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

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.

Introduction

Babesiosis is an emerging tick-borne disease affecting animals and humans which is caused by intraerythrocytic protozoans of the genus Babesia. The parasite infects a wide range of vertebrates and causes great economic losses in livestock. The burden of bovine babesiosis in tropical and subtropical regions is attributed to Babesia bovis and Babesia bigemina. While pathogenesis of B. bigemina is mainly related to intravascular hemolysis, sequestration of B. bovis-infected red blood cells (iRBCs) in internal organs and brain produces severe clinical symptoms which occasionally result in fatality [1]. Constraints against disease control include the low efficacy of available live vaccines for B. bovis, limited treatment options, and the emergence of drug and acaricide resistance of tick vectors [1-3]. The combination of new drugs and vaccine intervention are required to better control the disease.

Following RBC invasion of merozoites or sporozoites injected by ticks, B. bovis parasites modify the host cell via the export of numerous proteins to the RBC cytoplasm and surface, to facilitate metabolite exchange, increase RBC rigidity, and to mediate cytoadherence in deep tissues [4-6]. Modification of iRBCs results in the production of unique surface protrusions, ridges, which are the focal points for adhesion to endothelial cells [4,7,8]. Cytoadherence causes sequestration of iRBCs in the microvasculature of internal organs, thus avoiding spleen clearance. The binding of iRBCs to unknown receptor(s) on brain microvascular endothelial cells can cause cessation of blood flow and produce cerebral symptoms [8].

Although the mechanisms of protein export to the RBC cytoplasm and surface are not known, several exported proteins have been reported in B. bovis [9-15]. The majority of known exported proteins are the products of multigene families such as variant erythrocyte surface antigen 1 (VESA1), small open reading frame proteins (SmORFs), and spherical body protein 2 (SBP2). VESA1 proteins are heterodimeric proteins encoded by the largest multigene family, ves1, in B. bovis [16]. They cluster on the surface of ridges, undergo antigenic variation, and are responsible for host immune evasion and cytoadhesion [4,11]. SmORFs are produced from the second largest gene family, smorfs, and are exported to the RBC during parasite development [15,17]. While the function of SmORFs is unknown, their gene distribution and proximity to ves1 genes throughout the B. bovis genome indicates a role in VESA1 biology [17]. SBP2 is also encoded by a multigene family, sbp2. Unlike ves1 and smorfs, which are unique to B. bovis, sbp2 are conserved across the genus Babesia [17-19]. SBP2 are localized to spherical bodies, organelles analogous to dense granules in other apicomplexan parasites, and are released into the RBC cytoplasm upon invasion [9]. To date SBP1, SBP2, SBP3, and SBP4 have been characterized in B. bovis [9,10,12,13] but their functions during invasion and development in the RBC are unknown. Recently, Gohil et al. (2013) identified three novel exported proteins of B. bovis among 214 putative exported protein annotated for the presence of a signal peptide cleavage site but negative for transmembrane domains (TM) or glycosylphosphatidylinositol (GPI) anchor attachment motif [14]. Pellé et al. (2015) further refined this list to include 59 proteins harboring a PEXEL-like motif (PLM) preceded by a signal peptide sequence [15]. PEXEL (Plasmodium export element) is a unique conserved pentameric motif at the N-terminal of approximately 300 proteins in Plasmodium [20,21] which is cleaved within the endoplasmic reticulum by a protease and determines protein export [22,23]. A similar signal-mediated pathway was identified in Toxoplasma gondii, but as a sorting signal to dense granules before release into the parasitophorous vacuole (PV) space [24].

Despite their crucial role in B. bovis virulence and pathogenesis, iRBC surface exposed proteins have not been comprehensively characterized. In this study we performed biotinylation of iRBC surface proteins, allowing their extraction, purification, and proteomic analysis. We confirmed export of several candidate proteins and performed initial protein characterizations, including a family of multi-transmembrane integral membrane proteins and a novel virulence factor associated with iRBC cytoadhesion to endothelial cells.

Results

RBC surface proteomics

Biotinylation coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to obtain the first comprehensive surface proteome of B. bovis-iRBCs. We selected B. bovis for adhesion to bovine brain endothelial cells (BBECs) by panning uncloned parasites and the resulting cytoadherent parasite line was used for biotinylation (S1A Fig). The surface proteins of enriched iRBCs from in vitro cultured B. bovis were biotinylated, extracted, purified, and analyzed by LC-MS/MS in three biological replicates. Surface protein biotinylation was confirmed by live fluorescence microscopy. Biotinylation did not affect the integrity of iRBC membranes and the release of merozoites (S1B Fig). The extraction and purification of biotinylated proteins was confirmed by Western blot analysis (Fig 1A). A complete list of B. bovis proteins exported from the Scaffold software is provided in the supplemental information (S1 Table). In the first attempt, we lysed the iRBCs by a hypotonic solution followed by detergent protein extraction using the BugBuster reagent, which was shown to be efficient for P. falciparum-iRBCs surface proteins such as PfEMP1 [25]. LC-MS/MS analysis showed bovine hemoglobin contamination which might interfere with identification of B. bovis peptides. Therefore, in the second and third attempts, protein extraction was done following biotinylation and saponin treatment. This modification improved the detection of total B. bovis peptides and proteins. Mass spectra obtained from LC-MS/MS were searched against B. bovis and Bos taurus databases. Because parasite-encoded surface exposed proteins are low in abundance, we allowed protein identification by a single peptide. The number of unique peptides identified in the first, second, and third attempts from biotinylated samples were 805, 1600, and 948, respectively, in which 24–26% of identified peptides were from B. bovis and yielded 77, 163, and 130 proteins, respectively (Table 1). Out of the 255 unique B. bovis protein hits, 80 proteins detected from biotinylated samples by at least two MS analyses (Fig 1B) were further annotated for predicted export based upon the presence of a signal sequence, a transmembrane (TM) domain, or GPI anchor. This refinement resulted in a total of 38 putative secretory proteins of which 20 had no predicted function in the database (S2 Table). The well-known surface exposed B. bovis protein, VESA1, was identified in all three MS. VESA1 lacks an N-terminal signal sequence but has a C-terminal TM domain [11]. One SmORF and one SBP2, the products of two multigene families in B. bovis, SBP1 and SBP3 were also detected. These proteins have an N-terminal signal sequence and were shown to be exported into the iRBC cytoplasm [9,10,12,13,15]. Of the 38 identified proteins 32 possessed a PEXEL-like export motif (PLM; RxL or RxxL) proposed by Pellé et al. [15].

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.

Table 1

Summary of identified proteins.

