Molecular characterization of a novel microneme antigen in Neospora caninum

Sonda, Sabrina; Fuchs, Nicole; Gottstein, Bruno; Hemphill, Andrew

doi:10.1016/s0166-6851(00)00200-0

Cited by 41 publications

(42 citation statements)

References 44 publications

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“…Since N. caninum is very closed to T. gondii in morphology, it is expected that the same or very similar proteins of N. caninum tachyzoite participate in the invasion into host cells like in T. gondii. As the proteins related to cell invasion in N. caninum, Nc-p36 [10] and Ncp43 [9] existing on the surface of tachyzoite, NcMIC3 [12,17,19] in microneme, GRA2 [7] in dense glanules, and NC-SUB1 [13] which is one of the proteases were confirmed. In the inhibition assay using the MAbs obtained in this study, invasion by the tachyzoites into the host cells was highly inhibited, so that it is considered that the surface antigen identified might have the function of attachment, recognition or secretion.…”

Section: Resultsmentioning

confidence: 82%

“…Antigens of some protozoan parasites belonging to phylum Apicomplexa, such as Toxoplasma gondii participate in invading the host cells and were thought to be surface antigens, secretory proteins and those in the structures of apical complex such as microneme, rhoptry and dense granule [6,11,15,18]. Also in N. caninum, some antigens, possibly affecting the invasion, were studied, and the antigens on the body surface [9,10], dense granule [7], microneme [12,17,19] and rhoptry [1], glycoconjugates, glycoproteins [8] and subtilisin-like serineprotease [13] were reported until now. But the roles of these antigens in invasive process remain unclear in detail.…”

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confidence: 99%

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Monoclonal Antibodies Preventing Invasion of Neospora caninum Tachyzoites into Host Cells

Uchida

Ike

Kurotaki

et al. 2004

The Journal of Veterinary Medical Science

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ABSTRACT. Twelve monoclonal antibodies (MAbs) against Neospora caninum tachyzoites were produced to specify the antigens related to the invasion of tachyzoites into host cells. In the assay to evaluate the inhibition activity, all these MAbs prevented the cultured Vero cells from the invading by the tachyzoites. These MAbs recognized approximately a 73 kDa antigen in Western blot analysis. Immunofluorescence assay and immune electron microscopy revealed that this 73 kDa antigen is a part of the surface antigens of N. caninum tachyzoite, and that the tachyzoite antigen identified plays an important role for invasion of host cells. KEY WORDS: invasion process, monoclonal antibody, Neospora caninum, surface antigen.J. Vet. Med. Sci. 66(11): 1355-1358, 2004 Neospora caninum is a coccidian protozoon first isolated from the brain of a puppy by Dubey in 1988, which developed paralysis in the hind limbs [5]. Dogs infected with this parasite mainly show neurological symptoms and infected cows are attached with abortion and stillborn, so serious economical damages are produced [4]. In 1998, McAllister et al. reported that definitive host of the protozoon is dogs [14]. However, the life cycle of N. caninum is not completely clarified yet, and the procedures for prevention and treatment of neosporosis also are not established. The pathogenesis of N. caninum is known to be caused by multiplication of parasites resulting destruction of many kinds of host cells by invasion [2,3,20]. To develop the prevention and treatment of neosporosis, analyses of antigens related with the invasion process into host cells are required. Antigens of some protozoan parasites belonging to phylum Apicomplexa, such as Toxoplasma gondii participate in invading the host cells and were thought to be surface antigens, secretory proteins and those in the structures of apical complex such as microneme, rhoptry and dense granule [6,11,15,18]. Also in N. caninum, some antigens, possibly affecting the invasion, were studied, and the antigens on the body surface [9,10] In this study, we prepared monoclonal antibodies (MAbs) against N. caninum tachyzoites which obstruct the invasion of tachyzoites into host cells, and further, we tried to specify the antigen of N. caninum concerning with attachment and invasion into the host cells. MATERIALS AND METHODSParasites: Neospora caninum JPA-1 strain used was isolated and passaged by Yamane et al. in the National Institute of Animal Health, Japan [23]. The isolate was inoculated into monolayer Vero cells maintained in Eagle's minimum essential medium (MEM; NISSUI PHARMACEUTICAL Co., Japan) supplemented with 10% fetal calf serum (FCS; HARLAN SERA-LAB, England), 2 mM L-glutamine (Wako Pure Chemical Industries, Japan), 1% MEM Vitamin solution (GIBCO BRL, U.S.A.), 13 mM sodium hydrogen carbonate (NaHCO 3 ) (Wako Pure Chemical Industries), and 4 µl/ml Fungizone (GIBCO BRL). The tachyzoites cultured were used for examination. Purification of tachyzoites:The tachyzoites cultured were purified according to an improved metho...

