria mitochondrii" a very interesting tool to study the physiology of these cellular organelles. The interaction between the symbiont and the organelle has, however, been difficult to characterize. A parallelism with the predatory bacterium Bdellovibrio bacteriovorus has been drawn, suggesting the hypothesis that "Ca. Midichloria mitochondrii" could prey on mitochondria and consume them to multiply. We studied the life cycle of the bacterium within the host oocytes using a multidisciplinary approach, including electron microscopy, molecular biology, statistics, and systems biology. Our results were not coherent with a predatory-like behavior by "Ca. Midichloria mitochondrii" leading us to propose a novel hypothesis for its life cycle. Based on our results, we here present a novel model called the "mitochondrion-to-mitochondrion hypothesis." Under this model, the bacterium would be able to move from mitochondrion to mitochondrion, possibly within a mitochondrial network. We show that this model presents a good fit with quantitative electron microscopy data. IMPORTANCE Our results suggest that "Candidatus Midichloria mitochondrii," the intramitochondrial bacterium, does not invade mitochondria like predatory bacteria do but instead moves from mitochondrion to mitochondrion within the oocytes of Ixodes ricinus. A better understanding of the lifestyle of "Ca. Midichloria mitochondrii" will allow us to better define the role of this bacterial symbiont in the host physiology.

Dating Alphaproteobacteria evolution with eukaryotic fossils.

Elucidating the timescale of the evolution of Alphaproteobacteria, one of the most prevalent microbial lineages in marine and terrestrial ecosystems, is key to testing hypotheses on their co-evolution with eukaryotic hosts and Earth's systems, which, however, is largely limited by the scarcity of bacterial fossils. Here, we incorporate eukaryotic fossils to date the divergence times of Alphaproteobacteria, based on the mitochondrial endosymbiosis that mitochondria evolved from an alphaproteobacterial lineage. We estimate that Alphaproteobacteria arose ~1900 million years (Ma) ago, followed by rapid divergence of their major clades. We show that the origin of Rickettsiales, an order of obligate intracellular bacteria whose hosts are mostly animals, predates the emergence of animals for ~700 Ma but coincides with that of eukaryotes. This, together with reconstruction of ancestral hosts, strongly suggests that early Rickettsiales lineages had established previously underappreciated interactions with unicellular eukaryotes. Moreover, the mitochondria-based approach displays higher robustness to uncertainties in calibrations compared with the traditional strategy using cyanobacterial fossils. Further, our analyses imply the potential of dating the (bacterial) tree of life based on endosymbiosis events, and suggest that previous applications using divergence times of the modern hosts of symbiotic bacteria to date bacterial evolution might need to be revisited.

Mitofusin-2 boosts innate immunity through the maintenance of aerobic glycolysis and activation of xenophagy in mice.

Mitochondrial function and innate immunity are intimately linked; however, the mechanisms how mitochondrion-shaping proteins regulate innate host defense remains largely unknown. Herein we show that mitofusin-2 (MFN2), a mitochondrial fusion protein, promotes innate host defense through the maintenance of aerobic glycolysis and xenophagy via hypoxia-inducible factor (HIF)-1α during intracellular bacterial infection. Myeloid-specific MFN2 deficiency in mice impaired the antimicrobial and inflammatory responses against mycobacterial and listerial infection. Mechanistically, MFN2 was required for the enhancement of inflammatory signaling through optimal induction of aerobic glycolysis via HIF-1α, which is activated by mitochondrial respiratory chain complex I and reactive oxygen species, in macrophages. MFN2 did not impact mitophagy during infection; however, it promoted xenophagy activation through HIF-1α. In addition, MFN2 interacted with the late endosomal protein Rab7, to facilitate xenophagy during mycobacterial infection. Our findings reveal the mechanistic regulations by which MFN2 tailors the innate host defense through coordinated control of immunometabolism and xenophagy via HIF-1α during bacterial infection.