	1st MS				2nd MS				3rd MS
	Biotinylated		Non-biotinylated		Biotinylated		Non-biotinylated		Biotinylated		Non-biotinylated
	Peptides*	%	Peptides*	%	Peptides	%	Peptides	%	Peptides	%	Peptides	%
Bos taurus	612	76.0	179	89.9	1184	74.0	261	89.7	701	74.3	227	83.2
Babesia bovis	193	24.0	20	10.1	416	26.0	30	10.3	247	26.2	46	16.8
Total	805		199		1600		291		948		273
	Proteins	%	Proteins	%	Proteins	%	Proteins	%	Proteins	%	Proteins	%
Bos taurus	255	76.8	114	82.0	389	70.5	119	79.9	259	66.6	97	68.8
Babesia bovis	77	23.2	25	18.0	163	29.5	30	20.1	130	33.4	44	31.2
Total	332		139		552		149		389		141

* The peptide numbers are from BugBuster protein extract

Validation of localization for a subset of candidates

To evaluate the localization of putative exported proteins, we transformed parasites with plasmid constructs expressing target molecules fused with 2 myc epitopes (S2A Fig). We selected 10 proteins from the candidate list based on the three criteria for secretion and their abundance in the MS analysis (S3 Table) and further evaluated together with two positive control proteins, SBP3 and a variant of SmORF. Indirect immunofluorescence antibody test (IFAT) revealed that signals of three proteins were detected in iRBCs among selected 10 candidates. Two proteins showed signals inside the parasite and the edge of the iRBCs like SBP3 and SmORF, namely, BBOV_III000060 (Bb60-mtm) and BBOV_III011920 (Bb11920-mtm) (Fig 2A). These proteins possess ten TM domains each and are paralogs encoded by a multigene family (termed multi-transmembrane (mtm) family). Each possess an RxL motif at amino acid positions 24–27. The third protein, BBOV_III004280 (BbVEAP in the Fig 2A) showed signals inside iRBCs and parasites, but not from the edge of the RBC (Fig 2A). The deduced amino acid sequence of BBOV_III004280 possesses a signal peptide with an RxL motif at amino acid position 185–188 and is conserved among all piroplasms except B. microti. We designated BBOV_III004280 as VESA export-associated protein (VEAP) gene because knockdown of this gene suppressed VESA export as described below. The remaining seven candidates did not show signals in iRBCs by IFAT, suggesting that they were not exported to iRBCs (S2B Fig). Thus, we decided to further characterize three novel proteins that were exported to iRBC. Target protein expression in transgenic parasites was confirmed by Western blotting (Fig 2B). SBP3 and SmORF showed several bands indicating possible processing during export or degradation during protein extract preparation [15]. The products of the mtm genes Bb60-mtm and Bb11920-mtm showed bands at the expected size of 49 kDa plus band smears. BbVEAP showed a single band corresponding to the anticipated size of 100 kDa. Immunoelectron microcopy (IEM) revealed that Bb60-mtm is localized in the spherical bodies of merozoites and on the iRBC surface close to ridge structures (Fig 2C, S3 Fig). BbVEAP was detected in merozoite spherical bodies (Fig 2C, S3 Fig).

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.

A multigene family encoding Bbmtm is expanded in B. bovis

Of the three novel proteins that were exported into iRBC, Bb60-mtm and Bb11920-mtm are paralogous proteins with similar architectures of ten TM domains each and molecular weights of approximately 49 kDa. Via BLAST searches of PiroplasmaDB and GenBank, we found 44 total copies in the B. bovis genome and named this newly identified expanded gene family mtm (Fig 3A, S4 Table). The genes are typically telomeric and located in gene neighborhoods containing ves1 and smorf genes, two gene families encoding important exported proteins in B. bovis (Fig 3B). In P. falciparum, exported proteins are also typically localized to clusters within telomeric regions [26]. The family mtm exists in another Babesia species from sheep (Xinjiang), which is closely related to B. bovis [27], and to date the family is unique to these two species (Table 2). To examine in other Piroplasmida the possible expansion of gene families that encode multi-transmembrane proteins, we used TMHMM-2.0 to screen whole genome and proteome datasets (Table 2, Fig 3C and 3D). Specifically, a major facilitator superfamily (mfs)-like gene is greatly expanded in B. ovata and B. bigemina; while tpr (T. parva repeat) is significantly expanded in Theileria spp. [28,29], though several homologs of tpr exist in Babesia spp. Homology clustering shows that tpr in T. parva and T. annulata make one cluster while T. orientalis tpr forms a separate cluster (Fig 3C). Homology clustering of mtm revealed the existence of two clusters for B. bovis: A-type and B-type, and Bb60-mtm and Bb11920-mtm belong to B-type (Fig 3C, S4 Table). In summary, although orthologous relationships and expansions of genes encoding homologous multi-transmembrane proteins are not generally conserved across the Babesia and Theileria genera, what is conserved is a theme of lineage-specific expansions of gene families that encode multi-transmembrane proteins which are candidates for export to the iRBC.

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.

Table 2

Distribution of tpr-related, mtm and mfs genes in Piroplasmida.

Gene family	Species
	B. bovis	B. sp. (Xinjiang)	B. ovata	B. bigemina	B. microti	T. annulata	T. parva
tpr-related	7	4	3	3	5	50	44
mtm	44	7–8	-	-	-	-	-
mfs	2	2	28	41	-	1	1

Bbmtm is associated with BS uptake

The presence of expanded gene families encoding predicted exported multi-transmembrane proteins across piroplasmida suggests that intraerythrocytic development of these parasites requires de novo channel or transporter activity across the iRBC membrane. New permeability pathways have been described for the P. falciparum-iRBC membrane [30, 31], and the activity of a plasmodial surface anion channel (PSAC) was shown to be determined by the protein products of P. falciparum clag3.1 and clag3.2 genes [32] within the expanded clag gene family. Orthologs of clag genes are absent in piroplasmida, but Babesia parasites increase iRBC permeability to several organic solutes including sorbitol, suggesting the existence of channels or transporters [33]. Selection of P. falciparum under high BS concentration resulted in silencing of both clag3 genes, suggesting that resistance to BS happens through epigenetic downregulation of clag genes [34]. To gain insights into mtm function, we produced two BS-resistant lines of B. bovis by exposing the parasites to increasing concentrations of BS. These parasites showed increased half maximal inhibitory concentration (IC50) of BS compared to wild type (WT) (9.4 and 10.7 vs 2.3 μg/mL, Fig 4A), and delayed lysis in sorbitol lysis assays compared to WT (Fig 4B). RNA-seq analysis revealed that transcripts of several genes including one mtm (Bb60 for BS-resistant line 1 and BBOV_III000010 (Bb10) encoding an A-type Bbmtm for BS-resistant line 2) among many mtm family members was less abundant than in WT, suggesting that these mtms are potentially linked to BS uptake activity of iRBCs (Fig 4C, S5 Table). The sensitivity to BS was partially reversed in parasites cultured in the absence of BS for approximately two months (5 and 2.9 μg/mL, respectively), suggesting epigenetic regulation of this drug resistance. RNA-seq and qRT-PCR confirmed the recovery of downregulated Bb60 and Bb10 in the corresponding revertant parasite lines (Fig 4D, S4 Fig). Episomal overexpression of Bb60-mtm and Bb10-mtm in BS-resistant line 1 and 2 showed fluorescence signals inside the parasite and the edge of the iRBCs as expected (Fig 4E) and made these parasites more sensitive to BS (Fig 4F; IC50 of 7.8 and 2.3 μg/mL for BS-res1-Bb60 and BS-res2-Bb10, respectively), supporting our hypothesis on their role in BS uptake. However, episomally overexpressing Bb60-mtm with 10 or 100 nM WR99210 (Bb60-10 or Bb60-100 lines, respectively) in WT BS-sensitive line did not change IC50 to BS (2.8 or 2.5 μg/mL, respectively), perhaps indicating saturation of the channel activity (S5 Fig).

