Systematic characterization of all Toxoplasma gondii TBC domain-containing proteins identifies an essential regulator of Rab2 in the secretory pathway.

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide-dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii, precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC) domain-containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC domain-containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. Second, we use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein, which localizes to the endoplasmic reticulum (ER), is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and mitochondrial morphology. TgTBC9 knockdown also results in the formation of large lipid droplets (LDs) and multi-membranous structures surrounded by ER membranes, further indicating a disruption of ER functions. We show that the conserved dual-finger active site in the TBC domain of the protein is critical for its GTPase-activating protein (GAP) function and that the Plasmodium falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast 2 hybrid analyses that TgTBC9 preferentially binds Rab2, indicating that the TBC9-Rab2 pair controls ER morphology and vesicular trafficking in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan and provide new insight into intracellular vesicle trafficking in T. gondii.

PEXEL is a proteolytic maturation site for both exported and non-exported Plasmodium proteins.

Obligate intracellular malaria parasites dramatically remodel their erythrocyte host through effector protein export to create a niche for survival. Most exported proteins contain a pentameric Plasmodium export element (PEXEL)/host-targeting motif that is cleaved in the parasite ER by the aspartic protease Plasmepsin V (PMV). This processing event exposes a mature N terminus required for translocation into the host cell and is not known to occur in non-exported proteins. Here, we report that the non-exported parasitophorous vacuole protein UIS2 contains a bona fide PEXEL motif that is processed in the P. falciparum blood stage. While the N termini of exported proteins containing the PEXEL and immediately downstream ~10 residues are sufficient to mediate translocation into the RBC, the equivalent UIS2 N terminus does not promote the export of a reporter. Curiously, the UIS2 PEXEL contains an unusual aspartic acid at the fourth position, which constitutes the extreme N-terminal residue following PEXEL cleavage (P1', RIL↓DE). Using a series of chimeric reporter fusions, we show that Asp at P1' is permissive for PMV processing but abrogates export. Moreover, mutation of this single UIS2 residue to alanine enables export, reinforcing that the mature N terminus mediates export, not PEXEL processing per se. Prompted by this observation, we further show that PEXEL sequences in the N termini of other non-exported rhoptry proteins are also processed, suggesting that PMV may be a more general secretory maturase than previously appreciated, similar to orthologs in related apicomplexans. Our findings provide new insight into the unique N-terminal constraints that mark proteins for export.IMPORTANCEHost erythrocyte remodeling by malaria parasite-exported effector proteins is critical to parasite survival and disease pathogenesis. In the deadliest malaria parasite Plasmodium falciparum, most exported proteins undergo proteolytic maturation via recognition of the pentameric Plasmodium export element (PEXEL)/host-targeting motif by the aspartic protease Plasmepsin V, which exposes a mature N terminus that is conducive for export into the erythrocyte host cell. While PEXEL processing is considered a unique mark of exported proteins, we demonstrate that PEXEL motifs are present and processed in non-exported proteins. Importantly, we show that specific residues at the variable fourth position of the PEXEL motif inhibit export despite being permissive for processing, reinforcing that features of the mature N terminus, and not PEXEL cleavage, identify cargo for export. This opens the door to further inquiry into the nature and evolution of the PEXEL motif.

Characterization and functional analysis of Toxoplasma Golgi-associated proteins identified by proximity labelling.

