Next Article in Journal
A Regulatory SRNA Rli43 Is Involved in the Modulation of Biofilm Formation and Virulence in Listeria monocytogenes
Next Article in Special Issue
Detection of Theileria orientalis Genotypes from Cattle in Kyrgyzstan
Previous Article in Journal
The Enterotoxin Production and Antimicrobial Resistance of Campylobacter Strains Originating from Slaughter Animals
Previous Article in Special Issue
Complete Genomes of Theileria orientalis Chitose and Buffeli Genotypes Reveal within Species Translocations and Differences in ABC Transporter Content
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Pathogens
Volume 11
Issue 10
10.3390/pathogens11101135
pathogens-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Characterisation of the Theileria orientalis Piroplasm Proteome across Three Common Genotypes

by
Cheryl Jenkins
1,*,
Melinda L. Micallef
1,
Matthew P. Padula
2 and
Daniel R. Bogema
1
1
NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, NSW 2568, Australia
2
School of Life Sciences, Faculty of Sciences, University of Technology, Sydney, NSW 2007, Australia
*
Author to whom correspondence should be addressed.
Pathogens 2022, 11(10), 1135; https://doi.org/10.3390/pathogens11101135
Submission received: 24 August 2022 / Revised: 27 September 2022 / Accepted: 27 September 2022 / Published: 30 September 2022
(This article belongs to the Special Issue Bovine Theileriosis Caused by the Theileria orientalis Group)
Browse Figures
Versions Notes

Abstract

:
Theileria orientalis is an emerging apicomplexan pathogen of cattle occurring in areas populated by the principal vector tick, Haemaphysalis longicornis. Unlike transforming Theileria spp. that induce cancer-like proliferation of lymphocytes via their schizont stage, T. orientalis destroys host erythrocytes during its piroplasm phase resulting in anaemia. The underlying pathogenic processes of T. orientalis infection are poorly understood; consequently, there are no vaccines for prevention of T. orientalis infection and chemotherapeutic options are limited. To identify antigens expressed during the piroplasm phase of T. orientalis, including those which may be useful targets for future therapeutic development, we examined the proteome across three common genotypes of the parasite (Ikeda, Chitose and Buffeli) using preparations of piroplasms purified from bovine blood. A combination of Triton X-114 extraction, one-dimensional electrophoresis and LC-MS/MS identified a total of 1113 proteins across all genotypes, with less than 3% of these representing host-derived proteins. Just over three quarters of T. orientalis proteins (78%) identified were from the aqueous phase of the TX-114 extraction representing cytosolic proteins, with the remaining 22% from the detergent phase, representing membrane-associated proteins. All enzymes involved in glycolysis were expressed, suggesting that this is the major metabolic pathway used during the T. orientalis piroplasm phase. Proteins involved in binding and breakdown of haemoglobin were also identified, suggesting that T. orientalis uses haemoglobin as a source of amino acids. A number of proteins involved in host cell interaction were also identified which may be suitable targets for the development of chemotherapeutics or vaccines.

