Up-regulated expression of spherical body protein 2 truncated copy 11 in Babesia bovis is associated with reduced cytoadhesion to vascular endothelial cells

Gallego-López, Gina M; Lau, Audrey O.T.; O’Connor, Roberta M.; Ueti, Massaro W.; Cooke, Brian M.; Laughery, Jacob M.; Graça, Telmo; Madsen–Bouterse, Sally A.; Oldiges, Daiane P.; Allred, David R.; Suárez, Carlos E.

doi:10.1016/j.ijpara.2018.05.015

Cited by 18 publications

(25 citation statements)

References 40 publications

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“…The anti-BcSBP4 peptide antibodies react with an antigen that appears to be localized in internal parasite organelles and also in the erythrocyte cytoplasm. This pattern of reactivity is compatible with spherical body localization and comparable to what was observed for other spherical body proteins in the related parasites B. bovis, B. bigemina and B. orientalis [13,23,24].…”

Section: Resultssupporting

confidence: 88%

Identification and antigenicity of the Babesia caballi spherical body protein 4 (SBP4)

Mahmoud¹,

Kandil²,

El-Ezz³

et al. 2020

Parasites Vectors

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Background: The tick-borne intra-erythrocytic apicomplexan Babesia caballi is one of the etiological agents of equine babesiosis, an economically important disease of equids in most tropical and subtropical areas of the world. Discovering candidate antigens for improved diagnostic tools and vaccines remains needed for controlling equine babesiosis. This study describes the B. caballi sbp4 (Bcsbp4) gene and protein (BcSBP4) and analyzes its antigenicity in infected equids. Methods: BLAST searches of an uncurated B. caballi assembly genome using the B. bovis SBP4 as a query were carried out, followed by PCR amplification and sequencing of a newly identified BcSBP4. Characterization of this novel gene and protein was performed by bioinformatics analysis, western blots, immunofluorescence (IFA) and an in vitro neutralization test using anti SBP4 peptide antibodies. Antigenicity of recombinant BcSBP4 (rBcSBP4) was tested with sera from field animals (n = 18) using an indirect ELISA (iELISA). Results: Babesia caballi genome searches using B. bovis SBP4 as a query allowed identification of a novel gene termed Bcsbp4. The Bcsbp4 gene encodes for a protein of 30.58 kDa, which is fully conserved among B. caballi isolates from USA and Egypt. Bioinformatics analysis indicates that BcSBP4 contains a signal peptide and lacks additional transmembrane domains. Expression of BcSBP4 in blood stages of B. caballi was confirmed by western blot and IFA using antibodies against synthetic peptides representing putative B-cell epitopes of BcSBP4 predicted by in silico analysis. In vitro neutralization tests using anti-BcSBP4 peptide antibodies showed a marginal, but statistically significant inhibitory effect on the infectivity of B. caballi merozoites in horse red blood cells. Sera from eight B. caballi-infected equids, but none out of ten negative equid control sera, gave a positive signal in an rBcSBP4 based iELISA. Conclusions: The Bcsbp4 gene is expressed in B. caballi blood stages. The BcSBP4 protein is a potential candidate for developing a novel serological test that could detect B. caballi infection in equids in tropical and subtropical countries worldwide.

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Section: Resultssupporting

confidence: 88%

Identification and antigenicity of the Babesia caballi spherical body protein 4 (SBP4)

Mahmoud¹,

Kandil²,

El-Ezz³

et al. 2020

Parasites Vectors

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“…Because we could not co-precipitate these two proteins by immunoprecipitation experiments (S8B Fig), they may not directly interact. Recently it was shown that upregulation of SBP2 truncated copy 11 in B. bovis reduced binding of iRBCs to endothelial cells and its virulence in cattle [43,44]. However, it is unclear how SBP2 truncated copy 11 affects the export or surface expression of VESA1 or other putative cytoadhesion ligands.…”

Section: Plos Pathogensmentioning

confidence: 99%

Novel Babesia bovis exported proteins that modify properties of infected red blood cells

et al. 2020

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Babesia bovis causes a pathogenic form of babesiosis in cattle. Following invasion of red blood cells (RBCs) the parasite extensively modifies host cell structural and mechanical properties via the export of numerous proteins. Despite their crucial role in virulence and pathogenesis, such proteins have not been comprehensively characterized in B. bovis. Here we describe the surface biotinylation of infected RBCs (iRBCs), followed by proteomic analysis. We describe a multigene family (mtm) that encodes predicted multi-transmembrane integral membrane proteins which are exported and expressed on the surface of iRBCs. One mtm gene was downregulated in blasticidin-S (BS) resistant parasites, suggesting an association with BS uptake. Induced knockdown of a novel exported protein encoded by BBOV_III004280, named VESA export-associated protein (BbVEAP), resulted in a decreased growth rate, reduced RBC surface ridge numbers, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that BbVEAP is a novel virulence factor for B. bovis.

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“…A GFP expressing parasite is advantageous for the drug screening and in vivo tracking of the parasite location during infection. Studies have shown that transfection in B. bovis, to study the gene function, were helpful in developing vaccine against bovine babesiosis 29 , 30 . Therefore, a transgenic B. microti parasite will also be helpful in study of gene function and vaccine development.…”

Section: Discussionmentioning

confidence: 99%

“…Studies have shown that transfection in B. bovis, to study the gene function, were helpful in developing vaccine against bovine babesiosis 29,30 . Therefore, a transgenic B. microti parasite will also be helpful in study of gene function and vaccine development.…”

Section: Primers Sequencementioning

confidence: 99%

Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti

Jaijyan

Govindasamy²,

Singh

et al. 2020

Sci Rep

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Babesia microti, an emerging human pathogen, is primarily transmitted through a bite of an infected tick and blood transfusions in human. Stable transfection technique has been reported in many protozoan parasites over the past few years. However, in vivo transient and stable transfection method has not been established for Babesia microti. Here, for the first time, we present a method of transient as well as stable transfection of the Babesia microti (B. microti) in the in vivo conditions. We have identified a novel promoter of B. microti. We also demonstrated that Plasmodium berghei DHFR promoter is recognized and functional in B. microti. We show that BM-CTQ41297 promoter control the expression of two genes, which are present on either side and thus represents a bi-functional promoter in B. microti. The predicted promoter activity values using Promoter 2.0 program is higher for BM- CTQ41297 promoter than strong promoters such as β-actin, ef-1β, and many other promoters. Furthermore, we discovered a non-essential locus for the genetic manipulation of the parasite, allowing us to stably integrate foreign genes; GFP, mCherry, into the B. microti. The transfection using an electroporation method and genetic manipulation of B. microti is now achievable and it is possible to obtain transfected viable parasites under in vivo growing conditions. The growth curve analysis of transfected and WT B. microti are similar indicating no defects in the transgenic parasites. This study will enable other researchers in understanding the B. microti biology, host modulation and diverse parasite developmental stages using reverse genetics and holds great potential to identify novel drug targets and vaccine development.

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Up-regulated expression of spherical body protein 2 truncated copy 11 in Babesia bovis is associated with reduced cytoadhesion to vascular endothelial cells

Cited by 18 publications

References 40 publications

Identification and antigenicity of the Babesia caballi spherical body protein 4 (SBP4)

Identification and antigenicity of the Babesia caballi spherical body protein 4 (SBP4)

Novel Babesia bovis exported proteins that modify properties of infected red blood cells

Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti

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