Toxoplasma gondii possesses a highly polarized secretory pathway that contains both broadly conserved eukaryotic organelles and unique apicomplexan organelles which play essential roles in the parasite's lytic cycle. As in other eukaryotes, the T. gondii Golgi apparatus sorts and modifies proteins prior to their distribution to downstream organelles. Many of the typical trafficking factors found involved in these processes are missing from apicomplexan genomes, suggesting that these parasites have evolved unique proteins to fill these roles. Here we identify a novel Golgi-localizing protein (ULP1) which contains structural homology to the eukaryotic trafficking factor p115/Uso1. We demonstrate that depletion of ULP1 leads to a dramatic reduction in parasite fitness and replicative ability. Using ULP1 as bait for TurboID proximity labelling and immunoprecipitation, we identify eleven more novel Golgi-associated proteins and demonstrate that ULP1 interacts with the T. gondii COG complex. These proteins include both conserved trafficking factors and parasite-specific proteins. Using a conditional knockdown approach, we assess the effect of each of these eleven proteins on parasite fitness. Together, this work reveals a diverse set of novel T. gondii Golgi-associated proteins that play distinct roles in the secretory pathway. As several of these proteins are absent outside of the Apicomplexa, they represent potential targets for the development of novel therapeutics against these parasites. Importance: Apicomplexan parasites such as Toxoplasma gondii infect a large percentage of the world's population and cause substantial human disease. These widespread pathogens use specialized secretory organelles to infect their host cells, modulate host cell functions, and cause disease. While the functions of the secretory organelles are now better understood, the Golgi apparatus of the parasite remains largely unexplored, particularly regarding parasite-specific innovations that may help direct traffic intracellularly. In this work, we characterize ULP1, a protein that is unique to parasites but shares structural similarity to the eukaryotic trafficking factor p115/Uso1. We show that ULP1 plays an important role in parasite replication and demonstrate that it interacts with the conserved oligomeric Golgi (COG) complex. We then use ULP1 proximity labelling to identify eleven additional Golgi-associated proteins which we functionally analyze via conditional knockdown. This work expands our knowledge of the Toxoplasma Golgi apparatus and identifies potential targets for therapeutic intervention.

Wild-type C-Raf gene dosage and dimerization drive prostate cancer metastasis.

Mutated Ras and Raf kinases are well-known to promote cancer metastasis via flux through the Ras/Raf/MEK/ERK (mitogen-activated protein kinase [MAPK]) pathway. A role for non-mutated Raf in metastasis is also emerging, but the key mechanisms remain unclear. Elevated expression of any of the three wild-type Raf family members (C, A, or B) can drive metastasis. We utilized an in vivo model to show that wild-type C-Raf overexpression can promote metastasis of immortalized prostate cells in a gene dosage-dependent manner. Analysis of the transcriptomic and phosphoproteomic landscape indicated that C-Raf-driven metastasis is accompanied by upregulated MAPK signaling. Use of C-Raf mutants demonstrated that the dimerization domain, but not its kinase activity, is essential for metastasis. Endogenous Raf monomer knockouts revealed that C-Raf's ability to form dimers with endogenous Raf molecules is important for promoting metastasis. These data identify wild-type C-Raf heterodimer signaling as a potential target for treating metastatic disease.

ACD15, ACD21, and SLN regulate the accumulation and mobility of MBD6 to silence genes and transposable elements.

DNA methylation mediates silencing of transposable elements and genes in part via recruitment of the Arabidopsis MBD5/6 complex, which contains the methyl-CpG binding domain (MBD) proteins MBD5 and MBD6, and the J-domain containing protein SILENZIO (SLN). Here, we characterize two additional complex members: α-crystalline domain (ACD) containing proteins ACD15 and ACD21. We show that they are necessary for gene silencing, bridge SLN to the complex, and promote higher-order multimerization of MBD5/6 complexes within heterochromatin. These complexes are also highly dynamic, with the mobility of MBD5/6 complexes regulated by the activity of SLN. Using a dCas9 system, we demonstrate that tethering the ACDs to an ectopic site outside of heterochromatin can drive a massive accumulation of MBD5/6 complexes into large nuclear bodies. These results demonstrate that ACD15 and ACD21 are critical components of the gene-silencing MBD5/6 complex and act to drive the formation of higher-order, dynamic assemblies at CG methylation (meCG) sites.

Adenovirus E1A binding to DCAF10 targets proteasomal degradation of RUVBL1/2 AAA+ ATPases required for quaternary assembly of multiprotein machines, innate immunity, and responses to metabolic stress.