1. Introduction

The haemoprotozoan Theileria orientalis is an apicomplexan parasite with life stages that cycle between ticks and cattle. Cattle infected with high burdens of the Ikeda genotype often become anaemic with clinical signs including tachypnoea, lethargy, ataxia, abortion in pregnant animals and mortality in up to 10% of cases [1,2]. Stressors such as parturition, lactation and transport of infected animals exacerbate disease [1,2]. The geographical range of T. orientalis Ikeda has expanded significantly in the past two decades facilitated by the widespread occurrence of its principal tick vector, Haemaphysalis longicornis. Clinical disease outbreaks caused by T. orientalis Ikeda (formerly referred to as Theileria sergenti) have been reported in Japan and Korea for many years [3,4]; however recent incursions of T. orientalis Ikeda have been reported in the Asia-Pacific region [5,6,7,8] and the eastern United States [9].
Measures for control of this parasite are limited. In Australia and New Zealand, the parasite is now endemic across the range of the vector tick with widespread immunity observed in adult cattle [10,11,12]. However, mixing of naïve and endemic animals remains a major risk for clinical outbreaks of disease and passive immunity confers little protection on young calves, with high parasitaemias routinely observed between 3 and 12 weeks of age [13]. Control of vector ticks via acaricides is recommended as treatments and chemotherapeutics for T. orientalis infection are currently limited or unavailable. Buparvaquone, which is used for the treatment of Theileria parva infection (East Coast fever) in Africa, is also effective against T. orientalis if used during the earlier stages of infection; however, it is not approved for use in some countries and has a lengthy withholding period due to the retention of residues in meat and milk products [14]. Blood transfusions are sometimes used to treat anaemia due to T. orientalis infection; however, this process is costly and is not always successful [11].
While the “transforming” theilerias (such as Theileria parva and Theileria annulata) induce uncontrolled proliferation of B and T lymphocytes and/or macrophages via their schizont phase, it is the intraerythrocytic piroplasm phase of T. orientalis that is responsible for the observed pathology. In this respect, T. orientalis is more akin to Babesia spp. and haemosporidians such as Plasmodium spp. which cause destruction or sequestration of erythrocytes [15]. Nonetheless, the processes by which pathogenesis occur in this organism are poorly understood, hampering the development of chemotherapeutics or prophylactic measures such as vaccines. In this study, we conducted a proteomic analysis of T. orientalis piroplasms to identify expressed proteins that may form the basis of future treatment modalities.

2. Materials and Methods

2.1. Collection and Propagation of T. orientalis Strains

T. orientalis strains were sourced from Australian cattle testing PCR positive for a single major piroplasm surface protein (MPSP) genotype (Ikeda, Chitose or Buffeli) [5,16,17] and have been described in detail previously [18]. The strains are named according to their location of isolation: the Robertson strain was isolated from New South Wales is of the Ikeda genotype; the Fish Creek and Goon Nure strains were isolated from Victoria and are of the Chitose and Buffeli genotypes respectively. The strains were propagated in splenectomised calves inoculated with stabilates of each isolate at the Tick Fever Centre (Wacol, QLD, Australia) as previously described [18]. Details of blood testing and collection and the purification of parasites using the nitrogen cavitation method followed by differential centrifugation are also as previously described [18,19]. Purified piroplasm preparations were maintained at −80 °C until required for proteomic analysis.

2.2. Proteomics

2.2.1. Protein Purification

Proteins were purified from T. orientalis Ikeda, Chitose and Buffeli piroplasms using an in-house Triton X-114 method to separate aqueous and detergent soluble proteins. Approximately 0.1 g of piroplasms were thawed on ice and resuspended in 1 mL cold 1% triton buffer (1% TX-114, 10 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA). Samples were extracted overnight on a rotary shaker at 4 °C. Samples were centrifuged cold at 13,000 rpm for 15 min after which the supernatant was placed in a new tube and incubated for 10 min at 37 °C. The pellet represented TX-114 insoluble pellet. The supernatant was phase separated by centrifuging for 5 min at 10,000 rpm. The top phase (representing the aqueous phase) was pipetted into a new tube and 20 μL TX-114 was added. A 1 mL aliquot of 1% Triton buffer was added to the detergent phase and the samples were re-extracted on a rotary shaker as described above, but for 4 h. The phases were separated again with a 10 min incubation at 37 °C followed by centrifugation at 10,000 rpm for 5 min. The top and bottom phases (aqueous and detergent respectively) were separated into 2 × 50 mL centrifuge tubes and each sample was mixed with 10 volumes of ice-cold acetone to precipitate proteins. After an overnight incubation at −20 °C, precipitated proteins were pelleted by spinning at 13,000 rpm and 4 °C for 30 min. The acetone was decanted off each sample and the pellets air dried for a period of 30 min. The pellet from the aqueous phase was resuspended in 0.5 mL of SSS buffer (8 M Urea, 100 mM DTT, 4% chaps, 0.8% ampholytes, 40 mM Tris) and the pellet from the detergent phase was resuspended in 0.25 mL MSS buffer (5 M urea, 65 mM DTT, 2% chaps, 0.8% ampholytes, 40 mM tris, 2 M thiourea, 2% sulfobetaine). After 30 min incubation at RT with alternate cycles (×4) of sonication (30 s) and vortexing (15 s), samples were centrifuged at 13,000 rpm for 15 min. Supernatants were stored at −20 °C in fresh tubes.
The aqueous and detergent-phase proteins were passed through a 300 kDa filter (Pall Life Sciences, Port Washington, NY, USA) and were then desalted using Micro BioSpin P6 gel columns (BioRad Laboratories, Hercules, CA, USA). Aliquots of each fraction were analysed by electrophoresis on Criterion 12% TGX precast gels (BioRad Laboratories) at 200 V for 1 h. For visualisation of protein bands, gels were washed 3 × 10 min with MilliQ water and then stained with GelCode Blue stain reagent (ThermoFisher Scientific, Waltham, MA, USA). Destaining was achieved with MilliQ water.

