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

Rebecca R Pasquarelli / Peter S Back / Jihui Sha / James A Wohlschlegel / Peter J Bradley

PLoS Pathogens, Vol 19, Iss 10, p e

2023 Volume 1011707

Abstract: 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 ... ...

Abstract	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.
Keywords	Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
Subject code	570
Language	English
Publishing date	2023-10-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: RON5 is critical for organization and function of the Toxoplasma moving junction complex.

Josh R Beck / Allan L Chen / Elliot W Kim / Peter J Bradley

PLoS Pathogens, Vol 10, Iss 3, p e

2014 Volume 1004025

Abstract: Apicomplexans facilitate host cell invasion through formation of a tight-junction interface between parasite and host plasma membranes called the moving junction (MJ). A complex of the rhoptry neck proteins RONs 2/4/5/8 localize to the MJ during invasion ...

Abstract	Apicomplexans facilitate host cell invasion through formation of a tight-junction interface between parasite and host plasma membranes called the moving junction (MJ). A complex of the rhoptry neck proteins RONs 2/4/5/8 localize to the MJ during invasion where they are believed to provide a stable anchoring point for host penetration. During the initiation of invasion, the preformed MJ RON complex is injected into the host cell where RON2 spans the host plasma membrane while RONs 4/5/8 localize to its cytosolic face. While much attention has been directed toward an AMA1-RON2 interaction supposed to occur outside the cell, little is known about the functions of the MJ RONs positioned inside the host cell. Here we provide a detailed analysis of RON5 to resolve outstanding questions about MJ complex organization, assembly and function during invasion. Using a conditional knockdown approach, we show loss of RON5 results in complete degradation of RON2 and mistargeting of RON4 within the parasite secretory pathway, demonstrating that RON5 plays a key role in organization of the MJ RON complex. While RON8 is unaffected by knockdown of RON5, these parasites are unable to invade new host cells, providing the first genetic demonstration that RON5 plays a critical role in host cell penetration. Although invasion is not required for injection of rhoptry effectors into the host cytosol, parasites lacking RON5 also fail to form evacuoles suggesting an intact MJ complex is a prerequisite for secretion of rhoptry bulb contents. Additionally, while the MJ has been suggested to function in egress, disruption of the MJ complex by RON5 depletion does not impact this process. Finally, functional complementation of our conditional RON5 mutant reveals that while proteolytic separation of RON5 N- and C-terminal fragments is dispensable, a portion of the C-terminal domain is critical for RON2 stability and function in invasion.
Keywords	Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
Subject code	572
Language	English
Publishing date	2014-03-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Retraction Note

Caroline M. Mota / Allan L. Chen / Kevin Wang / Santhosh Nadipuram / Ajay A. Vashisht / James A. Wohlschlegel / Tiago W. P. Mineo / Peter J. Bradley

Scientific Reports, Vol 8, Iss 1, Pp 1-

New molecular tools in Neospora caninum for studying apicomplexan parasite proteins

2018 Volume 1

Abstract: This paper has been retracted at the request of the authors. ...

Abstract	This paper has been retracted at the request of the authors.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2018-07-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: RETRACTED ARTICLE

Caroline M. Mota / Allan L. Chen / Kevin Wang / Santhosh Nadipuram / Ajay A. Vashisht / James A. Wohlschlegel / Tiago W. P. Mineo / Peter J. Bradley

Scientific Reports, Vol 7, Iss 1, Pp 1-

New molecular tools in Neospora caninum for studying apicomplexan parasite proteins

2017 Volume 12

Abstract: Abstract The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. We have established a system specifically designed for Neospora caninum, and used this system ... ...