IMPORTANCE: Inactivation of EP300/CREBB paralogous cellular lysine acetyltransferases (KATs) during the early phase of infection is a consistent feature of DNA viruses. The cell responds by stabilizing transcription factor IRF3 which activates transcription of scores of interferon-stimulated genes (ISGs), inhibiting viral replication. Human respiratory adenoviruses counter this by assembling a CUL4-based ubiquitin ligase complex that polyubiquitinylates RUVBL1 and 2 inducing their proteasomal degradation. This inhibits accumulation of active IRF3 and the expression of anti-viral ISGs, allowing replication of the respiratory HAdVs in the face of inhibition of EP300/CBEBBP KAT activity by the N-terminal region of E1A.

A synthetic coolant (WS-23) in disposable electronic cigarettes impairs cytoskeletal function in EpiAirway microtissues exposed at the air liquid interface.

The design of popular disposable electronic cigarettes (ECs) was analyzed, and the concentrations of WS-23, a synthetic coolant, in EC fluids were determined for 22 devices from 4 different brands. All products contained WS-23 in concentrations that ranged from 1.0 to 40.1 mg/mL (mean = 21.4 ± 9.2 mg/mL). To determine the effects of WS-23 on human bronchial epithelium in isolation of other chemicals, we exposed EpiAirway 3-D microtissues to WS-23 at the air liquid interface (ALI) using a cloud chamber that generated aerosols without heating. Proteomics analysis of exposed tissues revealed that the cytoskeleton was a major target of WS-23. BEAS-2B cells were exposed to WS-23 in submerged culture to validate the main results from proteomics. F-actin, which was visualized with phalloidin, decreased concentration dependently in WS-23 treated BEAS-2B cells, and cells became immotile in concentrations above 1.5 mg/mL. Gap closure, which depends on both cell proliferation and migration, was inhibited by 0.45 mg/mL of WS-23. These data show that WS-23 is being added to popular EC fluids at concentrations that can impair processes dependent on the actin cytoskeleton and disturb homeostasis of the bronchial epithelium. The unregulated use of WS-23 in EC products may harm human health.

Unexpected metabolic rewiring of CO2 fixation in H2-mediated materials-biology hybrids.

A hybrid approach combining water-splitting electrochemistry and H2-oxidizing, CO2-fixing microorganisms offers a viable solution for producing value-added chemicals from sunlight, water, and air. The classic wisdom without thorough examination to date assumes that the electrochemistry in such a H2-mediated process is innocent of altering microbial behavior. Here, we report unexpected metabolic rewiring induced by water-splitting electrochemistry in H2-oxidizing acetogenic bacterium Sporomusa ovata that challenges such a classic view. We found that the planktonic S. ovata is more efficient in utilizing reducing equivalent for ATP generation in the materials-biology hybrids than cells grown with H2 supply, supported by our metabolomic and proteomic studies. The efficiency of utilizing reducing equivalents and fixing CO2 into acetate has increased from less than 80% of chemoautotrophy to more than 95% under electroautotrophic conditions. These observations unravel previously underappreciated materials' impact on microbial metabolism in seemingly simply H2-mediated charge transfer between biotic and abiotic components. Such a deeper understanding of the materials-biology interface will foster advanced design of hybrid systems for sustainable chemical transformation.

Identification of IMC43, a novel IMC protein that collaborates with IMC32 to form an essential daughter bud assembly complex in Toxoplasma gondii.

