Discovery of New Microneme Proteins in Cryptosporidium parvum and Implication of the Roles of a Rhomboid Membrane Protein (CpROM1) in Host–Parasite Interaction

Apicomplexan parasites possess several unique secretory organelles, including rhoptries, micronemes, and dense granules, which play critical roles in the invasion of host cells. The molecular content of these organelles and their biological roles have been well-studied in Toxoplasma and Plasmodium, but are underappreciated in Cryptosporidium, which contains many parasites of medical and veterinary importance. Only four proteins have previously been identified or proposed to be located in micronemes, one of which, GP900, was confirmed using immunogold electron microscopy (IEM) to be present in the micronemes of intracellular merozoites. Here, we report on the discovery of four new microneme proteins (MICs) in the sporozoites of the zoonotic species C. parvum, identified using immunofluorescence assay (IFA). These proteins are encoded by cgd3_980, cgd1_3550, cgd1_3680, and cgd2_1590. The presence of the protein encoded by cgd3_980 in sporozoite micronemes was further confirmed using IEM. Cgd3_980 encodes one of the three C. parvum rhomboid peptidases (ROMs) and is, thus, designated CpROM1. IEM also confirmed the presence of CpROM1 in the micronemes of intracellular merozoites, parasitophorous vacuole membranes (PVM), and feeder organelles (FO). CpROM1 was enriched in the pellicles and concentrated at the host cell–parasite interface during the invasion of sporozoites and its subsequent transformation into trophozoites. CpROM1 transcript levels were also higher in oocysts and excysted sporozoites than in the intracellular parasite stages. These observations indicate that CpROM1, an intramembrane peptidase with membrane proteolytic activity, is involved in host–parasite interactions, including invasion and proteostasis of PVM and FO.

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Biotinylation of the Neospora caninum parasitophorous vacuole reveals novel dense granule proteins

Parasites & Vectors ◽

10.1186/s13071-021-05023-7 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Congshan Yang ◽

Chenrong Wang ◽

Jing Liu ◽

Qun Liu

Keyword(s):

Neospora Caninum ◽

Gene Knockout ◽

Parasitophorous Vacuole ◽

Dense Granule ◽

Host Cells ◽

Spectrometric Analysis ◽

Data Set ◽

Dense Granules ◽

Granule Proteins

Abstract Background Neospora caninum is an obligate intracellular parasite that invades host cells and replicates within the parasitophorous vacuole (PV), which resists fusion with host cell lysosomal compartments. To modify the PV, the parasite secretes an array of proteins, including dense granule proteins (GRAs). The vital role of GRAs in the Neospora life cycle cannot be overestimated. Despite this important role, only a subset of these proteins have been identified, and most of their functions have not been elucidated. Our previous study demonstrated that NcGRA17 is specifically targeted to the delimiting membrane of the parasitophorous vacuole membrane (PVM). In this study, we utilize proximity-dependent biotin identification (BioID) to identify novel components of the dense granules. Methods NcGRA17 was BirA* epitope-tagged in the Nc1 strain utilizing the CRISPR/Cas9 system to create a fusion of NcGRA17 with the biotin ligase BirA*. The biotinylated proteins were affinity-purified for mass spectrometric analysis, and the candidate GRA proteins from BioID data set were identified by gene tagging. To verify the biological role of novel identified GRA proteins, we constructed the NcGRA23 and NcGRA11 (a–e) knockout strains using the CRISPR/Cas9 system and analyzed the phenotypes of these mutants. Results Using NcGRA17-BirA* fusion protein as bait, we have identified some known GRAs and verified localization of 11 novel GRA proteins by gene endogenous tagging or overexpression in the Nc1 strain. We proceeded to functionally characterize NcGRA23 and NcGRA11 (a–e) by gene knockout. The lack of NcGRA23 or NcGRA11 (a–e) did not affect the parasite propagation in vitro and virulence in vivo. Conclusions In summary, our findings reveal that BioID is effective in discovering novel constituents of N. caninum dense granules. The exact biological functions of the novel GRA proteins are yet unknown, but this could be explored in future studies. Graphical abstract

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Toxoplasma Effectors Targeting Host Signaling and Transcription

Clinical Microbiology Reviews ◽

10.1128/cmr.00005-17 ◽

2017 ◽

Vol 30 (3) ◽

pp. 615-645 ◽

Cited By ~ 142

Author(s):