Abstract	Abstract The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. We have established a system specifically designed for Neospora caninum, and used this system as a heterologous platform for the expression of foreign genes. Plasmid constructs containing fluorescent proteins or targeted genes of Toxoplasma gondii, driven by N. caninum promoters, have yielded robust expression and correct trafficking of target gene products as assessed by immunofluorescence assays and Western blot analyses. Using this approach, we here demonstrated that N. caninum expressing T. gondii’s GRA15 and ROP16 kinase are biologically active and induced immunological phenotypes consistent with T. gondii strains. N. caninum expressing TgGRA15 differentially disturbed the NF-κB pathway, inducing an increased IL-12 production. On the other hand, N. caninum expressing TgROP16 induced host STAT3 phosphorylation and consequent reduction of IL-12 synthesis. These results indicate that heterologous gene expression in N. caninum is a useful tool for the study of specific gene functions and may allow the identification of antigenic targets responsible for the phenotypic differences observed between these two closely related apicomplexan parasites. Additionally, these observations may prove to be useful for the development of vaccine protocols to control toxoplasmosis and/or neosporosis.
Keywords	Medicine ; R ; Science ; Q
Subject code	572
Language	English
Publishing date	2017-06-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: A Toxoplasma palmitoyl acyl transferase and the palmitoylated armadillo repeat protein TgARO govern apical rhoptry tethering and reveal a critical role for the rhoptries in host cell invasion but not egress.

Josh R Beck / Connie Fung / Kurtis W Straub / Isabelle Coppens / Ajay A Vashisht / James A Wohlschlegel / Peter J Bradley

PLoS Pathogens, Vol 9, Iss 2, p e

2013 Volume 1003162

Abstract: Apicomplexans are obligate intracellular parasites that actively penetrate their host cells to create an intracellular niche for replication. Commitment to invasion is thought to be mediated by the rhoptries, specialized apical secretory organelles that ... ...

Abstract	Apicomplexans are obligate intracellular parasites that actively penetrate their host cells to create an intracellular niche for replication. Commitment to invasion is thought to be mediated by the rhoptries, specialized apical secretory organelles that inject a protein complex into the host cell to form a tight-junction for parasite entry. Little is known about the molecular factors that govern rhoptry biogenesis, their subcellular organization at the apical end of the parasite and subsequent release of this organelle during invasion. We have identified a Toxoplasma palmitoyl acyltransferase, TgDHHC7, which localizes to the rhoptries. Strikingly, conditional knockdown of TgDHHC7 results in dispersed rhoptries that fail to organize at the apical end of the parasite and are instead scattered throughout the cell. While the morphology and content of these rhoptries appears normal, failure to tether at the apex results in a complete block in host cell invasion. In contrast, attachment and egress are unaffected in the knockdown, demonstrating that the rhoptries are not required for these processes. We show that rhoptry targeting of TgDHHC7 requires a short, highly conserved C-terminal region while a large, divergent N-terminal domain is dispensable for both targeting and function. Additionally, a point mutant lacking a key residue predicted to be critical for enzyme activity fails to rescue apical rhoptry tethering, strongly suggesting that tethering of the organelle is dependent upon TgDHHC7 palmitoylation activity. We tie the importance of this activity to the palmitoylated Armadillo Repeats-Only (TgARO) rhoptry protein by showing that conditional knockdown of TgARO recapitulates the dispersed rhoptry phenotype of TgDHHC7 knockdown. The unexpected finding that apicomplexans have exploited protein palmitoylation for apical organelle tethering yields new insight into the biogenesis and function of rhoptries and may provide new avenues for therapeutic intervention against Toxoplasma and related apicomplexan parasites.
Keywords	Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
Subject code	572
Language	English
Publishing date	2013-02-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis

Santhosh M. Nadipuram / Elliot W. Kim / Ajay A. Vashisht / Andrew H. Lin / Hannah N. Bell / Isabelle Coppens / James A. Wohlschlegel / Peter J. Bradley

mBio, Vol 7, Iss 4, p e00808-

2016 Volume 16

Abstract: Toxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent ...

Abstract	Toxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo. Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis.
Keywords	Science ; Q ; Microbiology ; QR1-502
Subject code	572
Language	English
Publishing date	2016-08-01T00:00:00Z
Publisher	American Society for Microbiology
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Identification of Novel O-Linked Glycosylated Toxoplasma Proteins by Vicia villosa Lectin Chromatography.