The inner membrane complex (IMC) of Toxoplasma gondii is essential for all phases of the parasite's life cycle. One of its most critical roles is to act as a scaffold for the assembly of daughter buds during replication by endodyogeny. While many daughter IMC proteins have been identified, most are recruited after bud initiation and are not essential for parasite fitness. Here, we report the identification of IMC43, a novel daughter IMC protein that is recruited at the earliest stages of daughter bud initiation. Using an auxin-inducible degron system we show that depletion of IMC43 results in aberrant morphology, dysregulation of endodyogeny, and an extreme defect in replication. Deletion analyses reveal a region of IMC43 that plays a role in localization and a C-terminal domain that is essential for the protein's function. TurboID proximity labelling and a yeast two-hybrid screen using IMC43 as bait identify 30 candidate IMC43 binding partners. We investigate two of these: the essential daughter protein IMC32 and a novel daughter IMC protein we named IMC44. We show that IMC43 is responsible for regulating the localization of both IMC32 and IMC44 at specific stages of endodyogeny and that this regulation is dependent on the essential C-terminal domain of IMC43. Using pairwise yeast two-hybrid assays, we determine that this region is also sufficient for binding to both IMC32 and IMC44. As IMC43 and IMC32 are both essential proteins, this work reveals the existence of a bud assembly complex that forms the foundation of the daughter IMC during endodyogeny.

ACD15, ACD21 and SLN regulate accumulation and mobility of MBD6 to silence genes and transposable elements.

DNA methylation mediates silencing of transposable elements and genes in part via recruitment of the Arabidopsis MBD5/6 complex, which contains the methyl-CpG-binding domain (MBD) proteins MBD5 and MBD6, and the J-domain containing protein SILENZIO (SLN). Here we characterize two additional complex members: α-crystalline domain containing proteins ACD15 and ACD21. We show that they are necessary for gene silencing, bridge SLN to the complex, and promote higher order multimerization of MBD5/6 complexes within heterochromatin. These complexes are also highly dynamic, with the mobility of complex components regulated by the activity of SLN. Using a dCas9 system, we demonstrate that tethering the ACDs to an ectopic site outside of heterochromatin can drive massive accumulation of MBD5/6 complexes into large nuclear bodies. These results demonstrate that ACD15 and ACD21 are critical components of gene silencing complexes that act to drive the formation of higher order, dynamic assemblies.

The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.

Toxoplasma gondii's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole (PV) in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both acute and chronic infections. Murine macrophages infected with ∆gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro, which was confirmed with reduced IL-12 and interferon-gamma in vivo. This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the nuclear factor-κB (NF-κB) complex. While GRA15 similarly regulates NF-κB, infection with ∆gra83/∆gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labeling experiments to reveal candidate GRA83 interacting T. gondii-derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit the parasite burden. Importance Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma's ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection is important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection.

FAP106 is an interaction hub for assembling microtubule inner proteins at the cilium inner junction.

Motility of pathogenic protozoa depends on flagella (synonymous with cilia) with axonemes containing nine doublet microtubules (DMTs) and two singlet microtubules. Microtubule inner proteins (MIPs) within DMTs influence axoneme stability and motility and provide lineage-specific adaptations, but individual MIP functions and assembly mechanisms are mostly unknown. Here, we show in the sleeping sickness parasite Trypanosoma brucei, that FAP106, a conserved MIP at the DMT inner junction, is required for trypanosome motility and functions as a critical interaction hub, directing assembly of several conserved and lineage-specific MIPs. We use comparative cryogenic electron tomography (cryoET) and quantitative proteomics to identify MIP candidates. Using RNAi knockdown together with fitting of AlphaFold models into cryoET maps, we demonstrate that one of these candidates, MC8, is a trypanosome-specific MIP required for parasite motility. Our work advances understanding of MIP assembly mechanisms and identifies lineage-specific motility proteins that are attractive targets to consider for therapeutic intervention.

Toxoplasma gondii encodes an array of TBC-domain containing proteins including an essential regulator that targets Rab2 in the secretory pathway.

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host-cell manipulation and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii , precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC)-domain containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC-domain containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. We then use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein that localizes to the ER is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and Golgi apparatus. We show that the conserved dual-finger active site in the TBC-domain of the protein is critical for its GTPase-activating protein (GAP) function and that the P. falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast two hybrid analyses that TgTBC9 directly binds Rab2, indicating that this TBC-Rab pair controls ER to Golgi traffic in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan, provide new insight into intracellular vesicle trafficking in T. gondii , and reveal promising targets for the design of novel therapeutics that can specifically target apicomplexan parasites.

