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Advances in understanding red blood cell modifications by Babesia [1]

['Hassan Hakimi', 'National Research Center For Protozoan Diseases', 'Obihiro University Of Agriculture', 'Veterinary Medicine', 'Obihiro', 'Hokkaido', 'Department Of Veterinary Pathobiology', 'College Of Veterinary Medicine', 'Texas A M University', 'College Station']

Date: 2022-11 

Babesia are tick-borne protozoan parasites that can infect livestock, pets, wildlife animals, and humans. In the mammalian host, they invade and multiply within red blood cells (RBCs). To support their development as obligate intracellular parasites, Babesia export numerous proteins to modify the RBC during invasion and development. Such exported proteins are likely important for parasite survival and pathogenicity and thus represent candidate drug or vaccine targets. The availability of complete genome sequences and the establishment of transfection systems for several Babesia species have aided the identification and functional characterization of exported proteins. Here, we review exported Babesia proteins; discuss their functions in the context of immune evasion, cytoadhesion, and nutrient uptake; and highlight possible future topics for research and application in this field.

Funding: This work was primarily supported by grants from Japan Society for the Promotion of Science ( https://www.jsps.go.jp/english/index.html ) to M.A. (16K08021, 19K06384, 22K05982), H.H. (15K18783, 19K15983), and S-I.K. (18K19258, 19H03120, JPJSBP120212501). This work was partially supported by National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine (NRCPD OUAVM, https://www.obihiro.ac.jp/facility/protozoa/en ) Joint Research grants to M.A. (28-11, 29-2, 30-1) and International Institute for Zoonosis Control, Hokkaido University ( https://www.czc.hokudai.ac.jp/en/ ) Joint Research grants to M.A (2021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Introduction

Babesia parasites modify the host red blood cell Babesia species are protozoan parasites belonging to the phylum Apicomplexa and are transmitted by ticks. More than 100 Babesia species have been reported, which infect mammalian and avian hosts, and several species are known to parasitize domestic animals such as cattle, horses, sheep, goats, as well as dogs [1]. Bovine Babesia species (B. bovis, B. bigemina, B. divergens, B. major, and B. ovata) capable of mediating disease are widely distributed in temperate and tropical regions in the world. It is estimated that 1.2 billion cattle are at risk of infection, and bovine babesiosis is a major cause of economic loss within the beef and dairy industries, thus highlighting the significance of this protozoan parasite in veterinary medicine [2]. Several Babesia species, including Babesia microti and B. divergens, have gained attention as pathogenic species for emerging zoonotic diseases [3]. Babesia sporozoite stage parasites are released from the salivary glands of infected vector ticks during a blood meal and directly invade red blood cells (RBCs) to become pear-shaped “pyriform” intraerythrocytic stage parasites [4,5,6]. The pyriform parasites rapidly replicate by binary fission, leading to paired parasites inside the RBC. Some clades of Babesia, such as B. microti and B. duncani, form 4 merozoites per division, which result in the appearance of the Maltese cross form [7]. This multiplication and the following destruction of the host RBC during parasite egress lead to pathologies including fever, anemia, jaundice, and hemoglobinuria. Although most apicomplexan parasites infect nucleated host cells, Babesia and a few closely related genera such as the malaria parasite Plasmodium have evolved to parasitize enucleated RBCs. During invasion and subsequent development, the parasite modifies host RBCs by exporting proteins. Modification of nucleated host cells is known for many apicomplexan parasites, and exported proteins manipulate host cell processes such as transcription and kinase activity [8]. Babesia and Plasmodium reside inside anucleate RBCs (Plasmodium also has a liver stage, but Babesia solely multiply inside RBCs), and, therefore, the role of exported proteins is likely different from apicomplexans, which infect nucleated cells. The repertoire of exported proteins is called the “exportome”, and in the instance of Plasmodium, hundreds of such proteins have been identified within numerous expanded gene families [9,10]. Extensive studies have described the role of Plasmodium exportome proteins in reinforcing the RBC cytoskeleton, altering the RBC surface to evade immune recognition, inducing cell adhesive properties, and changing the RBC membrane permeability to allow the uptake of nutrients and release of parasite metabolic waste products [11]. Since the exportome is important for the parasite survival and its pathogenicity, these proteins are considered to be drug or vaccine targets [11]. Despite the similarities to Plasmodium infection, less is known regarding the Babesia exportome, and the degree of overlap, if any, in the repertoire of exported proteins. While it is known that infected RBCs (iRBCs) are modified by Babesia parasites, such as to induce RBC adhesive properties and alter permeability, prior to the past few years, only a few exported proteins were identified and functional analysis was scarcely done [12,13]. However, the recent completion of genome sequences and the establishment of transfection systems for several Babesia species has aided characterization of the exportome, and the functions of several exported proteins have been partially revealed.