Neuronal mitochondrial dynamics coordinate systemic mitochondrial morphology and stress response to confer pathogen resistance in C. elegans.

Mitochondrial functions across different tissues are regulated in a coordinated fashion to optimize the fitness of an organism. Mitochondrial unfolded protein response (UPRmt) can be nonautonomously elicited by mitochondrial perturbation in neurons, but neuronal signals that propagate such response and its physiological significance remain incompletely understood. Here, we show that in C. elegans, loss of neuronal fzo-1/mitofusin induces nonautonomous UPRmt through multiple neurotransmitters and neurohormones, including acetylcholine, serotonin, glutamate, tyramine, and insulin-like peptides. Neuronal fzo-1 depletion also triggers nonautonomous mitochondrial fragmentation, which requires autophagy and mitophagy genes. Systemic activation of UPRmt and mitochondrial fragmentation in C. elegans via perturbing neuronal mitochondrial dynamics improves resistance to pathogenic Pseudomonas infection, which is supported by transcriptomic signatures of immunity and stress-response genes. We propose that C. elegans surveils neuronal mitochondrial dynamics to coordinate systemic UPRmt and mitochondrial connectivity for pathogen defense and optimized survival under bacterial infection.

Wolbachia affects mitochondrial population structure in two systems of closely related Palaearctic blue butterflies.

The bacterium Wolbachia infects many insect species and spreads by diverse vertical and horizontal means. As co-inherited organisms, these bacteria often cause problems in mitochondrial phylogeny inference. The phylogenetic relationships of many closely related Palaearctic blue butterflies (Lepidoptera: Lycaenidae: Polyommatinae) are ambiguous. We considered the patterns of Wolbachia infection and mitochondrial diversity in two systems: Aricia agestis/Aricia artaxerxes and the Pseudophilotes baton species complex. We sampled butterflies across their distribution ranges and sequenced one butterfly mitochondrial gene and two Wolbachia genes. Both butterfly systems had uninfected and infected populations, and harboured several Wolbachia strains. Wolbachia was highly prevalent in A. artaxerxes and the host's mitochondrial structure was shallow, in contrast to A. agestis. Similar bacterial alleles infected both Aricia species from nearby sites, pointing to a possible horizontal transfer. Mitochondrial history of the P. baton species complex mirrored its Wolbachia infection and not the taxonomical division. Pseudophilotes baton and P. vicrama formed a hybrid zone in Europe. Wolbachia could obscure mitochondrial history, but knowledge on the infection helps us to understand the observed patterns. Testing for Wolbachia should be routine in mitochondrial DNA studies.

Plant mitochondria and chloroplasts are targeted by the Rhizoctonia solani RsCRP1 effector.

The fungal species Rhizoctonia solani belongs to the Basidiomycota division and is a ubiquitous soil-borne pathogen. It is the main agent of the damping-off disease in seedlings and causes the root and crown rot disease in sugar beets. Plant pathogens deploy small secreted proteins, called effectors, to manipulate plant immunity in order to infect the host. Here, a gene (RsCRP1) encoded a putative effector cysteine-rich protein was cloned, expressed in Cercospora beticola and used for virulence assays. The RsCRP1 gene was highly induced upon the early-infection stage of sugar beet seedlings and disease was promoted. Confocal microscopy demonstrated localization to the chloroplasts and mitochondria upon transient expression of RsCRP1 in leaves of Nicotiana benthamiana. Further, this effector was unable to induce necrosis or to suppress hypersensitive response induced by the Avr4/Cf4 complex in N. benthamiana. Overall, these data indicate that RsCRP1 is a novel effector targeting distinct plant cell organelles in order to facilitate a successful infection at the early stages of the disease development.

Proteomic Identification of Coxiella burnetii Effector Proteins Targeted to the Host Cell Mitochondria During Infection.