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.

Characterization of BbVEAP

Our three attempts to disrupt the BbVEAP gene locus, the third protein in our list which is exported into iRBC, using CRISPR/Cas9 system were unsuccessful, suggesting possible essentiality for the parasite. To functionally characterize BbVEAP we inserted a glucosamine (GlcN)-inducible glmS riboswitch together with 2 myc epitopes at the 3’ end of the BbVEAP open reading frame (ORF) [35]. Integration of the glmS sequence into the endogenous locus was confirmed by PCR (Fig 5A) and the expression of the myc-tagged protein was confirmed by Western blot analysis with the predicted band size (Fig 5A and 5B). In the absence of GlcN, glmS-tagged parasites demonstrated a roughly 80% reduction in basal BbVEAP protein expression by Western blot analysis (Fig 5B and 5C). This reduction did not affect the growth of parasites compared to the control (Fig 5D). The reduction of BbVEAP protein expression without addition of GlcN could be due to leakiness of the glmS system, decrease of mRNA stability or translation due to glmS sequence at its 3’ end, or endogenously produced GlcN by B. bovis as is reported for Trypanosoma cruzi [36], an observation for future verification. Addition of GlcN resulted in a dose dependent parasite growth reduction of BbVEAP-myc-glmS lines compared to the control BbVEAP-myc parasite (S6 Fig). Significant reduction of BbVEAP protein expression (82–92% reduction) with 2.5 mM GlcN, the maximum concentration without effect on the control parasite, was confirmed by Western blot analysis (Fig 5B and 5C). This knockdown resulted in a significant decrease in the growth rate accompanied by a significant increase of ring stage (immature) parasites and a decrease of binary form (mature) parasites, suggesting a defect in parasite development (Fig 5D and 5E).

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).

Because BbVEAP is deposited in spherical bodies, we examined whether the knockdown of BbVEAP affected structure of these organelles by transmission electron microcopy (TEM). While no clear changes were seen in spherical body structure, we noticed fewer RBC surface ridges (Fig 5F). Scanning electron microscopy (SEM) images further revealed that the ridges were less protrusive with GlcN treatment in addition to a reduction in number (S7 Fig). To exclude the contribution of parasite stage on ridge numbers, we quantified the ridges in the mature stage of the parasite (binary form) using TEM images. GlcN treatment significantly reduced the number of ridges on the surface of iRBCs (Fig 5G). Because ridges are the focal point for adhesion of B. bovis-iRBCs to endothelial cells [8], we examined whether knockdown of BbVEAP affected the cytoadhesion of iRBCs to BBECs. For this purpose, we used a cloned parasite from the cytoadherent B. bovis line (S1A Fig) to generated additional transgenic parasite lines in which a myc-glmS sequence was inserted within the 3' end of the BbVEAP ORF the same way as the BbVEAP-myc-glmS line. The parasites were treated with GlcN for 3 days and cytoadhesion assays were conducted. BbVEAP expression was dramatically reduced in two BbVEAP-knockdown clones with GlcN treatment as determined by Western blot analysis (S8A Fig). While the addition of GlcN had no effect on the binding ability of WT parasites, cytoadhesion was abrogated in two BbVEAP-knockdown clones (Fig 6A). Although the expressed amount of VESA1 appears to be unchanged by Western blotting (S8A Fig), its distribution was affected; parasites without GlcN treatment showed a punctate pattern in the iRBC resembling the expression of VESA1 on ridges while in parasites with GlcN treatment signal was only detectable within the parasite cytoplasm (Fig 6B). Unlike VESA1, the expression and localization of SBP4, another exported protein into iRBC, was not affected when treated with GlcN (Fig 6B, S8A Fig).

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.

Discussion

In this study we used iRBC surface biotinylation coupled with mass spectrometry to characterize the surface proteome of B. bovis. This approach was successfully used to describe the Plasmodium parasite surface and iRBC surface parasite-encoded proteome [37,38]. We prioritized 38 proteins for potential export into iRBC, based upon their abundance in the above assay and subsequent annotation for export motifs. This list includes known exported proteins, such as VESA1, SBPs, and SmORF, thus validating the study methodology. Of the novel exported protein candidates, 2 Bbmtm proteins belong to a multigene family; and a protein we have termed BbVEAP which is largely conserved within piroplasma. It is noted that our method resulted in the biotinylation and identification of merozoite surface and abundant proteins (S2B Fig), possibly due to biotin entry into or lysis of RBCs and parasites.

The Bbmtm genes are expanded to 44 gene copies in the B. bovis genome, the same number as the expansion of smorf genes. Like smorfs, mtms are typically located within gene neighborhoods, often telomeric, containing ves1 multigene family members. VESA1, SmORFs, and Bbmtms are exported proteins and close association of their gene loci in the genome of B. bovis may suggest a common epigenetic control of expression. Although the mtm gene family is unique to B. bovis, proteins with a similar multi-transmembrane structure such as MFS and TPR are expanded in other piroplasms, indicating a conservation of lineage-specific expansion of multi-transmembrane proteins. Unlike Plasmodium and T. gondii, which maintain a PV membrane (PVM) through their developmental cycle in the host cell, the PVM ruptures within minutes of invasion by Babesia, like a related piroplasm, Theileria [39,40]. Therefore, these parasites are in direct contact with the host cell cytoplasm and this may allow the parasite to export proteins possessing multiple TM domains to the host cell. To our knowledge this is distinct from Plasmodium, for which no known multi-transmembrane proteins are exported across the PVM. The Plasmodium CLAG/RhopH1 proteins that is localized on the iRBC membrane possess two transmembrane domains, although lacking a ‘classical’ transporter or channel multi-TM structure, and are involved in the permeability pathway across the RBC membrane, also termed PSAC; but they are introduced via merozoite rhoptry secretion during the RBC invasion [32,41,42].

