Neospora caninum: Structure and Fate of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294

Winzer, Pablo; Anghel, Nicoleta; Imhof, Dennis; Balmer, Vreni; Ortega‐Mora, Luis Miguel; Ojo, Kayode K.; Voorhis, Wesley C. Van; Müller, Joachim; Hemphill, Andrew

doi:10.3390/pathogens9050382

Cited by 17 publications

(21 citation statements)

References 30 publications

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“…Immunofluorescence staining showed that the formation of MNCs during extended BKI-1294 treatment was accompanied by a lack of SAG1 surface expression and a decreased CDPK1 expression, and the formation of numerous zoites incapable of disjoining from each other. Following drug removal, proliferation continued, and zoites lacking NcSAG1 emerged from the periphery of these complexes, forming infective tachyzoites after 10 days [18]. These results suggested that MNCs could constitute a drug-induced resting stage that assures the survival of the parasite until the drug pressure is released.…”

Section: Introductionmentioning

confidence: 91%

Neospora caninum: Differential Proteome of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294

et al. 2020

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Background: the apicomplexan parasite Neospora caninum causes important reproductive problems in farm animals, most notably in cattle. After infection via oocysts or tissue cysts, rapidly dividing tachyzoites infect various tissues and organs, and in immunocompetent hosts, they differentiate into slowly dividing bradyzoites, which form tissue cysts and constitute a resting stage persisting within infected tissues. Bumped kinase inhibitors (BKIs) of calcium dependent protein kinase 1 are promising drug candidates for the treatment of Neospora infections. BKI-1294 exposure of cell cultures infected with N. caninum tachyzoites results in the formation of massive multinucleated complexes (MNCs) containing numerous newly formed zoites, which remain viable for extended periods of time under drug pressure in vitro. MNC and tachyzoites exhibit considerable antigenic and structural differences. Methods: Using shotgun mass spectrometry, we compared the proteomes of tachyzoites to BKI-1294 induced MNCs, and analyzed the mRNA expression levels of selected genes in both stages. Results: More than half of the identified proteins are downregulated in MNCs as compared to tachyzoites. Only 12 proteins are upregulated, the majority of them containing SAG1 related sequence (SRS) domains, and some also known to be expressed in bradyzoites Conclusions: MNCs exhibit a proteome different from tachyzoites, share some bradyzoite-like features, but may constitute a third stage, which remains viable and ensures survival under adverse conditions such as drug pressure. We propose the term “baryzoites” for this stage (from Greek βαρυσ = massive, bulky, heavy, inert).

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Section: Introductionmentioning

confidence: 91%

Neospora caninum: Differential Proteome of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294

et al. 2020

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“…It is possible that no tissue cysts were formed in BKI-1294 treated mice. In vitro, BKI-1294 induces the formation of MNCs, which contain newly formed zoites that undergo continuous DNA replication (21). A likely scenario is that MNCs were formed also in vivo, and this could be responsible for the observed reactivation of the live-vaccine and the increased vertical transmission upon re-infection.…”

Section: Discussionmentioning

confidence: 99%

“…Shotgun proteomics had shown that MNCs exhibited an altered proteome when compared to tachyzoites, and this drug-induced stage was termed baryzoite (22). Following in vitro treatment with BKI-1294 during 5 days, it took approximately 10 days of culture without drug for MNCs to disappear and tachyzoites with SAG1 surface expression to re-emerge (21). If similar MNCs were also formed in vivo, it is conceivable that this could interfere in the process of cerebral tissue cyst formation, and thus reactivation of the infection during pregnancy could be taking place in a different manner than in non-treated mice.…”

Section: Discussionmentioning

confidence: 99%

“…BKI-1294 interferes with host cell invasion and egress, but does not act parasiticidal in vitro (19). BKI-1294 also affects the intracellular tachyzoites by inducing the formation of multinucleated complexes (MNCs) (20), which are characterized by continued nuclear division, and the formation of intracellular zoites separated by the inner membrane complex, but lacking the major surface antigen 1 (SAG1) (21). However, these zoites failed to undergo disjunction and tachyzoite formation, remained trapped within the host cell cytoplasm, and exhibited altered mRNA and protein expression (19,22).…”

Section: Introductionmentioning

confidence: 99%

“…However, these zoites failed to undergo disjunction and tachyzoite formation, remained trapped within the host cell cytoplasm, and exhibited altered mRNA and protein expression (19,22). MNCs remained viable for up to 20 days under constant BKI-1294 drug pressure in vitro, and upon drug removal the block in zoite disjunction was alleviated and new infective tachyzoites were formed, which resumed proliferation (21).…”

Section: Introductionmentioning

confidence: 99%

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The Impact of BKI-1294 Therapy in Mice Infected With the Apicomplexan Parasite Neospora caninum and Re-infected During Pregnancy

et al. 2020

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Exposure of Neospora caninum tachyzoites to BKI-1294 in vitro results in the formation of long-lived multinucleated complexes (MNCs). However, in vivo treatment of BALB/c mice with BKI-1294 shortly after N. caninum infection during pregnancy was safe and profoundly reduced pup mortality and vertical transmission. We hypothesized that the formation of MNCs could trigger immune responses that contribute to BKI efficacy in vivo. In this study, mice were first vaccinated with a sublethal dose of N. caninum tachyzoites and were treated with BKI-1294. We then investigated the effects of these treatments after mating and re-infection during pregnancy. Effects on fertility, pup survival, vertical transmission, and parasite load in dams were evaluated. Cytokines in sera or splenocyte culture supernatants were assessed by either ELISA or the Luminex TM 200 system, and humoral immune responses against tachyzoite and MNC antigens were compared by ELISA, Western blotting and immunoproteomics. Our results showed that BKI-1294 treatment of live-vaccinated mice reduced the cerebral parasite load in the dams, but resulted in higher neonatal pup mortality and vertical transmission. In live-vaccinated mice, cytokine levels, most notably IFN-y, IL-10, and IL-12, were consistently lower in BKI-1294 treated animals compared to non-treated mice. In addition, comparative Western blotting identified two protein bands in MNC extracts that were only recognized by sera of live-vaccinated mice treated with BKI-1294, and were not found in tachyzoite extracts. We conclude that treatment of live-vaccinated mice with BKI-1294 influenced the cellular and humoral immune responses against infection, affected the safety of the live-vaccine, and decreased protection against re-infection and vertical transmission during pregnancy.

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Neospora caninum and Toxoplasma gondii infections in one-humped camels (Camelus dromedarius) in central desert of Iran

et al. 2023

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Neospora caninum: Structure and Fate of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294

Cited by 17 publications

References 30 publications

Neospora caninum: Differential Proteome of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294

Neospora caninum: Differential Proteome of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294

The Impact of BKI-1294 Therapy in Mice Infected With the Apicomplexan Parasite Neospora caninum and Re-infected During Pregnancy

Neospora caninum and Toxoplasma gondii infections in one-humped camels (Camelus dromedarius) in central desert of Iran

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