Modulation of the host cell is integral to the survival and replication of microbial pathogens. Several intracellular bacterial pathogens deliver bacterial proteins, termed "effector proteins" into the host cell during infection by sophisticated protein translocation systems, which manipulate cellular processes and functions. The functional contribution of individual effectors is poorly characterized, particularly in intracellular bacterial pathogens with large effector protein repertoires. Technical caveats have limited the capacity to study these proteins during a native infection, with many effector proteins having only been demonstrated to be translocated during over-expression of tagged versions. Here, we developed a novel strategy to examine effector proteins in the context of infection. We coupled a broad, unbiased proteomics-based screen with organelle purification to study the host-pathogen interactions occurring between the host cell mitochondrion and the Gram-negative, Q fever pathogen Coxiella burnetii. We identify four novel mitochondrially-targeted C. burnetii effector proteins, renamed Mitochondrial Coxiella effector protein (Mce) B to E. Examination of the subcellular localization of ectopically expressed proteins confirmed their mitochondrial localization, demonstrating the robustness of our approach. Subsequent biochemical analysis and affinity enrichment proteomics of one of these effector proteins, MceC, revealed the protein localizes to the inner membrane and can interact with components of the mitochondrial quality control machinery. Our study adapts high-sensitivity proteomics to study intracellular host-pathogen interactions, providing a robust strategy to examine the subcellular localization of effector proteins during native infection. This approach could be applied to a range of pathogens and host cell compartments to provide a rich map of effector dynamics throughout infection.

Symbiotic Origin of Apoptosis.

The progress of evolutionary biology has revealed that symbiosis played a basic role in the evolution of complex eukaryotic organisms, including humans. Mitochondria are actually simplified endosymbiotic bacteria currently playing the role of cellular organelles. Mitochondrial domestication occurred at the very beginning of eukaryotic evolution. Mitochondria have two different basic functions: they produce energy using oxidative respiration, and they initiate different forms of apoptotic programmed/regulated cell death. Apoptotic programmed cell death may have different cytological forms. Mechanisms of apoptotic programmed cell death exist even in the unicellular organisms, and they play a basic role in the development of complex multicellular organisms, such as fungi, green plants, and animals. Multicellularity was independently established many times among eukaryotes. There are indications that apoptotic programmed cell death is a trait required for the establishment of multicellularity. Regulated cell death is initiated by many different parallel biochemical pathways. It is generally accepted that apoptosis evolved during mitochondrial domestication. However, there are different hypothetical models of the origin of apoptosis. The phylogenetic studies of my group indicate that apoptosis probably evolved during an evolutionary arms race between host ancestral eukaryotic predators and ancestral prey mitochondria (named protomitochondria). Protomitochondrial prey produced many different toxins as a defense against predators. From these toxins evolved extant apoptotic factors. There are indications that aerobic respiration and apoptosis co-evolved and are functionally linked in extant organisms. Perturbations of apoptosis and oxidative respiration are frequently observed during neoplastic transition. Our group showed that perturbations of apoptosis in yeasts also cause perturbations of oxidative respiration.

Helicobacter pylori infection induces STAT3 phosphorylation on Ser727 and autophagy in human gastric epithelial cells and mouse stomach.

Helicobacter pylori (H. pylori) infection is considered as one of the principal risk factors of gastric cancer. Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) plays an important role in inflammation-associated gastric carcinogenesis. In the canonical STAT3 pathway, phosphorylation of STAT3 on Tyr705 is a major event of STAT3 activation. However, recent studies have demonstrated that STAT3 phosphorylated on Ser727 has an independent function in mitochondria. In the present study, we found that human gastric epithelial AGS cells infected with H. pylori resulted in localization of STAT3 phosphorylated on Ser727 (P-STAT3Ser727), predominantly in the mitochondria. Notably, H. pylori-infected AGS cells exhibited the loss of mitochondrial integrity and increased expression of the microtubule-associated protein light chain 3 (LC3), the autophagosomal membrane-associated protein. Treatment of AGS cells with a mitophagy inducer, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), resulted in accumulation of P-STAT3Ser727 in mitochondria. In addition, the elevated expression and mitochondrial localization of LC3 induced by H. pylori infection were attenuated in AGS cells harboring STAT3 mutation defective in Ser727 phosphorylation (S727A). We also observed that both P-STAT3Ser727 expression and LC3 accumulation were increased in the mitochondria of H. pylori-inoculated mouse stomach.