The function of Bbmtm, MFS, or TPR in piroplasms is not known but their structure suggests a transporter or channel activity. It was shown that B. divergens, a zoonotic Babesia species that infects cattle and immunocompromised humans, increases RBC permeability to various organic solutes, indicating the existence of parasite produced channels or transporters on the surface of iRBC [33]. Similarly, we have seen increased permeability of RBC to sorbitol when they were infected with B. bovis, which was reduced in BS-resistant lines. We found that only either Bb60 or Bb10 was downregulated in each of two BS-resistant lines among all mtm gene family members and revertants recovered the transcription level of respective mtm, indicating a link between expression of these mtms and BS resistance. These observations support the idea that Bbmtm acts as a channel or transporter, and if this is the case then Bb60-mtm or Bb10-mtm can transport BS across the RBC membrane. Although Bb60 and Bb10 were downregulated in BS-resistant lines, the expression of other mtms was unchanged in comparison to control parasites, suggesting stable epigenetic regulation, or a substrate specificity for each mtm. Transcripts of several other genes were also reduced in the BS-resistant parasite lines, which might contribute to this resistance, and needs future evaluation.

In this study we identified BbVEAP and found that this protein is involved in ridge formation, VESA1 expression on iRBC, and binding of iRBCs to endothelial cells, which establishes BbVEAP as a novel virulence factor of B. bovis. Although distribution of VESA1 in the iRBC was affected by BbVEAP knockdown, the mechanism behind this observation is unclear. 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. A knobless line of P. falciparum has been described, and though the surface expression of PfEMP1, the functional homolog of VESA1 in P. falciparum, is reduced in these parasites they remained able to bind to endothelial cells [45,46]. Ridgeless B. bovis has not been reported and VESA1 is the only known ligand for cytoadhesion of B. bovis iRBCs [4]. Depletion of BbVEAP abrogated cytoadhesion indicating its role in the export or distribution of VESA1 and/or other putative ligands to the iRBC surface. Additionally, BbVEAP has a single ortholog in all piroplasmida that includes parasite species lacking ridge-like structures on iRBCs, suggesting a piroplasm-conserved function other than cytoadhesion. Considering the essentiality of this gene and conservation across piroplasmida, including the genera Theileria, targeting this protein could be promising for the development of pan piroplasmida therapeutics.

Spherical bodies are large secretory organelles within the apical end of Babesia spp. [47]. The products of two multigene family in B. bovis, SBP2 and SmORFs, are localized in spherical bodies [9,15]. In this study we found two novel proteins released from spherical bodies to iRBC, Bbmtm and BbVEAP. It was proposed that the PLM in SBP2 and SmORFs works as a retention signal in spherical bodies [15]. Bbmtm and BbVEAP also have PLM, suggesting the importance of PLM for the protein export to iRBC via spherical bodies in Babesia parasites.

Surface exposed proteins are targets of immunity and thus are vaccine targets, such as PfEMP1-VAR2CSA which is expressed on the surface of P. falciparum iRBCs and is responsible for pathogenesis of pregnancy-associated malaria [48]. VAR2CSA-based vaccines are currently in clinical trials [49]. The Plasmodium new permeability pathway, PSAC, is produced by Plasmodium and is inserted into the iRBC membrane for nutrient acquisition. Since nutrient acquisition is essential for malaria parasite survival, PSAC is considered a promising target for the development of antimalarials [50]. Targeting channels or transporters on the Babesia-iRBC membrane may lead to the development of pan anti-babesiosis drugs. Further characterization of our proteome data set could pave the way for identification of novel vaccine and drug targets and further development of new control strategies for bovine babesiosis caused by B. bovis.

Methods

Parasite culture and transfection

B. bovis Texas strain was obtained from Washington State University and kept in continuous culture using a microaerophilic stationary-phase culture system composed of purified bovine RBCs at 10% hematocrit and GIT medium (Wako Pure Chemical Industries, Japan).

The transfection of B. bovis was done as described [51,52]. Briefly, 100 μL of B. bovis-iRBCs were mixed with 10 μg of plasmid constructs in 100 μL of Amaxa Nucleofector human T-cell solution. Transfection was done using a Nucleofector device, program v-024 (Amaxa Biosystems, Germany). Ten nanomolar WR99210 was added one day after the transfection to select a transgenic parasite population.

Enrichment of B. bovis-iRBCs

Enrichment of B. bovis-iRBCs was done using a Histodenz solution. The solution was prepared by dissolving 27.6 g of Histodenz (Sigma-Aldrich) in 100 mL of Tris-buffered solution (5 mM Tris-HCl, 3 mM KCl, and 0.3 mM Na2-EDTA, pH 7.5). The iRBCs were layered on the surface of 80% Histodenz solution in GIT medium and centrifuged for 30 min at 2500 x g with a swinging bucket rotor. The iRBCs at the bottom of the tube were used for biotinylation assays.

Biotinylation and protein extraction

Biotinylation of iRBC surface proteins was done as described [25,37]. In the first attempt, proteins were serially extracted using a hypotonic solution (20X diluted PBS) and BugBuster protein extraction reagent (Novagen) containing the endonuclease benzonase. MS showed high bovine hemoglobin contamination. Thus, in the second and third attempts biotinylated iRBCs were initially treated with 0.2% (w/v) saponin on ice for 15 min to remove hemoglobin. Protein extraction was done serially first by resuspending the parasite pellets in BugBuster protein extraction reagent containing the endonuclease benzonase. Following centrifugation and removing extracted proteins, the remaining parasite pellet was lysed with a solution containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris pH8.0, 1.0% Triton-X 100 (w/v), and protease inhibitor cocktail (Complete Mini, Roche) at 4°C for 1 h. The protein extract was incubated with Dynabeads MyOne Streptavidin C1 (Invitrogen) with rotation at 4°C for 1 h. The beads were washed 5 times with wash buffer containing 0.1% SDS, 400 mM urea, 150 mM NaCl, and 50 mM Tris-HCl (pH 8.0) to remove unbound proteins. Bound proteins were eluted from the beads by boiling for 5 min in 1x Sample Buffer (25 mM Tris (pH 6.8), 2.5% w/v SDS, 2.5% v/v glycerol, 0.08% w/v bromophenol blue, and 5% beta-mercaptoethanol). Proteins were assessed for biotinylation via Western blotting using horseradish peroxidase (HRP)-conjugated streptavidin (1:40,000, Invitrogen).

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The purified biotinylated proteins were subjected to LC-MS/MS as described [53]. The samples were briefly electrophoresed on SDS-polyacrylamide gel electrophoresis. The gel containing proteins were excised, fixed with acetic acid/methanol solution, and subjected to LC–MS/MS analysis at the W. M. Keck Biomedical Mass Spectrometry Laboratory, University of Virginia, USA. The data analysis was performed by database searching using the Sequest search algorithm against the Bos taurus and B. bovis reference strain in UniProt and SwissProt. Filtering and extraction of data was performed using Scaffold version 4.8.9 (Proteome Software Inc.). Protein identifications were accepted if they could be established at greater than 90% probability and contained at least 1 identified peptide. Quantitative value (normalized total spectra) was used to show an estimate of protein abundance between biotinylated and non-biotinylated samples. PiroplasmaDB-34 [54] was used for annotation of the target proteins.

