Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Oct 5;16(10):e1008917.
doi: 10.1371/journal.ppat.1008917. eCollection 2020 Oct.

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

Hassan Hakimi  1   2 , Thomas J Templeton  1 , Miako Sakaguchi  3 , Junya Yamagishi  4   5 , Shinya Miyazaki  1 , Kazuhide Yahata  1 , Takayuki Uchihashi  6 , Shin-Ichiro Kawazu  2 , Osamu Kaneko  1 , Masahito Asada  1   2
Affiliations

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

Hassan Hakimi et al. PLoS Pathog. .

Abstract

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.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Biotinylation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of B. bovis-iRBCs.
(A) Western blot analysis of sequentially extracted proteins from biotinylated and control (non-biotinylated) samples. The image is representative of three independent experiments done with an approximately two-month interval. The membrane was probed with horseradish peroxidase (HRP)-conjugated streptavidin. (B) Venn diagram showing the number of B. bovis proteins identified from biotinylated samples by three independent LC-MS/MS analyses.
Fig 2
Fig 2. Expression and localization analysis of candidate proteins determined by indirect immunofluorescence antibody test (IFAT), Western blotting, and immunoelectron microscopy (IEM).
(A) IFAT of parental wild type (WT) and transgenic B. bovis lines expressing myc-tagged target proteins. The parasites were reacted with anti-myc antibody (α-myc, green) and nuclei were stained with Hoechst 33342 (Hoechst, blue). Scale bar = 5 μm. (B) Western blot analysis of transgenic B. bovis expressing myc-tagged proteins and WT parasite (WT). The expected full-length bands of the proteins are indicated with black arrows. (C) Immunoelectron microscopic analysis of transgenic B. bovis expressing Bb60-mtm or BbVEAP tagged with myc epitopes. Anti-myc antibody shows concentration of Bb60-mtm and BbVEAP in spherical bodies (black arrows) and Bb60-mtm expression on the iRBC surface (red arrows). Scale bar = 1 μm.
Fig 3
Fig 3. Expansion and distribution of genes encoding multi-transmembrane proteins in piroplasms.
(A) Distribution of mtm and tpr-related genes in the B. bovis genome. (B) Arrangement of multigene families (ves1, mtm, smorf, and tpr-related genes) in the nuclear genome of B. bovis. (C) Homology clustering based on sequence similarities of genes with more than eight TM domains in piroplasms, Plasmodium falciparum, and T. gondii. (D) Schematics of selected gene products with multiple TM domains in piroplasms. Box indicates predicted TM domain.
Fig 4
Fig 4. Blasticidin S-resistance in B. bovis is linked with downregulation of Bb60.
(A) Growth inhibition curves of parasite lines in the presence of different concentrations of BS (μg/mL). All data are expressed as mean ± SEM of triplicate cultures. (B) Osmotic lysis of B. bovis wild type (WT) and BS-resistant line 1 in the presence of sorbitol. iRBCs were enriched and the lysis experiment was performed at 37°C. The graph is representative from two biological replicates done within a two-week interval. (C) The scatter diagram showing the differential expression of genes in B. bovis WT and BS-resistant lines. The horizontal and vertical axes represent the log 2-fold expression of WT and BS-resistant parasites, respectively. The upregulated and downregulated genes are shown in red and blue colors, respectively. (D) Scatter diagram showing the differential expression of genes in B. bovis WT and BS-sensitive revertant lines. The expression of downregulated mtms in BS-resistant lines, Bb60 and Bb10, recovered in revertant lines (green dots). (E) Indirect immunofluorescence antibody test of transgenic B. bovis BS-resistant lines episomally expressing myc-tagged Bb60-mtm or Bb10-mtm stained with anti-myc (green). The parasite nuclei were stained with Hoechst 33342 (Hoechst, blue). Scale bar = 5 μm. (F) Growth inhibition curves of a panel of parasite lines in the presence of different concentrations of BS (μg/mL). Bb60-mtm and Bb10-mtm are episomally overexpressed under 100 nM WR99210 in BS-resistant lines 1 and 2, respectively. All data are expressed as mean ± SEM.
Fig 5
Fig 5. Induced knockdown of BbVEAP using the glmS riboswitch system decreases growth rate and ridge numbers.