Mitochondrial Reactive Oxygen Species: Double-Edged Weapon in Host Defense and Pathological Inflammation During Infection.

Mitochondria are inevitable sources for the generation of mitochondrial reactive oxygen species (mtROS) due to their fundamental roles in respiration. mtROS were reported to be bactericidal weapons with an innate effector function during infection. However, the controlled generation of mtROS is vital for the induction of efficient immune responses because excessive production of mtROS with mitochondrial damage leads to sustained inflammation, resulting in pathological outcomes such as sepsis. Here, we discuss the beneficial and detrimental roles of mtROS in the innate immune system during bacterial, viral, and fungal infections. Recent evidence suggests that several pathogens have evolved multiple strategies to modulate mtROS for their own benefit. We are just beginning to understand the mechanisms by which mtROS generation is regulated and how mtROS affect protective and pathological responses during infection. Several agents/small molecules that prevent the uncontrolled production of mtROS are known to be beneficial in the maintenance of tissue homeostasis during sepsis. mtROS-targeted approaches need to be incorporated into preventive and therapeutic strategies against a variety of infections.

The transcription factor ANAC017 is a key regulator of mitochondrial proteotoxic stress responses in plants.

Impaired mitochondrial translation or reduced mitochondrial protein import can lead to imbalances in mitochondrial protein composition. Such mitochondrial proteotoxic stresses can trigger a nuclear transcriptional response commonly described as the mitochondrial unfolded protein response (UPRmt). Despite extensive studies of UPRmt pathways in animal and fungal systems, very little is known about how the UPRmt is regulated in plants. Through comparison of Arabidopsis thaliana whole-genome transcriptome data, it was found that most genes induced by mitochondrial ribosome inhibitor doxycycline are also induced by Complex III inhibitor antimycin A. We demonstrate that transcriptional responses to a wide range of mitochondrial proteotoxic stress-triggers are regulated by the transcription factor ANAC017, which was shown to reside in the endoplasmic reticulum (ER). By contrast, no consistent evidence was found for genes that are specifically induced by doxycycline but not antimycin A. Furthermore, ANAC017 gain- and loss-of-function mutants showed marked resistance or susceptibility, respectively, to mitochondrial stress-inducing treatments, demonstrating the physiological importance of ANAC017 during mitochondrial proteotoxic stress. Finally, it was shown that ethylene signalling promotes mitochondria-to-nucleus signalling, most likely independently of ANAC017. Overall, this study shows that in plants, the UPRmt is largely overlapping with, and perhaps identical to, 'classical' mitochondrial retrograde signalling, and is mediated by ER-anchored transcription factor ANAC017. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

When bacteria meet mitochondria: The strange case of the tick symbiont Midichloria mitochondrii†.

Mitochondria are key eukaryotic organelles that perform several essential functions. Not surprisingly, many intracellular bacteria directly or indirectly target mitochondria, interfering with innate immunity, energy production or apoptosis, to make the host cell a more hospitable niche for bacterial replication. The alphaproteobacterium Midichloria mitochondrii has taken mitochondrial targeting to another level by physically colonising mitochondria, as shown by transmission electron micrographs of bacteria residing in the mitochondrial intermembrane space. This unique localization provokes a number of questions around the mechanisms allowing, and reasons driving intramitochondrial tropism. We suggest possible scenarios that could lead to this peculiar localization and hypothesize potential costs and benefits of mitochondrial colonisation for the bacterium and its host.