Mohamed-Ali Hakimi ◽

Philipp Olias ◽

L. David Sibley

Keyword(s):

Host Cells ◽

Content Type ◽

Apicomplexan Parasites ◽

Small Noncoding Rnas ◽

Dense Granules ◽

Host Pathogen Interaction ◽

Morphological Studies ◽

Specific Host ◽

Host Pathogen ◽

Host Signaling

SUMMARY Early electron microscopy studies revealed the elaborate cellular features that define the unique adaptations of apicomplexan parasites. Among these were bulbous rhoptry (ROP) organelles and small, dense granules (GRAs), both of which are secreted during invasion of host cells. These early morphological studies were followed by the exploration of the cellular contents of these secretory organelles, revealing them to be comprised of highly divergent protein families with few conserved domains or predicted functions. In parallel, studies on host-pathogen interactions identified many host signaling pathways that were mysteriously altered by infection. It was only with the advent of forward and reverse genetic strategies that the connections between individual parasite effectors and the specific host pathways that they targeted finally became clear. The current repertoire of parasite effectors includes ROP kinases and pseudokinases that are secreted during invasion and that block host immune pathways. Similarly, many secretory GRA proteins alter host gene expression by activating host transcription factors, through modification of chromatin, or by inducing small noncoding RNAs. These effectors highlight novel mechanisms by which T. gondii has learned to harness host signaling to favor intracellular survival and will guide future studies designed to uncover the additional complexity of this intricate host-pathogen interaction.

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First Characterization of theNeospora caninumDense Granule Protein GRA9

BioMed Research International ◽

10.1155/2017/6746437 ◽

2017 ◽

Vol 2017 ◽

pp. 1-15 ◽

Cited By ~ 5

Author(s):

Margret Leineweber ◽

Katrin Spekker-Bosker ◽

Vanessa Ince ◽

Gereon Schares ◽

Andrew Hemphill ◽

...

Keyword(s):

Host Cell ◽

Cell Invasion ◽

Neospora Caninum ◽

Parasitophorous Vacuole ◽

Dense Granule ◽

Immunogold Electron Microscopy ◽

Host Cell Invasion ◽

Dense Granules ◽

Granule Protein

The obligate intracellular apicomplexan parasiteNeospora caninum (N. caninum)is closely related toToxoplasma gondii (T. gondii). The dense granules, which are present in all apicomplexan parasites, are important secretory organelles. Dense granule (GRA) proteins are released into the parasitophorous vacuole (PV) following host cell invasion and are known to play important roles in the maintenance of the host-parasite relationship and in the acquisition of nutrients. Here, we provide a detailed characterization of theN. caninumdense granule protein NcGRA9. The in silico genomic organization and key protein characteristics are described. Immunofluorescence-based localization studies revealed that NcGRA9 is located in the dense granules and is released into the interior of the PV following host cell invasion. Immunogold-electron microscopy confirmed the dense granule localization and showed that NcGRA9 is associated with the intravacuolar network. In addition, NcGRA9 is found in the “excreted secreted antigen” (ESA) fraction ofN. caninum. Furthermore, by analysing the distribution of truncated versions of NcGRA9, we provide evidence that the C-terminal region of this protein is essential for the targeting of NcGRA9 into the dense granules ofN. caninum, and the truncated proteins show reduced secretion.

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CRISPR/Cas9-Based Knockout of GNAQ Reveals Differences in Host Cell Signaling Necessary for Egress of Apicomplexan Parasites

mSphere ◽

10.1128/msphere.01001-20 ◽

2020 ◽

Vol 5 (6) ◽

pp. e01001-20

Author(s):

Paul-Christian Burda ◽

Hugo Bisio ◽

Jean-Baptiste Marq ◽

Dominique Soldati-Favre ◽

Volker T. Heussler

Keyword(s):