Kevin Wang / Eric D Peng / Amy S Huang / Dong Xia / Sarah J Vermont / Gaelle Lentini / Maryse Lebrun / Jonathan M Wastling / Peter J Bradley

PLoS ONE, Vol 11, Iss 3, p e

2016 Volume 0150561

Abstract: Toxoplasma gondii maintains its intracellular life cycle using an extraordinary arsenal of parasite-specific organelles including the inner membrane complex (IMC), rhoptries, micronemes, and dense granules. While these unique compartments play critical ... ...

Abstract	Toxoplasma gondii maintains its intracellular life cycle using an extraordinary arsenal of parasite-specific organelles including the inner membrane complex (IMC), rhoptries, micronemes, and dense granules. While these unique compartments play critical roles in pathogenesis, many of their protein constituents have yet to be identified. We exploited the Vicia villosa lectin (VVL) to identify new glycosylated proteins that are present in these organelles. Purification of VVL-binding proteins by lectin affinity chromatography yielded a number of novel proteins that were subjected to further study, resulting in the identification of proteins from the dense granules, micronemes, rhoptries and IMC. We then chose to focus on three proteins identified by this approach, the SAG1 repeat containing protein SRS44, the rhoptry neck protein RON11 as well as a novel IMC protein we named IMC25. To assess function, we disrupted their genes by homologous recombination or CRISPR/Cas9. The knockouts were all successful, demonstrating that these proteins are not essential for invasion or intracellular survival. We also show that IMC25 undergoes substantial proteolytic processing that separates the C-terminal domain from the predicted glycosylation site. Together, we have demonstrated that lectin affinity chromatography is an efficient method of identifying new glycosylated parasite-specific proteins.
Keywords	Medicine ; R ; Science ; Q
Subject code	572
Language	English
Publishing date	2016-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The moving junction protein RON8 facilitates firm attachment and host cell invasion in Toxoplasma gondii.

Kurtis W Straub / Eric D Peng / Bettina E Hajagos / Jessica S Tyler / Peter J Bradley

PLoS Pathogens, Vol 7, Iss 3, p e

2011 Volume 1002007

Abstract: The apicomplexan moving junction (MJ) is a highly conserved structure formed during host cell entry that anchors the invading parasite to the host cell and serves as a molecular sieve of host membrane proteins that protects the parasitophorous vacuole ... ...

Abstract	The apicomplexan moving junction (MJ) is a highly conserved structure formed during host cell entry that anchors the invading parasite to the host cell and serves as a molecular sieve of host membrane proteins that protects the parasitophorous vacuole from host lysosomal destruction. While recent work in Toxoplasma and Plasmodium has reinforced the composition of the MJ as an important association of rhoptry neck proteins (RONs) with micronemal AMA1, little is known of the precise role of RONs in the junction or how they are targeted to the neck subcompartment. We report the first functional analysis of a MJ/RON protein by disrupting RON8 in T. gondii. Parasites lacking RON8 are severely impaired in both attachment and invasion, indicating that RON8 enables the parasite to establish a firm clasp on the host cell and commit to invasion. The remaining junction components frequently drag in trails behind invading knockout parasites and illustrate a malformed complex without RON8. Complementation of Δron8 parasites restores invasion and reveals a processing event at the RON8 C-terminus. Replacement of an N-terminal region of RON8 with a mCherry reporter separates regions within RON8 that are necessary for rhoptry targeting and complex formation from those required for function during invasion. Finally, the invasion defects in Δron8 parasites seen in vitro translate to radically impaired virulence in infected mice, promoting a model in which RON8 has a crucial and unprecedented task in committing Toxoplasma to host cell entry.
Keywords	Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
Subject code	572
Language	English
Publishing date	2011-03-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Three-dimensional coherent X-ray diffractive imaging of whole frozen-hydrated cells