TBC1D9 regulates TBK1 activation through Ca2+ signaling in selective autophagy.

Invading microbial pathogens can be eliminated selectively by xenophagy. Ubiquitin-mediated autophagy receptors are phosphorylated by TANK-binding kinase 1 (TBK1) and recruited to ubiquitinated bacteria to facilitate autophagosome formation during xenophagy, but the molecular mechanism underlying TBK1 activation in response to microbial infection is not clear. Here, we show that bacterial infection increases Ca2+ levels to activate TBK1 for xenophagy via the Ca2+-binding protein TBC1 domain family member 9 (TBC1D9). Mechanistically, the ubiquitin-binding region (UBR) and Ca2+-binding motif of TBC1D9 mediate its binding with ubiquitin-positive bacteria, and TBC1D9 knockout suppresses TBK1 activation and subsequent recruitment of the ULK1 complex. Treatment with a Ca2+ chelator impairs TBC1D9-ubiquitin interactions and TBK1 activation during xenophagy. TBC1D9 is also recruited to damaged mitochondria through its UBR and Ca2+-binding motif, and is required for TBK1 activation during mitophagy. These results indicate that TBC1D9 controls TBK1 activation during xenophagy and mitophagy through Ca2+-dependent ubiquitin-recognition.

Interferon Gamma Reprograms Host Mitochondrial Metabolism through Inhibition of Complex II To Control Intracellular Bacterial Replication.

The mechanisms by which interferon gamma (IFN-γ) controls the replication of cytosolic pathogens independent of responses, such as the generation of reactive oxygen species/reactive nitrogen species (ROS/RNS), have not been fully elucidated. In the current study, we developed a model using Francisella tularensis, the causative agent of tularemia, in which pathways triggered by IFN-γ commonly associated with bacterial control were not required. Using this model, we demonstrated that IFN-γ-mediated production of itaconate and its ability to impair host mitochondrial function, independent of activity on the pathogen, were central for the restriction of bacterial replication in vitro and in vivo We then demonstrate that IFN-γ-driven itaconate production was dispensable, as directly targeting complex II using cell membrane-permeable metabolites also controlled infection. Together, these findings show that while reprogramming of mitochondrial metabolism is a key factor in IFN-γ control of intracellular bacteria, the development of antimicrobial strategies based on targeting host mitochondrial metabolism independent of this cytokine may be an effective therapeutic approach.

Mitofusin 2-Deficiency Suppresses Mycobacterium tuberculosis Survival in Macrophages.

Apoptosis is an important host defense mechanism against mycobacterial infection. However, the molecular mechanisms regulating apoptosis during mycobacterial infection are not well known. Recent reports suggest that bacterial infection regulates mitochondrial fusion and fission in various ways. Here, we investigated the role of mitochondria in Mycobacterium tuberculosis (Mtb)-infected macrophages. Mtb H37Rv (Rv) infection induced mitofusin 2 (MFN2) degradation, leading to mitochondrial fission. Interestingly, Mtb H37Ra (Ra) infection induced significantly greater mitochondrial fragmentation than Rv infection. Mtb-mediated Parkin, an E3 ubiquitin ligase, contributed to the degradation of MFN2. To evaluate the role of endoplasmic reticulum stress in the production of Parkin during Mtb infection, we analyzed Parkin production in 4-phenylbutyric acid (4-PBA)-pretreated macrophages. Pretreatment with 4-PBA reduced Parkin production in Mtb-infected macrophages. In contrast, the level of MFN2 production recovered to a level similar to that of the unstimulated control. In addition, Ra-infected macrophages had reduced mitochondrial membrane potential (MMP) compared to those infected with Rv. Interestingly, intracellular survival of mycobacteria was decreased in siMFN2-transfected macrophages; in contrast, overexpression of MFN2 in macrophages increased Mtb growth compared with the control.

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

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.