Host Cell ◽

Parasitophorous Vacuole ◽

Host Cells ◽

Infected Host ◽

Protein Subunit ◽

Host Cell Membrane ◽

Guanine Nucleotide Binding Protein ◽

Liver Stage ◽

Content Type ◽

Apicomplexan Parasites

ABSTRACTToxoplasma gondii and members of the genus Plasmodium are obligate intracellular parasites that leave their infected host cell upon a tightly controlled process of egress. Intracellular replication of the parasites occurs within a parasitophorous vacuole, and its membrane as well as the host plasma membrane need to be disrupted during egress, leading to host cell lysis. While several parasite-derived factors governing egress have been identified, much less is known about host cell factors involved in this process. Previously, RNA interference (RNAi)-based knockdown and antibody-mediated depletion identified a host signaling cascade dependent on guanine nucleotide-binding protein subunit alpha q (GNAQ) to be required for the egress of Toxoplasma tachyzoites and Plasmodium blood stage merozoites. Here, we used CRISPR/Cas9 technology to generate HeLa cells deficient in GNAQ and tested their capacity to support the egress of T. gondii tachyzoites and Plasmodium berghei liver stage parasites. While we were able to confirm the importance of GNAQ for the egress of T. gondii, we found that the egress of P. berghei liver stages was unaffected in the absence of GNAQ. These results may reflect differences between the lytic egress process in apicomplexans and the formation of host cell-derived vesicles termed merosomes by P. berghei liver stages.IMPORTANCE The coordinated release of apicomplexan parasites from infected host cells prior to reinvasion is a critical process for parasite survival and the spread of infection. While Toxoplasma tachyzoites and Plasmodium blood stages induce a fast disruption of their surrounding membranes during their egress from host cells, Plasmodium liver stages keep the host cell membrane intact and leave their host cell in host cell-derived vesicles called merosomes. The knockout of GNAQ, a protein involved in G-protein-coupled receptor signaling, demonstrates the importance of this host factor for the lytic egress of T. gondii tachyzoites. Contrastingly, the egress of P. berghei is independent of GNAQ at the liver stage, indicating the existence of a mechanistically distinct strategy to exit the host cell.

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Microneme Protein 6 Is Involved in Invasion and Egress by Neospora caninum

Pathogens ◽

10.3390/pathogens10020201 ◽

2021 ◽

Vol 10 (2) ◽

pp. 201

Author(s):

Xianmei Wang ◽

Di Tang ◽

Fei Wang ◽

Gaowei Jin ◽

Lifang Wang ◽

...

Keyword(s):

Neospora Caninum ◽

Parasite Load ◽

Host Cells ◽

Host Cell Invasion ◽

Caninum Infection ◽

Apicomplexan Parasites ◽

Key Steps ◽

Microneme Protein ◽

Adhesion And Invasion ◽

Microneme Proteins

Background: Neospora caninum, is the etiological agent of neosporosis, an infection that causes abortions in cattle and nervous system dysfunction in dogs. Invasion and egress are the key steps of the pathogenesis of N. caninum infection. Microneme proteins (MICs) play important roles in the recognition, adhesion, and invasion of host cells in other apicomplexan parasites. However, some MICs and their functions in N. caninum infection have rarely been reported. Methods: The homologous recombination strategy was used to investigate the function of MIC6 in N. caninum infection. Results: ΔNcMIC6 showed a smaller plaque size and weakened capacities of invasion and egress than Nc1. Transcription levels of the egress-related genes CDPK1, PLP1, and AMA1 of ΔNcMIC6 were downregulated. Due to the lack of NcMIC6, virulence of the pathogen in the infected mouse was weakened. The subcellular localization of NcMIC1 and NcMIC4 in ΔNcMIC6, however, did not change. Nevertheless, the transcription levels of MIC1 and MIC4 in ΔNcMIC6 were downregulated, and the expression and secretion of MIC1 and MIC4 in ΔNcMIC6 were reduced compared with that in Nc1. Furthermore, the absence of NcMIC6 weakened the virulence in mice and lower parasite load detected in mice brains. Conclusions: NcMIC6 is involved in host cell invasion and egress in N. caninum and may work synergistically with other MICs to regulate the virulence of the pathogen. These data lay a foundation for further research into the function and application of NcMIC6.

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Characterization of the Tubovesicular Network in Plasmodium vivax Liver Stage Hypnozoites and Schizonts

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.687019 ◽

2021 ◽

Vol 11 ◽

Author(s):

Kayla Sylvester ◽

Steven P. Maher ◽

Dora Posfai ◽

Michael K. Tran ◽

McKenna C. Crawford ◽

...

