The tissue cyst of Toxoplasma gondii, found in latent infection, serves a critical role in both transmission and reactivation of this organism. Within infected cells, slowly replicating parasites (bradyzoites) are surrounded by a cyst matrix, cyst wall, and cyst membrane. The cyst wall is clearly delineated by ultrastructural analysis; however, the composition and function of this layer in host-parasite interactions are not fully understood. In order to understand the composition of the cyst wall, a proteomic analysis of purified cyst wall fragments, that were enriched with Percoll gradients and subsequently immunoprecipitated with CST1 antibody, was performed. Known cyst wall proteins, such as CST1, BPK1, MCP4, MAG1, GRA2, GRA3, and GRA5, were identified in this preparation by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, dense granule proteins (GRAs) not previously shown to associate with the cyst wall, as well as uncharacterized hypothetical proteins, were identified in this cyst wall preparation. Several of these hypothetical cyst wall (CST) proteins were epitope tagged, and immunofluorescence assays confirmed their localization as novel cyst matrix and cyst wall proteins. Expression of two of these newly identified cyst wall proteins was eliminated by gene knockout (CST2-KO and CST3-KO). CST2-KO parasites were highly attenuated in virulence and did not establish detectable cyst burdens. This targeted proteomic approach allowed the identification of new components of the cyst wall that probably have roles in the parasite/host interface. IMPORTANCE Toxoplasma gondii is a highly prevalent parasite worldwide that presents life-threatening risks to immunocompromised and pregnant individuals. Whereas the life stage responsible for acute infection can be treated, the life stage responsible for chronic infection is refractory to currently available therapeutics. Little is known about the protein composition of the cyst wall, an amorphous structure formed by parasites that is suspected to facilitate persistence within muscle and nervous tissue during chronic (latent) infection. By implementing a refined approach to selectively purify cyst wall fragments, we identified several known and novel cyst wall proteins from our sample preparations. We confirmed the localizations of several proteins from this data set and identified one that is involved in parasite virulence. These data will propel further studies on cyst wall structure and function, leading to therapeutic strategies that can eliminate the chronic infection stage.

Sporadic colon cancer accounts for approximately 80% of colorectal cancer (CRC) with high incidence in Western societies strongly linked to long-term dietary patterns. A unique mouse model for sporadic CRC results from feeding a purified rodent Western-style diet (NWD1) recapitulating intake for the mouse of common nutrient risk factors each at its level consumed in higher risk Western populations. This causes sporadic large and small intestinal tumors in wild-type mice at an incidence and frequency similar to that in humans. NWD1 perturbs intestinal cell maturation and Wnt signaling throughout villi and colonic crypts and decreases mouse Lgr5hi intestinal stem cell contribution to homeostasis and tumor development. Here we establish that NWD1 transcriptionally reprograms Lgr5hi cells, and that nutrients are interactive in reprogramming. Furthermore, the DNA mismatch repair pathway is elevated in Lgr5hi cells by lower vitamin D3 and/or calcium in NWD1, paralleled by reduced accumulation of relevant somatic mutations detected by single-cell exome sequencing. In compensation, NWD1 also reprograms Bmi1+ cells to function and persist as stem-like cells in mucosal homeostasis and tumor development. The data establish the key role of the nutrient environment in defining the contribution of two different stem cell populations to both mucosal homeostasis and tumorigenesis. This raises important questions regarding impact of variable human diets on which and how stem cell populations function in the human mucosa and give rise to tumors. Moreover, major differences reported in turnover of human and mouse crypt base stem cells may be linked to their very different nutrient exposures.

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole (PV), the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV-1 virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV-1 Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of TgGRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PV membrane (PVM). These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission.

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.

A characteristic of the latent cyst stage of Toxoplasma gondii is a thick cyst wall that forms underneath the membrane of the bradyzoite vacuole. Previously, our laboratory group published a proteomic analysis of purified in vitro cyst wall fragments that identified an inventory of cyst wall components. To further refine our understanding of the composition of the cyst wall, several cyst wall proteins were tagged with a promiscuous biotin ligase (BirA*), and their interacting partners were screened by streptavidin affinity purification. Within the cyst wall pulldowns, previously described cyst wall proteins, dense granule proteins, and uncharacterized hypothetical proteins were identified. Several of the newly identified hypothetical proteins were validated to be novel components of the cyst wall and tagged with BirA* to expand the model of the cyst wall interactome. Community detection of the cyst wall interactome model revealed three distinct clusters: a dense granule, a cyst matrix, and a cyst wall cluster. Characterization of several of the identified cyst wall proteins using genetic strategies revealed that MCP3 affects in vivo cyst sizes. This study provides a model of the potential protein interactions within the cyst wall and the groundwork to understand cyst wall formation. IMPORTANCE A model of the cyst wall interactome was constructed using proteins identified through BioID. The proteins within this cyst wall interactome model encompass several proteins identified in a prior characterization of the cyst wall proteome. This model provides a more comprehensive understanding of the composition of the cyst wall and may lead to insights on how the cyst wall is formed.