Immune evasion and cytoadhesion To establish an infection in the host, Babesia parasites must evade immune recognition, including avoidance of an antibody-mediated immune response or direct clearance of iRBCs in the spleen. The spleen plays a critical role in recognizing and clearing abnormal and aged RBCs, as well as RBCs altered due to infection. In the case of B. bovis, avoidance of splenic clearance is achieved by the display of parasite-encoded ligands on the surface of the iRBCs, which confer capillary endothelial adhesion and sequestration in internal organs [45]. However, in this manner, the parasite exposes itself to a possible antibody-mediated immune response, which it in turn evades over the course of an infection by sequentially switching the expression of the surface ligands with other members of a repertoire of antigenically variant surface proteins. Antigenic variation in Babesia parasites was first described in the rodent parasite B. rodhaini [46], and following in B. bigemina [47], and was suggested to occur in B. bovis by the parasite-encoded proteins on the surface of iRBC that were named VESA1 [31,48,49]. VESA1 is a heterodimeric protein encoded by the multicopy ves1α and ves1β gene families in B. bovis [31,50]. The first genome sequence of B. bovis revealed 119 copies of ves1 genes [40], which, with improvement of genome assembly, increased to 133 genes consisting of 81 ves1α, 48 ves1β, and 4 unclassified ves1 [51]. In the B. bovis genome, ves1α and ves1β gene pairs are located in a divergent orientation and are expressed at a site called locus of active transcription (LAT) [52]. It is believed that ves1 genes show monoallelic expression [53] in which an active LAT containing a ves1α and ves1β pair are simultaneously expressed by a bidirectional promoter located within the LAT intergenic region [54]. Antigenic variation and the resulting immune evasion in B. bovis occur through epigenetic in situ switching of transcribed ves1 and genomic recombination, which results in mosaicism of ves1 genes [55]. Sequestration of B. bovis is mediated by interaction of iRBC surface ridges with bovine endothelial cells (Fig 1C) [16,56,57]. The sequestered iRBCs can disrupt blood flow in internal organs and in the brain, which causes cerebral babesiosis [16]. It was proposed that VESA1 is responsible for cytoadhesion of iRBCs based upon the observations that VESA1 proteins are clustered on ridges, and specific monoclonal antibodies against VESA1 inhibited binding of iRBCs to endothelial cells and reversed binding of cytoadhered iRBCs [57]. Additionally, chemical disruption of VESA1 export or trypsin treatment to remove surface proteins resulted in inhibition of binding, which was regained when VESA1 was repopulated on the RBC [56]. Further transcriptomics analysis supported a VESA1 role in pathogenesis [58]. VESA1 has a single transmembrane domain, a large extracellular domain, and a short cytoplasmic tail [31,50], a structure that superficially resembles the P. falciparum RBC surface adhesin, erythrocyte membrane protein 1 (PfEMP1). The extracellular region of VESA1 proteins possess a cysteine- and lysine-rich domain and variant domain conserved sequences [31,59]. While the role of PfEMP1, the specific binding domains, and the host receptors are well documented for cerebral and pregnancy-associated malaria [60–62], the role of binding domains, receptors, and the impact of VESA1 for sequestration remain to be determined for Babesia. Two additional exported proteins were shown to be involved in cytoadhesion of B. bovis iRBC, SBP2t11, and VESA1 export-associated protein, BbVEAP ([33,63]; Fig 2). Up-regulation of SBP2t11 was associated with low virulence of B. bovis, and its overexpression reduced