Jose A. Rodriguez / Rui Xu / Chien-Chun Chen / Zhifeng Huang / Huaidong Jiang / Allan L. Chen / Kevin S. Raines / Alan Pryor Jr / Daewoong Nam / Lutz Wiegart / Changyong Song / Anders Madsen / Yuriy Chushkin / Federico Zontone / Peter J. Bradley / Jianwei Miao

IUCrJ, Vol 2, Iss 5, Pp 575-

2015 Volume 583

Abstract: A structural understanding of whole cells in three dimensions at high spatial resolution remains a significant challenge and, in the case of X-rays, has been limited by radiation damage. By alleviating this limitation, cryogenic coherent diffractive ... ...

Abstract	A structural understanding of whole cells in three dimensions at high spatial resolution remains a significant challenge and, in the case of X-rays, has been limited by radiation damage. By alleviating this limitation, cryogenic coherent diffractive imaging (cryo-CDI) can in principle be used to bridge the important resolution gap between optical and electron microscopy in bio-imaging. Here, the first experimental demonstration of cryo-CDI for quantitative three-dimensional imaging of whole frozen-hydrated cells using 8 keV X-rays is reported. As a proof of principle, a tilt series of 72 diffraction patterns was collected from a frozen-hydrated Neospora caninum cell and the three-dimensional mass density of the cell was reconstructed and quantified based on its natural contrast. This three-dimensional reconstruction reveals the surface and internal morphology of the cell, including its complex polarized sub-cellular structure. It is believed that this work represents an experimental milestone towards routine quantitative three-dimensional imaging of whole cells in their natural state with spatial resolutions in the tens of nanometres.
Keywords	coherent diffractive imaging ; cryo-CDI ; three-dimensional imaging ; three-dimensional cellular structure ; coherent diffraction ; X-ray imaging ; Neospora caninum ; Science ; Q
Subject code	500 ; 535
Language	English
Publishing date	2015-09-01T00:00:00Z
Publisher	International Union of Crystallography
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Identification of novel proteins in Neospora caninum using an organelle purification and monoclonal antibody approach.

Catherine S Sohn / Tim T Cheng / Michael L Drummond / Eric D Peng / Sarah J Vermont / Dong Xia / Stephen J Cheng / Jonathan M Wastling / Peter J Bradley

PLoS ONE, Vol 6, Iss 4, p e

2011 Volume 18383

Abstract: Neospora caninum is an important veterinary pathogen that causes abortion in cattle and neuromuscular disease in dogs. Neospora has also generated substantial interest because it is an extremely close relative of the human pathogen Toxoplasma gondii, yet ...

Abstract	Neospora caninum is an important veterinary pathogen that causes abortion in cattle and neuromuscular disease in dogs. Neospora has also generated substantial interest because it is an extremely close relative of the human pathogen Toxoplasma gondii, yet does not appear to infect humans. While for Toxoplasma there are a wide array of molecular tools and reagents available for experimental investigation, relatively few reagents exist for Neospora. To investigate the unique biological features of this parasite and exploit the recent sequencing of its genome, we have used an organelle isolation and monoclonal antibody approach to identify novel organellar proteins and develop a wide array of probes for subcellular localization. We raised a panel of forty-six monoclonal antibodies that detect proteins from the rhoptries, micronemes, dense granules, inner membrane complex, apicoplast, mitochondrion and parasite surface. A subset of the proteins was identified by immunoprecipitation and mass spectrometry and reveal that we have identified and localized many of the key proteins involved in invasion and host interaction in Neospora. In addition, we identified novel secretory proteins not previously studied in any apicomplexan parasite. Thus, this organellar monoclonal antibody approach not only greatly enhances the tools available for Neospora cell biology, but also identifies novel components of the unique biological characteristics of this important veterinary pathogen.
Keywords	Medicine ; R ; Science ; Q
Subject code	572
Language	English
Publishing date	2011-04-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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