Cytoadhesion assay

Cytoadhesion assays were done as described [55]. Briefly, bovine brain endothelial cells (BBECs; Cell Applications Inc., USA) were seeded in 6 well plates containing cover glasses (Matsunami Glass, Japan). B. bovis-iRBCs with 2–5% parasitemia and 1% hematocrit were added to the BBEC culture. The cells were incubated for 90 min with gentle agitation every 15 min. Nonadherent iRBCs were washed away with Hanks-balanced salt solution. Cells on the cover glasses were fixed with methanol and stained with Giemsa's solution and the number of bound iRBCs were counted for 500 BBECs.

Plasmid construction

The schematic of the plasmid expressing myc-tagged target proteins is shown in S2 Fig. The primers used for plasmid construction are listed in S6 Table. B. bovis elongation factor-1α intergenic region-B (ef-1α IG-B), ORF of the gene of interest (GOI), and thioredoxine peroxidase-1 (tpx-1) 3’ noncoding region (NR) were PCR-amplified from B. bovis genomic DNA. A DNA fragment containing B. bovis actin 5’ NR (act 5’NR), human dihydrofolate reductase (hdhfr), and B. bovis rhoptry associated protein 3’ NR (rap 3’NR) was amplified from a B. bovis green fluorescent protein (GFP)-expressing plasmid [51]. ef-1α IG-B was cloned into the EcoRI site of pBluescript SK using In-Fusion HD Cloning Kit (Takara Bio Inc., Japan). Subsequently, the ORF of GOI tagged with 2 myc epitopes, tpx-1 3’NR, and act 5’NR-hdhfr-rap 3’NR were cloned into SmaI to make the final plasmid for episomal expression of myc-tagged proteins.

The CRISPR/Cas9 system was employed to delete BBOV_III004280 or insert myc and glmS sequences into the 3' end of the original locus [56]. Briefly, homologous regions (HRs) were PCR-amplified from B. bovis genomic DNA and inserted into the BamHI site of the BbU6-Cas9-hDHFR plasmid. Single guide RNA (sgRNA) was inserted into the AarI site of BbU6-Cas9-hDHFR using T4 DNA ligase (New England Biolabs, USA). Parasites were transfected as described [51,52] and the obtained transgenic parasites were cloned by limiting dilution before analysis. Diagnostic PCR was done using glms-F-IF and 4280-3NR-integR (S6 Table) to confirm insertion of myc-glmS.

Development of BS-resistant B. bovis and comparative transcriptomics

B. bovis WT parasites were initially cultured with 1 μg/mL blasticidin-S solution (BS; Thermo Fisher Scientific, USA), and the BS concentration was increased stepwise. Two independent resistant lines that propagate under 4 μg/mL BS were produced. To produce revertant parasites, BS-resistant lines were cultured without BS for two months. Comparative transcriptomics between B. bovis WT and its BS-resistant derivatives or revertant parasites were performed by RNA-seq. RNA was extracted from parasites using TRIzol reagent (Invitrogen). Libraries were constructed using a TruSeq Stranded mRNA Library Preparation Kit (Illumina, USA), according to the manufacturer’s protocol and the products were subjected to Novaseq6000 (Illumina) with the 150-bp paired-end protocol. Acquired reads were mapped against B. bovis T2Bo reference genome obtained from PiroplasmaDB-37 using HISAT2 [57]. Read data normalization and differential expression were obtained using Cufflinks with the default parameters [58]. Complementary DNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen) with random primers.

Bioinformatics analysis

SignalP-5.0 and -3.0 were used to predict putative signal peptides [59]. TMHMM-2.0 was used to predict TM domains [60]. GPI anchors were predicted using PredGPI [61]. To identify multigene families encoding multiple TM domains, whole genome sets of protein sequences for B. bovis, B. bigemina, B. ovata, B. microti, Theileria annulata, T. parva, T. orientalis, P. falciparum, and T. gondii were evaluated using TMHMM-2.0 and proteins were selected having equal to or more than eight TM domains and at least one TM domain average per 100 amino acids. Mutual homology among the selected proteins were identified by BLASTP [62] and proteins with more than a 200 bit-score and by eye were regarded as homologs. The overall relationships were visualized with Gephi [63] using a Fruchterman–Reingold layout.

SDS-PAGE and Western blotting

Parasite proteins were extracted using 1.0% Triton-X 100 (w/v) in PBS and protease inhibitor cocktail (Complete Mini, Roche) at 4°C for 1 h. The protein fractions were separated by electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were probed with mouse anti-myc monoclonal antibody (1:500; 9B11, Cell Signaling Technology, USA), rabbit anti-TPx-1 polyclonal antibody (1:250 [52]), rabbit anti-VESA1 polyclonal antibody (1:100), or rabbit anti-SBP4 polyclonal antibody (1:1000 [13]) at 4°C overnight. Washing was done in PBS supplemented with 0.05% Tween-20 (TPBS). Secondary probe of membranes was done with HRP-conjugated goat anti-mouse or rabbit IgG (1:8,000; Promega, USA). Protein bands were visualized using ECL Select Western Blotting Detection Reagent (GE healthcare) and detected by a chemiluminescence detection system (LAS-4000 mini; Fujifilm, Japan).

Immunoprecipitations were performed as described [64]. Parasite pellets were prepared by saponin treatment of BbVEAP-myc tagged parasites and cross-linked with 1 mM DSP (Sigma). Proteins were extracted with 1% Triton X-100 (w/v) in PBS (containing 1 mM EDTA, 10% glycerol, and protease inhibitors) at 4°C for 1 h. The extracted proteins were incubated with anti-myc mouse mAb (9B11, Cell Signaling) at room temperature for 4 h with gentle rotation. The solution was then mixed with protein G Sepharose 4 fast flow (GE Healthcare) and incubated with rotation at 4°C overnight. The mixture was centrifuged and the beads were washed with 0.5% Triton X-100 (w/v) in PBS (containing 1 mM EDTA, 10% glycerol, and protease inhibitors). To elute proteins, the beads were mixed with 2.4 μg/μL of c-myc peptide (Thermo Fisher Scientific) and incubated at 4°C for 12 h. The beads were centrifuged and the supernatant was collected as an immunoprecipitated (IP) fraction.

Indirect immunofluorescence antibody test (IFAT)

IFAT was done on thin blood smears from cultured parasites that had been air-dried and fixed in a 1:1 acetone:methanol mixture at −20°C for 5 min [65]. Smears were immunostained with mouse anti-myc monoclonal antibody (9B11) at 1:500 dilution in TPBS and incubated at 37°C for 60 min. Double immunostaining of smears was done with rabbit anti-VESA1α (antisera against peptide YNQVVHYIRALFYQLYFLRK; Medical & Biological Laboratories Co., ltd, Japan) at 1:50 dilution, or rabbit anti-SBP4 at 1:1000 dilution. The smears were incubated with Alexa fluor 488- or 594-conjugated goat anti-mouse or Alexa fluor 488-conjugated goat anti-rabbit IgG antibody (1:500; Invitrogen) at 37°C for 30 min. For staining the nuclei, the smears were incubated with 1 μg/mL Hoechst 33342 solution at 37°C for 20 min. The smears were examined using a confocal laser-scanning microscope (A1R; Nikon, Japan).