The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.

Toxoplasma gondii 's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both the acute and chronic infection. Murine macrophages infected with Δ gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro , which was confirmed with reduced IL-12 and interferon gamma (IFN-γ) in vivo . This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the NF-κB complex. While GRA15 similarly regulates NF-κB, infection with Δ gra83/ Δ gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labelling experiments to reveal candidate GRA83 interacting T. gondii derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit parasite burden. Importance: Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma' s ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection are important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection.

PEXEL is a proteolytic maturation site for both exported and non-exported Plasmodium proteins.

Obligate intracellular malaria parasites dramatically remodel their erythrocyte host through effector protein export to create a niche for survival. Most exported proteins contain a pentameric P lasmodium ex port el ement (PEXEL)/Host Targeting Motif that is cleaved in the parasite ER by the aspartic protease Plasmepsin V (PMV). This processing event exposes a mature N-terminus required for translocation into the host cell and is not known to occur in non-exported proteins. Here we report that the non-exported parasitophorous vacuole protein UIS2 contains a bona fide PEXEL motif that is processed in the P. falciparum blood-stage. While the N-termini of exported proteins containing the PEXEL and immediately downstream ∼10 residues is sufficient to mediate translocation into the RBC, the equivalent UIS2 N-terminus does not promote export of a reporter. Curiously, the UIS2 PEXEL contains an unusual aspartic acid at the fourth position which constitutes the extreme N-terminal residue following PEXEL cleavage (P1', RILτDE). Using a series of chimeric reporter fusions, we show that Asp at P1' is permissive for PMV processing but abrogates export. Moreover, mutation of this single UIS2 residue to alanine enables export, reinforcing that the mature N-terminus mediates export, not PEXEL processing per se . Prompted by this observation, we further show that PEXEL sequences in the N-termini of other non-exported rhoptry proteins are also processed, suggesting that PMV may be a more general secretory maturase than previously appreciated, similar to orthologs in related apicomplexans. Our findings provide new insight into the unique N-terminal constraints that mark proteins for export. Importance: Host erythrocyte remodeling by malaria parasite exported effector proteins is critical to parasite survival and disease pathogenesis. In the deadliest malaria parasite Plasmodium falciparum , most exported proteins undergo proteolytic maturation via recognition of the pentameric P lasmodium ex port el ement (PEXEL)/Host Targeting motif by the aspartic protease Plasmepsin V (PMV) which exposes a mature N-terminus that is conducive for export into the erythrocyte host cell. While PEXEL processing is considered a unique mark of exported proteins, we demonstrate PEXEL motifs are present and processed in non-exported proteins. Importantly, we show that specific residues at the variable fourth position of the PEXEL motif inhibit export despite being permissive for processing by PMV, reinforcing that features of the mature N-terminus, and not PEXEL cleavage, identify cargo for export cargo. This opens the door to further inquiry into the nature and evolution of the PEXEL motif.

Role of cytoneme structures and extracellular vesicles in Trichomonas vaginalis parasite-parasite communication.

Trichomonas vaginalis, the etiologic agent of the most common non-viral sexually transmitted infection worldwide. With an estimated annual prevalence of 276 million new cases, mixed infections with different parasite strains are expected. Although it is known that parasites interact with their host to enhance their own survival and transmission, evidence of mixed infections call into question the extent to which unicellular parasites communicate with each other. Here, we demonstrated that different T. vaginalis strains can communicate through the formation of cytoneme-like membranous cell connections. We showed that cytonemes formation of an adherent parasite strain (CDC1132) is affected in the presence of a different strain (G3 or B7RC2). Our findings provide evidence that this effect is contact-independent and that extracellular vesicles (EVs) are responsible, at least in part, of the communication among strains. We found that EVs isolated from G3, B7RC2, and CDC1132 strains contain a highly distinct repertoire of proteins, some of them involved in signaling and communication, among other functions. Finally, we showed that parasite adherence to host cells is affected by communication between strains as binding of adherent T. vaginalis CDC1132 strain to prostate cells is significantly higher in the presence of G3 or B7RC2 strains. We also observed that a poorly adherent parasite strain (G3) adheres more strongly to prostate cells in the presence of an adherent strain. The study of signaling, sensing, and cell communication in parasitic organisms will enhance our understanding of the basic biological characteristics of parasites, which may have important consequences in pathogenesis.

