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

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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Identification of a Neospora caninum Microneme Protein (NcMIC1) Which Interacts with Sulfated Host Cell Surface Glycosaminoglycans

Infection and Immunity ◽

10.1128/iai.70.6.3187-3198.2002 ◽

2002 ◽

Vol 70 (6) ◽

pp. 3187-3198 ◽

Cited By ~ 34

Author(s):

Nadine Keller ◽

Arunasalam Naguleswaran ◽

Angela Cannas ◽

Nathalie Vonlaufen ◽

Marianne Bienz ◽

...

Keyword(s):

Cell Surface ◽

Host Cell ◽

Solid Phase ◽

Neospora Caninum ◽

Host Cells ◽

Sequence Motif ◽

Host Cell Surface ◽

Adhesive Proteins ◽

Microneme Protein ◽

Microneme Proteins

ABSTRACT The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37°C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.

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CHARACTERIZATION OF PATHOLOGY AND PARASITE LOAD IN OUTBRED AND INBRED MOUSE MODELS OF CHRONIC NEOSPORA CANINUM INFECTION

Journal of Parasitology ◽

10.1645/ge-3290 ◽

2004 ◽

Vol 90 (3) ◽

pp. 579-583 ◽

Cited By ~ 23

Author(s):

Esther Collantes-Fernández ◽

Gema Álvarez-García ◽

Valentín Pérez-Pérez ◽

Juana Pereira-Bueno ◽

Luis Miguel Ortega-Mora

Keyword(s):

Mouse Models ◽

Neospora Caninum ◽

Inbred Mouse ◽

Parasite Load ◽

Caninum Infection

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A new microneme protein of Neospora caninum , NcMIC8 is involved in host cell invasion

Experimental Parasitology ◽

10.1016/j.exppara.2017.01.004 ◽

2017 ◽

Vol 175 ◽

pp. 21-27 ◽

Cited By ~ 3

Author(s):

Jing Wang ◽

Di Tang ◽

Wensheng Li ◽

Jianhai Xu ◽

Qun Liu ◽

...

Keyword(s):

Host Cell ◽

Cell Invasion ◽

Neospora Caninum ◽

Host Cell Invasion ◽

Microneme Protein

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Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail

mBio ◽

10.1128/mbio.00754-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 13

Author(s):

Shruthi Krishnamurthy ◽

Bin Deng ◽

Roxana del Rio ◽

Kerry R. Buchholz ◽

Moritz Treeck ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Transmembrane Domain ◽

Critical Role ◽

Host Cells ◽

Receptor Protein ◽

Host Cell Invasion ◽

Cell Binding ◽

Apicomplexan Parasites

ABSTRACT Apical membrane antigen 1 (AMA1) is a receptor protein on the surface of Toxoplasma gondii that plays a critical role in host cell invasion. The ligand to which T . gondii AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the “moving junction,” a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion. IMPORTANCE Nearly one-third of the world’s population is infected with the protozoan parasite Toxoplasma gondii , which causes life-threatening disease in neonates and immunocompromised individuals. T. gondii is a member of the phylum Apicomplexa, which includes many other parasites of veterinary and medical importance, such as those that cause coccidiosis, babesiosis, and malaria. Apicomplexan parasites grow within their hosts through repeated cycles of host cell invasion, parasite replication, and host cell lysis. Parasites that cannot invade host cells cannot survive or cause disease. AMA1 is a highly conserved protein on the surface of apicomplexan parasites that is known to be important for invasion, and the work presented here reveals new and unexpected insights into AMA1 function. A more complete understanding of the role of AMA1 in invasion may ultimately contribute to the development of new chemotherapeutics designed to disrupt AMA1 function and invasion-related signaling in this important group of human pathogens.

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RecNcMIC3-1-R is a microneme- and rhoptry-based chimeric antigen that protects against acute neosporosis and limits cerebral parasite load in the mouse model for Neospora caninum infection

Vaccine ◽

10.1016/j.vaccine.2011.07.038 ◽

2011 ◽

Vol 29 (40) ◽

pp. 6967-6975 ◽

Cited By ~ 24

Author(s):

Thierry Monney ◽

David Rütti ◽

Michelle Schorer ◽

Karim Debache ◽

Denis Grandgirard ◽

...