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Section: Resultsmentioning

confidence: 82%

mentioning

confidence: 99%

Monoclonal Antibodies Preventing Invasion of Neospora caninum Tachyzoites into Host Cells

Uchida

Ike

Kurotaki

et al. 2004

The Journal of Veterinary Medical Science

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“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting (50) were used to assess the differences in protein expression by N. caninum tachyzoites and bradyzoites. Equal numbers of both parasite stages were loaded, and silver staining revealed partially distinct banding patterns for bradyzoite and tachyzoite extracts (Fig.…”

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confidence: 99%

In Vitro Induction of Neospora caninum Bradyzoites in Vero Cells Reveals Differential Antigen Expression, Localization, and Host-Cell Recognition of Tachyzoites and Bradyzoites

et al. 2004

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We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum bradyzoites in Vero cells and the separation of viable parasites from host cells. Treatment of tachyzoite-infected Vero cell cultures with 17 M sodium nitroprusside for 8 days severely scaled down parasite proliferation, led to reduced expression of tachyzoite surface antigens, and induced the expression of the bradyzoite marker NcBAG1 and the cyst wall antigen recognized by the monoclonal antibody MAbCC2. Transmission electron microscopy demonstrated that intracellular parasites were located within parasitophorous vacuoles that were surrounded by a cyst wall-like structure, and the dense granule antigens NcGRA1, NcGRA2, and NcGRA7 were incorporated into the cyst wall. Adhesion-invasion assays employing purified tachyzoites and bradyzoites showed that tachyzoites adhered to, and invaded, Vero cells with higher efficiency than bradyzoites. However, removal of terminal sialic acid residues from either the host cell or the parasite surface increased the invasion of Vero cells by bradyzoites, but not tachyzoites.

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“…Microneme proteins include potentially adhesive soluble components such as NcMIC1 , NcMIC2 (Lovett et al 2000), NcMIC4 , and membrane-bound microneme proteins such as NcMIC3 (Sonda et al 2000, Naguleswaran et al 2001. While proteolytic processing of the soluble microneme proteins has been shown to occur, there is no indication that the membrane-bound NcMIC3 is undergoing modification.…”

Section: Micronemal Proteins and Host Cell Surface Receptorsmentioning

confidence: 99%

“…Several N. caninum microneme proteins identified to date possess adhesive domains that could interact with receptors on the surface of target cells, similar to related domains found in vertebrate extracellular matrix proteins. These adhesive motifs include thrombospondin-(TSP)-like domains in NcMIC1 , integrin-and TSP-type I-like domains in NcMIC2 (Lovett et al 2000), and epidermal growth factor (EGF)-like domains in NcMIC3 (Sonda et al 2000).…”

Section: Micronemal Proteins and Host Cell Surface Receptorsmentioning

confidence: 99%

Neospora caninum and neosporosis — recent achievements in host and parasite cell biology and treatment

Hemphill

Gottstein

2006

Acta Parasitologica

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Neospora caninum is an apicomplexan parasite, which owes its importance to the fact that it represents the major infectious cause of bovine abortion worldwide. Its life cycle is comprised of three distinct stages: Tachyzoites, representing the proliferative and disease-causing stage, bradyzoites, representing a slowly replicating, tissue cyst-forming stage, and sporozoites, which represent the end product of a sexual process taking place within the intestinal tissue of the final canine host. Tachyzoites are capable of infecting a large variety of host cells in vitro and in vivo, while bradyzoites have been found mainly within the central nervous system. In order to survive, proliferate, and proceed in its life cycle, N. caninum has evolved some amazing features. First, the parasite profits immensely from its ability to interact with, and invade, a large number of host cell types. Secondly, N. caninum exploits its capability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite or vice versa). Thirdly, this parasite has evolved mechanisms that modulate its host cells according to its own requirements, and these must, especially in the case of the bradyzoite stage, involve mechanisms that ensure long term survival of not only the parasite but also of the host cell. These three key events (host cell invasion -stage conversion -host cell modulation) represent potential targets for intervention. In order to elucidate the molecular and cellular bases of these important features of N. caninum, cell culture-based approaches and laboratory animal models are extensively exploited. In this review, we will summarize the present knowledge and achievements related to host cell and parasite cell biology.

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Molecular characterization of a novel microneme antigen in Neospora caninum

Cited by 41 publications

References 44 publications

Monoclonal Antibodies Preventing Invasion of Neospora caninum Tachyzoites into Host Cells

Monoclonal Antibodies Preventing Invasion of Neospora caninum Tachyzoites into Host Cells

In Vitro Induction of Neospora caninum Bradyzoites in Vero Cells Reveals Differential Antigen Expression, Localization, and Host-Cell Recognition of Tachyzoites and Bradyzoites

Neospora caninum and neosporosis — recent achievements in host and parasite cell biology and treatment

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