2.2.2. Trypsin In-Gel Digestion “Slice and Dice” Protocol

Gel slices were taken from each of the stained gels with a scalpel blade, were diced into 1 mm2 cubes and kept moist in individual 0.6 mL microfuge tubes with deionised water. Excess water was removed, and the gel pieces were equilibrated with bicarbonate solution (100 mM NH4HCO3). A 200 µL volume of 50% acetonitrile-50 mM NH4HCO3 was added to each tube to destain the gel; samples were vortexed and incubated for 10 min. To dehydrate the samples, excess liquid was removed, 200 µL of 100% acetonitrile was added, and samples were vortexed and incubated at room temperature for 10 min.
To reduce and alkylate the proteins, the gel pieces were rehydrated with 50 µL of 5 mM tributylphosphine/20 mM acrylamide in NH4HCO3 and incubated for 90 min. Gel pieces were then washed with 100 mM NH4HCO3 for 5 min, followed by 50% acetonitrile-50 mM NH4HCO3 for 5 min and finally were dehydrated with 200 µL of acetonitrile. Following dehydration, any remaining liquid was removed and to generate peptides, the gel pieces were rehydrated with 12.5 ng/µL of trypsin in 100 mM NH4HCO3 at 4 °C for 30 min. Following re-swelling of the gel, NH4HCO3 was added as required to keep the gel covered with liquid. Protein digestion was achieved overnight at 37 °C.

2.2.3. Mass Spectrometry

Following trypsin digestion, the samples were sonicated in a waterbath for 10 min to extract the peptides and centrifuged to release the supernatant. The resulting digest solution was transferred to a clean tube. A 50% acetonitrile-2% formic acid solution (30 µL) was added to the gel pieces with sonication and centrifugation repeated as above. The supernatant was then added to the previous supernatant sample. This step was repeated again yielding a peptide extract volume of >60 µL. Concentration of the samples to a volume of 15 µL was achieved using a SpeedVac vacuum concentrator. Prior to analysis, microparticulates were removed by centrifuging the samples at 14,000 rpm for 10 min. The samples were then loaded into an autosampler tube and into a QSTAR Elite Liquid chromatography-tandem mass spectrometry (LC-MS/MS) system for analysis [20]. MS/MS MASCOT searches were performed against a composite database consisting of the Bos taurus proteome and the known sequences of apicomplexan parasite proteins to overcome any omissions in the available T. orientalis protein sequences. Scores were assigned to protein matches and peptide matches with an E-value of <0.05 were considered statistically significant and the peptide identification deemed valid. The relative abundance of proteins was calculated using the exponentially modified protein abundance index (emPAI) [21] within MASCOT. Duplicate matches were removed from the final numbers.