Electron microscopy

For preparation of transmission electron microcopy (TEM) samples, iRBCs were fixed with 2% glutaraldehyde (Nacalai Tesque, Japan) in 0.1 M sodium cacodylate buffer at 4°C for 60 min. The samples were rinsed, and then post-fixed with 1% OsO4 (Nacalai Tesque) at 4°C for 60 min. The samples were washed, dehydrated in a graded series of ethanol and acetone, and embedded in Quetol 651 epoxy resin (Nisshin EM, Japan). Ultra-thin sections were stained and examined at 80 kV under a transmission electron microscope (JEM-1230; JEOL, Japan).

Immunoelectron microcopy samples were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (PB) on ice for 15 min. Cells were dehydrated with 30%, 50%, 70%, and 95% ethanol each time at 4°C for 5 min, and embedded in LR White resin (London Resin Company, UK). Thin sections were blocked with 5% non-fat milk (Becton, Dickinson and Company, USA) and 0.0001% Tween 20 (Wako, Japan) in PBS (PBS-MT) at 37°C for 30 min and incubated with rabbit anti-myc polyclonal antibody (1:100; ab9106, Abcam, UK) or control normal rabbit IgG at 4°C overnight. After washing the sections were incubated with goat anti-rabbit IgG conjugated to 15 nm gold particles (1:20; EY Laboratories Inc., USA), and fixed with 0.5% OsO4 at room temperature for 5 min. The sections were stained and observed by TEM.

For scanning electron microscopy (SEM), the iRBCs were enriched by density gradient separation using a Percoll and sorbitol solution [33]. Samples were fixed with 1.2% glutaraldehyde in 0.1 M PB at room temperature for 20 min. They were then rinsed and post-fixed with 1% OsO4 at room temperature for 10 min. After dehydration in graded series of ethanol, the samples were immersed in t-butyl alcohol and placed at -20°C overnight, and then freeze-dried. The samples were sputter-coated with gold and palladium and imaged on a scanning electron microscope (JSM-840; JEOL).

Statistical analyses

The parasitemia and proportion of parasite stages were plotted using Prism 6 (GraphPad Software, USA) and evaluated using multiple t-test. The ridge numbers on the surface of iRBCs were compared using Mann-Whitney U test. The difference in number of cytoadhered iRBCs per 500 BBECs was evaluated using a two-tailed paired Student’s t-test. The values were considered significantly different if P-value was below 0.05.

Panning of B. bovis and live fluorescence microscopy of biotinylated iRBCs.

(A) Selection of cytoadherent B. bovis to BBECs. The number of bound iRBCs per 100 BBECs were counted. (B) Live fluorescence microscopy of biotinylated iRBCs reacted with streptavidin-conjugated Alexa Fluor 488 (green). The parasite nuclei were stained with Hoechst 33342 (Hoechst, blue). No released merozoites were seen. Scale bar = 10 μm.

(TIF)

Click here for additional data file.

Schematic of a plasmid expressing myc-tagged candidate proteins and fluorescence microscopy images.

(A) Schematic of a plasmid expressing myc-tagged candidate proteins. ef-1αIG-B, elongation factor-1α intergenic region B; GOI orf, gene of interest ORF; tpx-3’NR, thioredoxine peroxidase-1 3’ noncoding region; hdhfr orf, human dihydrofolate reductase ORF; rap-3’NR, rhoptry associated protein 3’ noncoding region. (B) Indirect immunofluorescence antibody test of transgenic B. bovis expressing myc-tagged target proteins stained with anti-myc (green). The parasite nuclei were stained with Hoechst 33342 (Hoechst, blue). Scale bar = 5 μm.

(TIF)

Click here for additional data file.

Quantification of gold particles in immunoelectron micrographs.

The number of gold particles were quantified in B. bovis with Bb60-mtm and BbVEAP tagged with myc epitopes. Gold particle numbers were counted in spherical bodies and iRBC surface only in parasites with clear spherical bodies in electron micrograph sections (****, P < 0.0001; determined by Mann-Whitney U test).

(TIF)

Click here for additional data file.

Quantitative reverse transcription PCR of Bb60 and Bb10.

Relative transcript levels of Bb60 and Bb10 in WT, BS-resistant lines and BS-sensitive revertants. Transcript levels are normalized against methionyl-tRNA synthetase (Gene ID: BBOV_I001970).

(TIF)

Click here for additional data file.

Overexpression of Bb60-mtm in WT line did not affect the BS resistance.

Growth inhibition curves of two Bb60-overexpressing parasite lines in the presence of different concentrations of BS (μg/mL). Bb60-mtm is episomally overexpressed in WT parasites under 10 or 100 nM WR99210 in Bb60-10 or Bb60-100 lines, respectively. All data are expressed as mean ± SEM.

(TIF)

Click here for additional data file.

Growth of BbVEAP-myc-glmS parasite under increasing concentrations of GlcN.

Growth of BbVEAP-myc-glmS and control parasites (BbVEAP-myc) in the absence or presence of 1, 2.5, 5, and 10 mM GlcN. Initial parasitemia was 0.05% and parasitemia was monitored for 3 days with daily culture medium replacement. The data are shown as mean ± S.D. from technical triplicates.

(TIF)

Click here for additional data file.

Scanning electron microscopy of BbVEAP-myc-glmS parasite in the absence or presence of GlcN.

Scanning electron microscopy showing ridges on the surface of RBC infected with BbVEAP-myc-glmS parasites or a control parasite following 3 days exposure to GlcN. Scale bar = 1 μm.

(TIF)

Click here for additional data file.

Western blot analysis of BbVEAP-myc-glmS and WT parasites and immunoprecipitation of BbVEAP-myc parasite.

(A) Western blot analysis of two clones (tg1, tg2) of BbVEAP-myc-glmS and WT parasites in the presence or absence of GlcN. TPx-1 detected with anti-TPx-1 antibody was used as a loading control. (B) Immunoprecipitation with anti-myc for BbVEAP-myc parasite.

(TIF)

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List of identified proteins by mass spectrometry.

(XLSX)

Click here for additional data file.

List of predicted secretory proteins.

(XLSX)

Click here for additional data file.

List of putative exported proteins confirmed for their localization.

(XLSX)

Click here for additional data file.

List of mtm in the B. bovis genome.

(XLSX)

Click here for additional data file.

Genes up- or downregulated in BS-resistant parasites.

(XLSX)

Click here for additional data file.

List of primers used in this study.

(XLSX)

Click here for additional data file.