binding of iRBCs to endothelial cells [63,64]. While the cleavage at PLM and the export of SBP2t11 has been demonstrated [63], it is likely involved in cytoadhesion indirectly, as it is not expressed on the surface of iRBC. It remains unclear whether SBP2t11 affects VESA1 export, ridge formation, or other factors responsible for cytoadhesion. Knockdown of BbVEAP using the glmS ribozyme system disrupted the export of VESA1, decreased ridge numbers, and abrogated cytoadhesion of iRBCs [33]. BbVEAP may function as a chaperone for the export of VESA1 as an integral protein and ridge-forming proteins; however, immunoprecipitation of BbVEAP did not confirm a direct interaction with VESA1 [33]. Given that BbVEAP knockdown did not affect SBP4 export, BbVEAP expression is necessary for the export and correct localization of a subset of proteins including VESA1 and ridge-forming proteins. Additionally, it was shown that BbVEAP is indispensable for parasite development in the RBC, making it the only known essential exportome protein [33,65]. The existence of VEAP among piroplasma parasites indicates a piroplasma-specific conserved function. The essentiality of VEAP could be due to its role in the export of other essential proteins such as channels or transporters, a hypothesis that needs future confirmation. Although ves1α and ves1β genes are unique for B. bovis, ves-like genes are found in homologous genomic regions in all Babesia species for which genome sequence is available (Fig 4A). Ves1a and ves1b genes are found in B. bigemina, B. divergens, and B. ovata and encode proteins that contain a single transmembrane domain and a short cytoplasmic region at C-terminus similar to the products of ves1α and ves1β genes of B. bovis [66,67]. A shorter ves gene group called ves2 encode proteins lacking the C-terminal transmembrane domain and the cytoplasmic region and were found based on their homology to the 5′ end of ves1 in B. bigemina, B. divergens, and B. ovata [66,67]. The expanded ves2 genes of B. ovata cluster together with B. bigemina ves2 based upon high sequence identity (Fig 4A). Given the fact that ves2 exists in the homologous positions of smorf, it was suggested that ves2 is analogous to smorf in these parasites [66]; however, the function of both gene families is unknown. VESA1 from B. bovis was experimentally confirmed to be expressed on the iRBCs surface [28], and VESA1 from B. orientalis was shown to be exported to iRBC [68]. While in B. bovis VESA1 is responsible for cytoadhesion and antigenic variation [69], cytoadhesion has not been documented for other Babesia spp., despite the presence of expanded ves gene families in their genomes. It is likely that VESA from other Babesia species are also surface proteins responsible for antigenic variation [66]. Future experiments are needed to characterize the functions of VESA in Babesia spp. in the mammalian host and tick vector, as the expression of some ves1 genes were up-regulated in the kinetes, the invasive stage of parasites in the tick hemolymph, of B. bovis [51]. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Homology clustering based on sequence similarities of ves and multi-transmembrane protein encoding genes. (A) ves and smorf genes sequence were extracted from piroplasmaDB (https://piroplasmadb.org/piro/app), and a sequence similarity network was visualized by Gephi. (B) The figure was reproduced from Hakimi and colleagues [33]. The genes encoding proteins with more than 8 TM domains were extracted from piroplasmaDB and clustered. SmORF, Small Open Reading Frame; TM, transmembrane. https://doi.org/10.1371/journal.ppat.1010770.g004

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010770

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