3. Results and Discussion

3.1. Electrophoretic Analysis of Purified T. orientalis Proteins

Aqueous and detergent soluble T. orientalis proteins were electrophoresed on 1D SDS-PAGE gels (Criterion 12% TGX precast gels, BioRad). The aqueous and detergent fractions for each T. orientalis genotype are shown in Figure S1A,B. respectively.

3.2. LC MS/MS Identification of Purified T. orientalis Proteins

Eight, eleven and twelve individual gel slices were analysed from the aqueous phase of Chitose, Ikeda and Buffeli genotype piroplasms respectively. When examining the detergent phase, twelve slices for Chitose and eleven for both Ikeda and Buffeli were analysed (highlighted in Figure S1). All samples were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Spectra for individual gel slices were then converted into a peak list and Mascot MS/MS searches were performed. A total of 1113 proteins were identified across all T. orientalis genotypes. Of these proteins only 2.8% (n = 31) represented Bos taurus proteins and a further 1.9% (n = 21) represented other contaminating proteins, indicating efficient purification of T. orientalis piroplasms from bovine blood.
A total of 1061 target proteins (95.3%) were identified that matched either the reference T. orientalis Shintoku genome or related apicomplexan parasites and other protozoans. This represents approximately one quarter of the total predicted proteome of T. orientalis with 4058, 3980 and 3924 genes predicted in the Ikeda, Chitose and Buffeli genomes respectively [22,23]. Approximately three quarters (78%; n = 829) of the proteins identified were aqueous phase proteins, while 22% (n = 232) were from the detergent phase of the TX-114 extraction, representing membrane-associated and other hydrophobic proteins. This proportion of detergent phase proteins was similar to those recovered from Theileria annulata schizonts using TX114 [24]. The total number of proteins recovered from the T. orientalis Ikeda and Buffeli genotypes was greater than for the Chitose genotype (Table 1). Indeed, one limitation of this study was that the number of proteins identified per genotype was not sufficient to allow for meaningful cross-comparison of all proteins expressed between pathogenic and apathogenic types. The inability to culture T. orientalis in vitro under standard conditions, with experimental infection of animals used for propagation, may also have influenced the protein profile across genotypes.