28 Mar 2020

Dear Dr. Asada,

Thank you very much for submitting your manuscript "Novel Babesia bovis exported proteins that modify properties of infected red blood cells" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Your submission was evaluated by three independent reviewers and found to contain novel information of potential significance to our understanding of Babesia spp. parasites. However, significant concerns were raised, including the starting point of surface biotinylation. Of particular significance are concerns regarding the veracity of some conclusions that were not felt to be adequately supported by the data provided. For example, stronger rationale is needed justifying the focus on Bb60 and Bb10 when numerous genes underwent significant transcriptional changes. It was felt that conclusions about function of Bbmtm in nutrient acquisition, and of BbVEAP in cytoadhesion, were of potential significance but inadequately supported. If you choose to respond by minimizing your claims most of these issues may be dealt with by rewriting. If you choose to gain more experimental evidence to support your claims this could amount to a significant amount of effort. Your revision should address the specific points raised by each reviewer.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

David R. Allred, Ph.D.

Guest Editor

PLOS Pathogens

Kirk Deitsch

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

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Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: (No Response)

Reviewer #2: Hakimi et al. show in their manuscript entitled 'Novel Babesia bovis exported proteins that modify properties of infected red blood cells' by a combination of surface biotinylation/LcMSMS and follow-up studies involving inducible knockouts the identification of a new group of proteins, presumed transporters in the RBC membrane and another, independent protein essential for host cell modifications resulting in cytoadherence.

A large amount of work clearly went into this manuscript. The manuscript is well written, the experiments well conducted and the results are interesting, not only to people in the field of Babesia biology, but also for people interested in host cell modifications.

However, the manuscript could improve in the following ways:

Firstly, the authors do not make it clear that the surface biotinylation approach resulted, like it did previously in other species, in leakiness of the RBC and/or stickiness of other, non-surface proteins, requiring drastical filtering of the 255 B. bovis hits, to come to a list of 38 proteins. This list still includes a number of known merozoite surface proteins, not only parasite proteins translocated to the RBC surface. Indeed, the authors show in S2 Fig, that 7 of the 10 uncharacterised proteins from that list, are localised to either apical organelles or the merozoite surface. Only 3 show the targeted export into the host cell the authors were looking for in their biotinylation experiment. Therefore, the authors should make it clear that the surface labelling did in fact label most abundant proteins, both within the RBC and probably also within the parasite and therefore the list of 38 resembles a list of proteins for which masspec evidence is available in blood stages and which have additionally one of the following criteria: signal sequence, TM domain or ER retention signal.

Secondly, the flow of the manuscript is lacking. The authors could improve this by highlighting that once they generated the list of expressed proteins in B.bovis which have one of the 3 criteria stated above, that they focussed on 2 of the genes from their list of 38 which showed export into the RBC. One of these is a member of the novel multigene family of potential transporters, which is a very interesting finding. The authors state that in B. bovis this list has 44 members but fail to include this list in the supplementary materials. In order to demonstrate that at least some of these MTMs are not only localized to the RBC membrane but also function as transporters the authors conduct RNA-seq analysis on BSD resistant B. bovis lines, identifying reduced expression of Bb60 and Bb10. This is comparable to studies in Plasmodium falciparum where the same has been described. Presumably Bb10 is a member of the MTM family which was not identified my the masspec approach? Also, presumably the authors tried to generate knockouts of Bb60 without detecting a growth or adhesion phenotype? Did the authors follow up on the function of Bb11920, but failed to find a phenotype and hence dropped the characterisation?

Following on from two MTM family members the authors then started to characterise VEAP, again without a clear link why the reader has to jump to this molecule next. The authors should clearly state that the chosen proteins for characterisation are the ones which they demonstrated earlier of being exported.

To characterise the function of VEAP, the authors use very elegantly the GlmS system to inducibly down-regulate the transcript levels upon glucosamine addition. Unclear is however why even before GlcN addition the protein levels are reduced by 80% compared to wt. Could the authors please address this and speculate? 20% of protein level of VEAP clearly was sufficient to perform a wildtype-like function, and the further reduction by GlcN addition resulted in the recorded phenotype which is striking and an important contribution to RBC surface modification mechanisms in Babesia.

Reviewer #3: Review for Hakimi et al: this manuscript describes the surface biotinylation of Babesia bovis infected erythrocytes and the validation of a selection of the identified hits. It reveals 3 new exported proteins from B. bovis. Two belong to a group of multi transmembrane host cell surface proteins encoded by a gene family termed Bbmtm and one that was termed VEAP, a protein without transmembrane domain that localizes to the host cell cytoplasm. The authors go on to present functional data for both, the Bbmtm and VEAP. This includes the analysis of a conditional VEAP knock down that indicates a role of this protein in cytoadhesion of B. bovis infected erythrocytes and a role in formation of host cell ridges. The authors also postulate that Bbmtm are involved in the nutrient permeability of the membrane of infected erythrocytes.

Overall this is a very interesting paper that identifies novel exported proteins that play (or in the case of Bbmtm products may play) important roles for B. bovis. However, there are a number of concerns that need to be considered. While the identification of Bbmtm gene products as novel host cell membrane proteins is convincing and the comparative analysis of this family in Babesia and related parasites is important, the functional insight into these proteins is very preliminary. These findings should either be substantiated (overexpression of Bb60 or Bb10 in the blasticidine resistant lines would be an easy experiment to do, see major points) or the conclusions on the functon of these proteins needs to be toned down or even removed. The data on VEAP is stronger but the influence on VESA1 is not very broadly documented and should also be substantiated. If these points can be thoroughly addressed, this will result in a very nice study that will be of high interest to the field.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: Major points:

1. The data on a role of Bbmtm gene products in nutrient permeability of the host cell is not very conclusive. The authors raise parasites resistant to blasticidine and show that this is due to a reduced permeability of the membrane of infected erythrocytes. Transcriptomics with these parasites showed that in the two blasticidine resistant clones either Bb60 or Bb10 (both Bbmtm members) were downregulated - but among quite a large list of other genes, some of which were much more downregulated. Causality between downregulation of a Bbmtm and the phenotype is therefore not established. For instance VESA1 is downregulated much more in both cases, but likely is not the reason for the observed phenotype. The same is the case for many other downregulated genes. Bb10 and Bb60 are also not the most dramatically regulated genes.

Figure S3 shows that Bb10 is expressed more in Res1 than in the corresponding reverted parasites and this difference seems to be in a similar fold level to the upregulation of Bb10 in the Reverted 2 vs Res2 line, indicating that a certain level of randomness is involved.

To establish the role of the Bbmtm in the nutrient permeability of the infected erythrocyte more experiments are needed. Instead of overexpressing Bb60 in wild type parasites, overexpression of the downregulated Bb60 or Bb10 should be carried out in the R1 and R2 lines. Only if this reverts the phenotype, a function of these proteins in the channel activity can be postulated. This experiment is essential to draw the conclusion that these proteins are involved in this activity, otherwise these statements need to be removed from the paper (e.g. such as line 385...: 'This resistance was linked to the expression of one mtm (Bb10 and Bb60 in each line) indicating that mtm is responsible for BSD uptake and may form a channel or transporter on the surface of iRBC...').

Some other remarks related to this point:

- If Bbmtm indeed turn out to play a role in the nutrient channel function of the host cell, I wonder why only very few Babesia species have an expanded family of these proteins if they have this function.