(A) Schematic of CRISPR/Cas9 plasmid to insert myc-glmS sequences within the BbVEAP gene locus and agarose gel electrophoresis of diagnostic PCR to confirm integration of myc-glmS sequence at the 3' end of the BbVEAP ORF. rap-3’NR, rhoptry associated protein 3’ noncoding region; hdhfr orf, human dihydrofolate reductase ORF; ef-1a IG, elongation factor-1α intergenic region; tpx-3’NR, thioredoxine peroxidase-1 3’ noncoding region; U6-3’NR, B. bovis U6 spliceosomal RNA 3’ noncoding region; gRNA, guide RNA; and HR, homologous region. glmS-C1 and glmS-C2 indicate transgenic lines independently generated and following "-1" and "-2" indicate 2 independent clones. (B) Western blot analysis of 2 independently generated BbVEAP-myc-glmS clones and a control BbVEAP-myc parasite line expressing myc-tagged BbVEAP without the glmS element from the endogenous gene locus in the presence or absence of glucosamine (GlcN). Anti-TPx-1 antibody was used to detect TPx-1 protein as a loading control. The image is representative of three independent experiments done within an approximately one-week interval. (C) Densitometry of BbVEAP protein levels in all conditions measured relative to the control parasite (GlcN-untreated BbVEAP-myc line) incubated in the presence or absence of GlcN. (D) Growth of BbVEAP-myc-glmS and control BbVEAP-myc lines in the presence or absence of GlcN. Initial parasitemia was 0.1% and parasitemia was monitored for 3 days with daily culture medium replacement. The data are shown as mean ± S.D. for three independent experiments performed with a one-week interval. (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001; determined by multiple t test). (E) Proportion of ring, binary, and multiple stages in different parasite lines in the presence or absence of GlcN on day 3 post GlcN introduction. The data are shown as mean ± S.D. (*, P < 0.05; **, P < 0.01; ns, not significant, P ≥ 0.05; determined by multiple t test). (F) Transmission electron microscopy images of BbVEAP-myc-glmS in the presence (+) or absence (-) of GlcN. Black arrows indicate spherical bodies and red arrow shows ridges. Scale bar = 0.5 μm. (G) Quantification of ridge numbers on the surface of iRBCs of BbVEAP-myc-glmS parasites in the presence or absence of GlcN at day 3 post GlcN introduction. Ridge numbers per 10 μm of iRBC circumference were quantified only in mature stage iRBCs (binary form) (****, P< 0.0001; determined by Mann-Whitney U test).
Fig 6
Fig 6. BbVEAP knockdown abrogates binding of iRBCs to endothelial cells.
(A) Cytoadhesion assay of BbVEAP-myc-glmS and WT parasites in the presence (+) or absence (-) of GlcN. All data are expressed as mean ± SEM of triplicate assay (****, P< 0.0001; determined by paired Student’s t test). (B) Indirect immunofluorescence microscopy test of BbVEAP-myc-glmS parasite in the presence (+) or absence (-) of GlcN (α-myc, red; α-VESA1 and α-SBP4, green). The parasite nuclei were stained with Hoechst 33342 (Hoechst, blue). Scale bar = 5 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bock R, Jackson L, de Vos A, Jorgensen W. Babesiosis of cattle. Parasitology. 2004; 129: S247–S249. 10.1017/s0031182004005190 - DOI - PubMed
    1. Florin-Christensen M, Suarez CE, Rodriguez AE, Flores DA, Schnittger L. Vaccines against bovine babesiosis: where we are now and possible roads ahead. Parasitology. 2014; 141:1563–1592. - PubMed
    1. Vudriko P, Okwee-Acai J, Byaruhanga J, Tayebwa DS, Omara R, Muhindo JB, et al. Evidence-based tick acaricide resistance intervention strategy in Uganda: concept and feedback of farmers and stakeholders. Ticks Tick Borne Dis. 2018; 9:254.–. 10.1016/j.ttbdis.2017.09.011 - DOI - PubMed
    1. O’Connor RM, Allred DR. Selection of Babesia bovis infected erythrocytes for adhesion to endothelial cells coselects for altered variant erythrocyte surface antigen isoforms. J Immunol. 2000; 64:2037–2045. - PubMed
    1. Hutchings CL, Li A, Fernandez KM, Fletcher T, Jackson LA, Molloy JB, et al. New insights into the altered adhesive and mechanical properties of red blood cells parasitized by Babesia bovis Mol Microbiol. 2007; 65:1092–1105. 10.1111/j.1365-2958.2007.05850.x - DOI - PubMed

Publication types

Grants and funding

This study was supported partly by grants from Japan Society for the Promotion of Science (https://www.jsps.go.jp/english/) to H.H. (15K18783, 19K15983), M.A. (16K08021, 19K06384), S.K. (18K19258, 19H03120) and O.K. (16F16105). This work was supported by NRCPD OUAVM Joint Research Grant of NRCPD, Obihiro University of Agriculture and Veterinary Medicine (https://www.obihiro.ac.jp/facility/protozoa/ento) to M.A. (28-11, 29-2, 30-1). H.H. is a recipient of the JSPS Postdoctoral Fellowship for foreign researchers from the Japan Society for the Promotion of Science. T.J.T. was supported by a visiting professorship to the Institute of Tropical Medicine, Nagasaki University (http://www.tm.nagasaki-u.ac.jp/nekken/en/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.