3.3. Protein Functional Groups and Relative Abundance

Across all genotypes, approximately 40% of the proteins identified were yet uncharacterised proteins with unknown functions; however putative functions could be assigned to the remaining 60% of proteins. Where putative functions could be assigned, the most common functional designations were metabolism, translation, transcription, regulation and signalling, protein processing and turnover and cellular organisation and transport (Figure 1 and Table S1). As expected, proteins with cytosolic functions, such as those involved in translation or metabolism, were generally identified in the aqueous fractions; while membrane-associated proteins, such as transporters and major surface proteins were located in the detergent fractions. The detergent phases also contained a high proportion of uncharacterised proteins (Table S2).
The relative abundance of proteins as estimated via emPAI indicated similarities between the most abundant proteins across T. orientalis genotypes. In the aqueous phase fractions, proteins involved in translation, such as elongation factor Tu and elongation factor 1α and ribosomal proteins, proteins involved in homeostasis such as heat shock proteins and redox proteins, metabolic enzymes and cytoskeletal proteins such as actin and tubulin were amongst the most abundant. A number of proteins were also identified that have putative roles in host cell attachment and interaction. Proteins with emPAI scores >1.0 are shown in Table 2.
Proteins involved in redox homeostasis (thioredoxin, peroxiredoxin) were identified as highly expressed, which is unsurprising given the oxidative stresses imposed by the haem-rich intraerythrocytic environment. Heat shock proteins (HSPs) 70 and 90 were abundant in all genotypes of T. orientalis (Table 2). HSPs also play an important role in parasite homeostasis by maintaining the integrity of protein folding under thermal stress. This is thought to be particularly critical in apicomplexans that have lifecycle stages in both poikilothermic and homeothermic hosts, such as Theileria, Babesia and Plasmodium spp. In P. falciparum, HSPs are also involved in export of parasite proteins to the erythrocyte surface and regulation of pathogenesis, and consequently both HSP 70 and HSP 90 are major targets for antimalarial compounds [25]. Novobiocin has also been shown to inhibit Babesia caballi and Theileria equi in in vitro culture by targeting HSP 90 [26] suggesting that HSPs may also be suitable therapeutic targets for T. orientalis.
Table
Table 2. Most abundant (emPAI > 1.0) aqueous phase proteins by T. orientalis genotype.
Table 2. Most abundant (emPAI > 1.0) aqueous phase proteins by T. orientalis genotype.
GenotypeProtein NameAccessionemPAIFunctionReference
IkedaToLocg 1 protein Q75R3452.06Unknown[27]
Uncharacterised proteinJ4DPH810.83Unknown[23]
Uncharacterised proteinJ4CCC55.06Unknown[23]
Uncharacterised proteinJ7M4S64.93Unknown[23]
Uncharacterised proteinJ4C2Q43.36Unknown[23]
Uncharacterised proteinJ4C9B13.06Unknown[23]
Uncharacterised proteinJ4D7C01.88Unknown[23]
ActinS7VNC91.67Cellular organisation and transport[23]
Elongation factor 1 alphaJ4D5 × 21.46Translation[23]
Heat shock protein 70J4D7001.35Protein processing and turnover[25]
Microneme-rhoptry related proteinD0FY421.33Host cell interactions[28]
Heat shock protein 90J4CCT61.29Protein processing and turnover[26]
Peptidyl-prolyl cis-trans isomeraseJ7MC361.29Homeostasis[27]
Uncharacterised proteinJ4DNX91.24Unknown[23]
Nucleoside diphosphate kinase J4C3B51.11Metabolism—nucleotide synthesis[29]
Uncharacterised proteinJ4C3S51.05Unknown[23]
Deoxyuridine 5’-triphosphate nucleotidohydrolaseJ4D5D21.04Metabolism—nucleotide synthesis[30]
ThioredoxinJ7MGV81.02Redox homeostasis[31]
BuffeliElongation factor 1-alphaL1LF411.99Translation[22]
Heat shock protein 70J4D7001.81Protein processing and turnover[25]
Glyceraldehyde 3-phosphate dehydrogenaseJ4C4H61.59Metabolism—glycolysis[32]
Uncharacterised proteinJ4C2Q41.67Unknown[22]
Uncharacterised proteinJ4CCC51.46Unknown[22]
Heat shock protein 90J4CCT61.19Protein processing and turnover[26]
GMP synthaseJ4C7N61.09Metabolism—nucleotide synthesis[33]
Deoxyuridine 5’-triphosphate nucleotidohydrolaseJ4D5D21.04Metabolism—nucleotide synthesis[30]
PeroxiredoxinJ7MCG41.04Redox homeostasis[34]
ChitoseActinS7VNC98.54Cellular organisation and transport[22]
Beta tubulin1106449851.07Cell division[22]
Heat shock protein 90J4CCT61.04Protein processing and turnover[26]
Uncharacterised proteins were amongst the most abundant proteins in the aqueous phase fractions. Interestingly, in the pathogenic genotype, T. orientalis Ikeda, ToLocg 1 (Theileria orientalis low copy number gene 1) protein was the most abundant protein identified and was approximately 5× more abundant than the next most abundant protein. The function of this protein is unknown but homologs of ToLocg 1 are present in Theileria parva and Theileria uilenbergi, with the homlog in T. uilenbergi being used for ELISA development due to the fact that it is immunodominant in this species [27]. ToLocg 1 was also relatively abundant, although much less so (emPAI = 0.55) in the T. orientalis Buffeli aqueous phase. This protein was not identified in the Chitose genotype, and the gene encoding ToLocg 1 is absent from the Chitose genome [22]. Further investigation of the uncharacterised proteins revealed that the top 7 most abundant aqueous phase proteins in T. orientalis Ikeda (including ToLocg 1), belong to the same orthogroup as defined by Orthofinder [22]. Further searches with the EggNOG database v5.0.0 reveal that these seven proteins plus one additional (J4DNX9) match to two EggNOG orthologous groups (ENOG503KE5M and ENOG503KDN3; Figure S3). Both EggNOG groups are specific to Theileria spp., and higher in copy number compared to T. parva and T. annulata. All eight proteins have predicted signal peptides or transmembrane domains (Table S3). Only two of these proteins (J4CCC5 and J4C2Q4) were found to be expressed in Buffeli piroplasms and none were expressed in the Chitose genotype. Furthermore, the T. orientalis Chitose genome only contains genes for J4CCC5 and J4C2Q4, and a truncated gene for J4C9B1 (truncated by ~45%) but completely lacks the remaining five homologs. The difference in the presence and expression of these genes between the pathogenic and apathogenic genotypes is striking and the respective functions of members of this ortholog group is worthy of further investigation.
The most abundant proteins in the detergent phases of each T. orientalis genotype were proteins involved in interactions with host cells (Table 3). The major piroplasm surface protein (MPSP) and another well-defined piroplasm membrane protein, P23, dominated the detergent fraction of all three genotypes. These proteins are known to be immunodominant in piroplasm phase of T. orientalis [35,36] but are also expressed in other lifecycle stages [37]. In T. orientalis Ikeda, a variant of the protein tocp1, a cysteine protease homolog, was also identified, as was a sodium/glutamate symporter which is believed to be involved in fuelling the TCA cycle (see Section 3.4.1). ADP ribosylation factor (Arf) was also relatively abundant in T. orientalis Ikeda. Arf proteins are GTPases important in intracellular signalling events and in T. gondii mediate the release of effector molecules from dense granules [38]. Theileria spp. contain microspheres, equivalent to the dense granules of T. gondii, during the sporozoite phase but not the schizont phase [39]. It is unclear whether these are present during the piroplasm phase [40].