Keyword(s):

Plasmodium Vivax ◽

Molecular Mechanisms ◽

Parasitophorous Vacuole ◽

Clinical Relapse ◽

Liver Stage ◽

Apicomplexan Parasites ◽

Stage Development ◽

Host Parasite ◽

High Resolution Microscopy

Plasmodium is a genus of apicomplexan parasites which replicate in the liver before causing malaria. Plasmodium vivax can also persist in the liver as dormant hypnozoites and cause clinical relapse upon activation, but the molecular mechanisms leading to activation have yet to be discovered. In this study, we use high-resolution microscopy to characterize temporal changes of the P. vivax liver stage tubovesicular network (TVN), a parasitophorous vacuole membrane (PVM)-derived network within the host cytosol. We observe extended membrane clusters, tubules, and TVN-derived vesicles present throughout P. vivax liver stage development. Additionally, we demonstrate an unexpected presence of the TVN in hypnozoites and observe some association of this network to host nuclei. We also reveal that the host water and solute channel aquaporin-3 (AQP3) associates with TVN-derived vesicles and extended membrane clusters. AQP3 has been previously shown to localize to the PVM of P. vivax hypnozoites and liver schizonts but has not yet been shown in association to the TVN. Our results highlight host-parasite interactions occur in both dormant and replicating liver stage P. vivax forms and implicate AQP3 function during this time. Together, these findings enhance our understanding of P. vivax liver stage biology through characterization of the TVN with an emphasis on the presence of this network in dormant hypnozoites.

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The Toxoplasma gondii Dense Granule Protein GRA7 Is Phosphorylated upon Invasion and Forms an Unexpected Association with the Rhoptry Proteins ROP2 and ROP4

Infection and Immunity ◽

10.1128/iai.01667-07 ◽

2008 ◽

Vol 76 (12) ◽

pp. 5853-5861 ◽

Cited By ~ 59

Author(s):

Joe Dan Dunn ◽

Sandeep Ravindran ◽

Seon-Kyeong Kim ◽

John C. Boothroyd

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Parasitophorous Vacuole ◽

Opportunistic Pathogen ◽

Dense Granule ◽

Host Cells ◽

Obligate Intracellular ◽

Dense Granules ◽

Obligate Intracellular Parasite ◽

Granule Protein

ABSTRACT The obligate intracellular parasite Toxoplasma gondii infects warm-blooded animals throughout the world and is an opportunistic pathogen of humans. As it invades a host cell, Toxoplasma forms a novel organelle, the parasitophorous vacuole, in which it resides during its intracellular development. The parasite modifies the parasitophorous vacuole and its host cell with numerous proteins delivered from rhoptries and dense granules, which are secretory organelles unique to the phylum Apicomplexa. For the majority of these proteins, little is known other than their localization. Here we show that the dense granule protein GRA7 is phosphorylated but only in the presence of host cells. Within 10 min of invasion, GRA7 is present in strand-like structures in the host cytosol that contain rhoptry proteins. GRA7 strands also contain GRA1 and GRA3. Independently of its phosphorylation state, GRA7 associates with the rhoptry proteins ROP2 and ROP4 in infected host cells. This is the first report of interactions between proteins secreted from rhoptries and dense granules.

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Intervacuolar Transport and Unique Topology of GRA14, a Novel Dense Granule Protein in Toxoplasma gondii

Infection and Immunity ◽

10.1128/iai.00782-08 ◽

2008 ◽

Vol 76 (11) ◽

pp. 4865-4875 ◽

Cited By ~ 60

Author(s):

Michael E. Rome ◽

Josh R. Beck ◽

Jay M. Turetzky ◽

Paul Webster ◽

Peter J. Bradley

Keyword(s):

Toxoplasma Gondii ◽

Parasitophorous Vacuole ◽

Dense Granule ◽

Obligate Intracellular ◽

Dense Granules ◽

Granule Protein ◽

Host Cytoplasm ◽

C Terminus ◽

Membrane Bound ◽

Host Parasite

ABSTRACT Toxoplasma gondii is an obligate intracellular parasite that resides in the cytoplasm of its host in a unique membrane-bound vacuole known as the parasitophorous vacuole (PV). The membrane surrounding the parasite is remodeled by the dense granules, secretory organelles that release an array of proteins into the vacuole and to the PV membrane (PVM). Only a small portion of the protein constituents of the dense granules have been identified, and little is known regarding their roles in infection or how they are trafficked within the infected host cell. In this report, we identify a novel secreted dense granule protein, GRA14, and show that it is targeted to membranous structures within the vacuole known as the intravacuolar network and to the vacuolar membrane surrounding the parasite. We disrupted GRA14 and exploited the knockout strain to show that GRA14 can be transferred between vacuoles in a coinfection experiment with wild-type parasites. We also show that GRA14 has an unexpected topology in the PVM with its C terminus facing the host cytoplasm and its N terminus facing the vacuolar lumen. These findings have important implications both for the trafficking of GRA proteins to their ultimate destinations and for expectations of functional domains of GRA proteins at the host-parasite interface.