Keyword(s):

Mouse Model ◽

Neospora Caninum ◽

Parasite Load ◽

Caninum Infection

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

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.778560 ◽

2021 ◽

Vol 8 ◽

Author(s):

Xin Gao ◽

Jigang Yin ◽

Dongqiang Wang ◽

Xiaohui Li ◽

Ying Zhang ◽

...

Keyword(s):

Parasitophorous Vacuole ◽

Immunofluorescence Assay ◽

Host Cells ◽

Immunogold Electron Microscopy ◽

Apicomplexan Parasites ◽

Subsequent Transformation ◽

Dense Granules ◽

Veterinary Importance ◽

Host Parasite ◽

Microneme Proteins

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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Identification and Characterization of a Neospora caninum Microneme-Associated Protein (NcMIC4) That Exhibits Unique Lactose-Binding Properties

Infection and Immunity ◽

10.1128/iai.72.8.4791-4800.2004 ◽

2004 ◽

Vol 72 (8) ◽

pp. 4791-4800 ◽

Cited By ~ 22

Author(s):

Nadine Keller ◽

Michèle Riesen ◽

Arunasalam Naguleswaran ◽

Nathalie Vonlaufen ◽

Rebecca Stettler ◽

...

Keyword(s):

Host Cell ◽

Gel Electrophoresis ◽

Neospora Caninum ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Vero Cells ◽

Host Cells ◽

Immunogold Electron Microscopy ◽

Binding Properties ◽

Microneme Protein

ABSTRACT Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of Neospora caninum tachyzoites which was purified by α-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the Neospora microneme protein family; the protein was named NcMIC4 (N. caninum microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37°C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced M r but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.

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Temporal Distribution and Parasite Load Kinetics in Blood and Tissues during Neospora caninum Infection in Mice

Infection and Immunity ◽

10.1128/iai.74.4.2491-2494.2006 ◽

2006 ◽

Vol 74 (4) ◽

pp. 2491-2494 ◽

Cited By ~ 54

Author(s):

Esther Collantes-Fernández ◽

Inmaculada López-Pérez ◽

Gema Álvarez-García ◽

Luis M. Ortega-Mora

Keyword(s):

Acute Phase ◽

Neospora Caninum ◽

Clinical Signs ◽

Chronic Phase ◽

Temporal Distribution ◽

Parasite Load ◽

Caninum Infection ◽

Kinetics Of ◽

The Brain

ABSTRACT The kinetics of Neospora caninum loads in mice inoculated with NC-Liv or NC-1 isolates were studied. The acute phase was characterized by parasitemia and the detection of parasite DNA in several organs, whereas during the chronic phase, the parasite was detected mainly in the brain. Mice infected with NC-Liv developed clinical signs, showing higher brain parasite burdens than NC-1-infected mice.

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High Resolution Low Voltage Field Emission Scanning Electron Microscopy on De-Embedded Thick Sections Reveals Structural Interactions of the Surface of the Apicomplexan Parasite Toxoplasma Gondii with the Host Cell in the Parasitophorous Vacuole

Microscopy and Microanalysis ◽

10.1017/s143192760003573x ◽

2000 ◽

Vol 6 (S2) ◽

pp. 648-649

Author(s):

Heide Schatten ◽

David Sibley ◽

Hans Ris

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Biochemical Characterization ◽

Low Voltage ◽

Cytoskeletal Proteins ◽

Host Cells ◽

Farm Animals ◽

Host Cell Invasion ◽

Apicomplexan Parasites

The protozoan parasite Toxoplasma gondii represents a large group of Apicomplexan parasites with a highly unusual motility system that is crucial for cell locomotion and host cell invasion. Studying the motility system and interactions with host cells will increase our knowledge on how to prevent infection. Apicomplexan parasites can cause considerable health problems to animals including sheep, goats, pigs, and chicken. Eimeria is known as a pathogen of coccidiosis in chicken, and Cryptosporidium causes cryptospiridiosis in cattle and other farm animals. Toxoplasma gondii can cause infection that results in abortion, central nervous disorders and death of stressed and immunocompromised farm animals. Common to all Apicomplexan parasites is an unconventional motility system that is thought to be actively involved in host-cell invasion. However, the structural and biochemical characterization of Apicomplexan parasites has proven more difficult than previously thought because of genetic and behavioral differences of the parasites’ cytoskeletal proteins that differ from those in well studied mammalian systems.

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