3.4. Metabolic Pathways

3.4.1. Glycolysis and the Citric Acid (TCA) Cycle

Across all genotypes of T. orientalis, all enzymes involved in glycolysis were identified (Figure 2). Based on the presence of the substrate determining “GGDG” motif (Supplementary Figure S2), T. orientalis appears to use ATP-dependent phosphofructokinase (ATP-PFK) for the committed step of glycolysis which converts fructose-6-phosphate to fructose-1,6-bisphosphate, while in several other apicomplexans, pyrophosphate-dependent PFK (PPi-PFK) performs this function [32,46]. One key difference between these enzymes is that PPi-PFK can also catalyse this reaction in the reverse (gluconeogenic) direction. All Theileria spp. sequenced to date [24], along with some other apicomplexans such as Plasmodium spp. [47] lack key enzymes involved in gluconeogenesis, instead relying on the host for certain sugars. T. orientalis also appears to lack these enzymes including fructose bisphosphatase which catalyses the reverse reaction to ATP-dependent PFK, glucose-6-phosphatase, pyruvate carboxylase and phosphoenolpyruvate carboxylase, none of which were detected in this study and genes encoding these enzymes are also absent in the T. orientalis genome (Figure 2). Interestingly, enzymes involved in glycerol metabolism (glycerol kinase and glycerol-3-phosphate dehydrogenase) were also expressed in T. orientalis and were also relatively abundant despite being undetected in the T. parva sporozoite proteome [32] and detected at only very low levels in the T. annulata schizont proteome [24]. Glycerol may enter the glycolytic pathway or may be involved in glycerolipid synthesis. Indeed, diacylglycerol kinase (DGK) was also relatively abundant in T. orientalis Ikeda. DGK is involved in the production of phosphatidic acid, an important molecule in exocytosis.
Glycolysis is the major metabolic pathway used by the erythrocyte stages of a number of Plasmodium spp. [48] and this may also be true for T. orientalis. While most of the enzymes involved in the TCA cycle were identified as expressed in this study (Figure 2), their relative abundance was generally lower than seen for the glycolytic enzymes (Table S2). The lack of a mitochondrial pyruvate dehydrogenase complex in a number of apicomplexans [49] including Theileria spp. suggests that these pathways are not linked via the classical route involving acetyl CoA [44]. It has been suggested that glutamate may supplement the TCA cycle in T. parva [44] and T. annulata [24] and this may also be the case in T. orientalis piroplasms (Figure 2), supported by the relative abundance of a membrane-associated sodium/glutamate transporter in T. orientalis Ikeda (Table 3).