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Disrupting Plasmodium UIS3–host LC3 interaction with a small molecule causes parasite elimination from host cells

Communications Biology ◽

10.1038/s42003-020-01422-1 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Sonali Setua ◽

Francisco J. Enguita ◽

Ângelo Ferreira Chora ◽

Harish Ranga-prasad ◽

Aparajita Lahree ◽

...

Keyword(s):

Host Cell ◽

Small Molecule ◽

Parasitophorous Vacuole ◽

Normal Function ◽

Host Cells ◽

Dependent Manner ◽

Docking Analysis ◽

Phenotypic Screen ◽

Autophagy Pathway ◽

Host Parasite

AbstractThe malaria parasite Plasmodium obligatorily infects and replicates inside hepatocytes surrounded by a parasitophorous vacuole membrane (PVM), which is decorated by the host-cell derived autophagy protein LC3. We have previously shown that the parasite-derived, PVM-resident protein UIS3 sequesters LC3 to avoid parasite elimination by autophagy from hepatocytes. Here we show that a small molecule capable of disrupting this interaction triggers parasite elimination in a host cell autophagy-dependent manner. Molecular docking analysis of more than 20 million compounds combined with a phenotypic screen identified one molecule, C4 (4-{[4-(4-{5-[3-(trifluoromethyl) phenyl]-1,2,4-oxadiazol-3-yl}benzyl)piperazino]carbonyl}benzonitrile), capable of impairing infection. Using biophysical assays, we established that this impairment is due to the ability of C4 to disrupt UIS3–LC3 interaction, thus inhibiting the parasite’s ability to evade the host autophagy response. C4 impacts infection in autophagy-sufficient cells without harming the normal autophagy pathway of the host cell. This study, by revealing the disruption of a critical host–parasite interaction without affecting the host’s normal function, uncovers an efficient anti-malarial strategy to prevent this deadly disease.

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An Uninvited Seat at the Dinner Table: How Apicomplexan Parasites Scavenge Nutrients from the Host

Microorganisms ◽

10.3390/microorganisms9122592 ◽

2021 ◽

Vol 9 (12) ◽

pp. 2592

Author(s):

Federica Piro ◽

Riccardo Focaia ◽

Zhicheng Dou ◽

Silvia Masci ◽

David Smith ◽

...

Keyword(s):

Plasma Membrane ◽

Host Cell ◽

Defense Mechanisms ◽

Parasitophorous Vacuole ◽

Host Cells ◽

Metabolic Demand ◽

Host Cell Membrane ◽

Apicomplexan Parasites ◽

Extracellular Milieu ◽

Cell Defense

Obligate intracellular parasites have evolved a remarkable assortment of strategies to scavenge nutrients from the host cells they parasitize. Most apicomplexans form a parasitophorous vacuole (PV) within the invaded cell, a replicative niche within which they survive and multiply. As well as providing a physical barrier against host cell defense mechanisms, the PV membrane (PVM) is also an important site of nutrient uptake that is essential for the parasites to sustain their metabolism. This means nutrients in the extracellular milieu are separated from parasite metabolic machinery by three different membranes, the host plasma membrane, the PVM, and the parasite plasma membrane (PPM). In order to facilitate nutrient transport from the extracellular environment into the parasite itself, transporters on the host cell membrane of invaded cells can be modified by secreted and exported parasite proteins to maximize uptake of key substrates to meet their metabolic demand. To overcome the second barrier, the PVM, apicomplexan parasites secrete proteins contained in the dense granules that remodel the vacuole and make the membrane permissive to important nutrients. This bulk flow of host nutrients is followed by a more selective uptake of substrates at the PPM that is operated by specific transporters of this third barrier. In this review, we recapitulate and compare the strategies developed by Apicomplexa to scavenge nutrients from their hosts, with particular emphasis on transporters at the parasite plasma membrane and vacuolar solute transporters on the parasite intracellular digestive organelle.

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