IMC29 Plays an Important Role in Toxoplasma Endodyogeny and Reveals New Components of the Daughter-Enriched IMC Proteome.

The Toxoplasma inner membrane complex (IMC) is a unique organelle that plays critical roles in parasite motility, invasion, egress, and replication. The IMC is delineated into the apical, body, and basal regions, defined by proteins that localize to these distinct subcompartments. The IMC can be further segregated by proteins that localize specifically to the maternal IMC, the daughter bud IMC, or both. While the function of the maternal IMC has been better characterized, the precise roles of most daughter IMC components remain poorly understood. Here, we demonstrate that the daughter protein IMC29 plays an important role in parasite replication. We show that Δimc29 parasites exhibit severe replication defects, resulting in substantial growth defects and loss of virulence. Deletion analyses revealed that IMC29 localization is largely dependent on the N-terminal half of the protein containing four predicted coiled-coil domains while IMC29 function requires a short C-terminal helical region. Using proximity labeling, we identify eight novel IMC proteins enriched in daughter buds, significantly expanding the daughter IMC proteome. We additionally report four novel proteins with unique localizations to the interface between two parasites or to the outer face of the IMC, exposing new subregions of the organelle. Together, this work establishes IMC29 as an important early daughter bud component of replication and uncovers an array of new IMC proteins that provides important insights into this organelle. IMPORTANCE The inner membrane complex (IMC) is a conserved structure across the Apicomplexa phylum, which includes obligate intracellular parasites that cause toxoplasmosis, malaria, and cryptosporidiosis. The IMC is critical for the parasite to maintain its intracellular lifestyle, particularly in providing a scaffold for daughter bud formation during parasite replication. While many IMC proteins in the later stages of division have been identified, components of the early stages of division remain unknown. Here, we focus on the early daughter protein IMC29, demonstrating that it is crucial for faithful parasite replication and identifying specific regions of the protein that are important for its localization and function. We additionally use proximity labeling to reveal a suite of daughter-enriched IMC proteins, which represent promising candidates to further explore this IMC subcompartment.

High-Field Asymmetric Waveform Ion Mobility Spectrometry Interface Enhances Parallel Reaction Monitoring on an Orbitrap Mass Spectrometer.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) enables gas-phase separations on a chromatographic time scale and has become a useful tool for proteomic applications. Despite its emerging utility, however, the molecular determinants underlying peptide separation by FAIMS have not been systematically investigated. Here, we characterize peptide transmission in a FAIMS device across a broad range of compensation voltages (CVs) and used machine learning to identify charge state and three-dimensional (3D) electrostatic peptide potential as major contributors to peptide intensity at a given CV. We also demonstrate that the machine learning model can be used to predict optimized CV values for peptides, which significantly improves parallel reaction monitoring workflows. Together, these data provide insight into peptide separation by FAIMS and highlight its utility in targeted proteomic applications.

A genetic model for in vivo proximity labelling of the mammalian secretome.

Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalysed proximity labelling enables the identification of proteins within a specific cellular compartment. Here, we report a BirA*G3 mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mouse, widespread labelling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) proteomics revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, highlighting both conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte-specific activation of BirA*G3 highlighted liver-specific biotinylated secretome profiles. The BirA*G3 mouse model demonstrates enhanced labelling efficiency and tissue specificity over viral transduction approaches and will facilitate a deeper understanding of secretory protein interplay in development, and in healthy and diseased adult states.

Iron-regulated assembly of the cytosolic iron-sulfur cluster biogenesis machinery.

The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.