- The fact that (line 364...): 'VESA1, SmORFs, and Bbmtms are exported proteins and close association of their gene loci in the genome of B. bovis may suggest a common epigenetic control of expression.' might also be used for a reversed argument and could explain why Bbmtms were downregulated, even though they may not have a function in this activity (if another neighbouring gene is involved in this activity and all other genes in the vicinity are epigenitically co-regulated).

2. The functional data on BbVEAP is more direct and more broadly founded than that on Bbmtm. The conditional knock down of this protein convincingly shows a reduced presence of ridges and reduced cytoadherence. However, the validation of the knock down is only shown for the non-cytoadherent parasites. The full validation should also be shown for the cytoadherent parasites (this could for instance be provided as a supplementary figure). More information should also be given about this cytoadherent line (origin; characteristics; was it used before?). Furthermore the effect on VESA1 needs to be more thoroughly analysed. In Fig. 6b, in the GlcN+ parasites the VESA1 signal simply seems very faint. Is there a real difference in the location? Can the change in distribution (or expression levels?) of VESA1 be substantiated and quantified (IFA and Westernblot)? Is there a change in the distribution of VESA1 on the erythrocyte surface in EM?

How do the authors explain that VEAP appears to be soluble in the host cell but was detected by the surface biotinylation? What does this mean for the other hits found in their mass spectrometry analysis? How can VEAP1 influence VESA1 and ridge formation in this location?

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: - Include number of unique peptides identified per protein in table S1

- Line 126 states that a complete list of all identified proteins is given, it should read all identified B. bovis proteins as the host proteins are not listed.

- Include a list of all 44 B.bovis MTM genes

- It would be desirable to verify that other MTM proteins identified by masspec in this study also localize to the RBC membrane. Why choose Bb60 and Bb11920 out of the list of 7 mtm family proteins?

Reviewer #3: Minor points:

- The authors report that 32 of the proteins identified by the surface biotinylation possessed a

PEXEL-like export motif (RxLx or RxxLx). Please clarify where in the protein these motifs were found. In malaria parasites these motifs are only functional if in a specific range downstream of a signal peptide but this is different in T. gondii. What were the criteria for calling of such a motifs in this study? I could not find the information in the M&M how this was done.

- Twelve proteins, including two positive controls, were selected for validation (line 168... and Figure S2). Can the authors explain in the text how exactly these proteins were selected?

- Please provide statistics for the gold particle counts shown in Fig. 2c (how cells were selected; number of cells inspected; location of gold etc)

- Why do Bbmtm A and B cluster so far apart in 3c? Does this mean they are two independent families? What is the level of sequence homology?

- Figure 5a PCR gel. I assume these are cloned parasites. If not, it should be shown that there are no parasites without the correct genomic insertion using a PCR to detect the unmodified locus (to exclude the presence of parasites with incorrect insertions). What is tg1-1 and tg1-2 etc in this figure part?

- there is a 'data not shown' in the discussion, please either provide the data or remove

- indicate at least fixative used for the IFA procedure (line 557 'air-dried and fixed as described [62]'.), so the reader does not need to consult other publicatons to know the type of IFA that was used.

- please provide the source of rabbit anti-SBP4 antibody (or provide validation if newly generated)

- is this a commercial rabbit anti-VESA1α antibody by MBL? If it is a custom made antibody, again validation of these antibodies needs to be provided

- How were the CRISPR lines obtained? Were parasites cloned? If yes, how?

- exact information how often experiments were done and if technical or biological replicas are shown is often missing. Just as an example, the legend of figure 6 A says that all data are 'triplicate assays'. Are these independent experiments or technical replicas?

- please remove the statement in line 390-392: if this is true, this manuscript at least does not show it.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Submitted filename: PPathogens.D-20-00279.review.docx

Click here for additional data file.

9 Jul 2020

Submitted filename: Answers to reviewers_R1.docx

Click here for additional data file.

29 Jul 2020

Dear Dr. Asada,

Thank you very much for submitting your manuscript "Novel Babesia bovis exported proteins that modify properties of infected red blood cells" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please note that Reviewer 3 has identified a few remaining points in need of correction before the manuscript could be accepted. Of particular importance are two points: (i) The reviewer makes a significant point that the data in S5 Figure is important to your arguments. Please try either to combine it into one of the main figures, or make it a main figure. (ii) Please respond to the comment regarding correction of labeling in S8 Figure to avoid any confusion. Upon inspection, I observed two issues in S5 Figure: (i) the x-axes are labeled in “log ug/mL”, whereas in the figure legend the concentrations are provided in nM. Please make the units of concentration consistent throughout the manuscript (either system, but preferably in molarity). (ii) In the lower panel of Figure S5B there are 4 curves but only 3 identified samples; please correct this. In addition, Figure S1B shows evidence of significant pixel shift between the DIC and fluorescent images. Please realign the superimposed images for correct alignment. Thank you for your attention to these matters.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

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Sincerely,

David R. Allred, Ph.D.

Guest Editor

PLOS Pathogens

Kirk Deitsch

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #2: (No Response)

Reviewer #3: The revised manuscript satisfactorily deals with the reviewer concerns. It contains new data that increases the support for the conclusions of the paper, including basic data on the adherent lines and overexpression data of Bb10 and Bb60 in the BS-resistant line.

I have only a few minor points left that I recommend to consider:

- The data in Fig. S5 is important (without it the evidence for Bb10 and Bb60 in BSD transport is rather weak). Could it also be part of a main figure?

- + and - GlcN is mixed up in Fig S8

- one further explanation for what the authors call 'leakiness' of the glmS line could be that insertion of the glmS sequence in the 3'-region affected the stability or translation of the target mRNA independent of its interaction with GlcN.

- Please get the manuscript checked by a native speaker: Some examples:

line 174: Two proteins showed signals inside parasite... add 'the' before parasite

line 185: Remaining seven candidates did not show signals... add 'the' at start of sentence

line 363: inserted to the 3' end of the BbVEAP ORF the same as BbVEAP-myc-glmS line. 'the same way as'?

line 366: with GlcN treatment by Western blot analysis (S8A Fig). 'as determined by Western blot analysis'?

line 426: These observation... observation should be plural

line 429 the expression of other mtms were unchanged... 'was unchaged'

**********

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Reviewer #2: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at .

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see

11 Aug 2020

Submitted filename: Answers to reviewers_R1.docx

Click here for additional data file.

20 Aug 2020

Dear Dr. Asada,

We are pleased to inform you that your manuscript 'Novel Babesia bovis exported proteins that modify properties of infected red blood cells' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

David R. Allred, Ph.D.

Guest Editor

PLOS Pathogens

Kirk Deitsch

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

This manuscript provides some very intriguing new characters in Babesia biology that are worthy of attention and followup.

Reviewer Comments (if any, and for reference):

25 Sep 2020

Dear Dr. Asada,

We are delighted to inform you that your manuscript, "Novel Babesia bovis exported proteins that modify properties of infected red blood cells," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064