3.4.2. Purine Metabolism

Apicomplexans, including Theileria spp., lack the ability to synthesise purine rings de novo and these are instead salvaged from the host. Because Theileria spp. do not reside intracellularly within a parasitophorous vacuole (this is degraded rapidly following cell entry [50]), such metabolites are readily available [51]. Based on genome sequences, Theileria spp. contain a limited number of enzymes that are involved in interconversion of purines and some of these were identified as expressed in the piroplasm phase of T. orientalis.
Adenylosuccinate lyase and adenylosuccinate synthetase, involved in the conversion of inosine monophosphate (IMP) to adenosine monophosphate (AMP), were both identified in the Ikeda genotype, as were IMP dehydrogenase and guanosine monophosphate (GMP) synthase, which are involved in the conversion of IMP to GMP.

3.4.3. Amino Acid Metabolism

Apicomplexans also rely on the host for uptake of amino acids and a range of integral membrane proteins known as the Apicomplexan Amino acid Transporters (ApiATs) have been identified in Toxoplasma and Plasmodium spp. that are integral to the plasma membrane [51]. Based on amino acid homology there are 9 ApiATs in T. orientalis genome (J4CDF9, J4DPP7, J4C3V0, J4C3H6, J4D7Y9, J4DPC3, J4DNH3, J4D773, J4D8Y0) each with between 10 and 12 predicted transmembrane domains. Interestingly, none of these genes were detected in any T. orientalis genotypes in this study. In Toxoplasma gondii, expression of many of the ApiATs during the tachyzoite phase was limited or not detectable, suggesting that expression of these transporters might be specific to particular life cycle stages [52]. Ultrastructural studies show that Theileria spp. contain food vacuoles formed by endocytosis of host cell cytoplasm [53]. In the blood stage of Plasmodium spp., the food vacuole contains large quantities of haemoglobin which are broken down to provide the majority of amino acids for replication [54] and this may also be the case for the piroplasm phase of Theileria parasites. The Tocp1 protein is identified as relatively abundant in the T. orientalis Ikeda membrane fraction (Table 3), a cysteine protease-like protein with known haemoglobin-binding activity [45]. Furthermore, the papain family cysteine proteases (falcipains), falcilysin and aminopeptidases have all been implicated in the breakdown of haemoglobin in Plasmodium spp. [54,55,56,57,58], and all three types of proteases were found to be expressed in T. orientalis piroplasms in this study (Table 4). Falcilysin is being investigated as a potential drug target for Plasmodium as this metalloprotease is essential for viability of the blood stage of the parasite [59]; therefore, this may also be worth investigating as a chemotherapeutic target for Theileria spp.
T. orientalis also possesses a number of enzymes for the interconversion of amino acids including serine hydroxymethyltransferase (catalysing the interconversion of serine and glycine) and aspartate aminotransferase (catalyses conversion of aspartate to glutamate) and adenosylhomocysteinase (involved in methionine metabolism). A number of these enzymes have also been targeted for chemotherapeutic development in Plasmodium [61,62] and could be of utility in development of drugs for Theileria given the similar reliance of these parasites on a very limited repertoire of enzymes for amino acid metabolism.

3.5. Proteins Involved in Host Cell Interactions

A number of proteins were identified that are known to be involved in host cell adherence or other interactions, either in Theileria spp. or in other apicomplexans. These, along with other potential virulence factors, are listed in Table 5. Peptidyl prolyl cis/trans isomerases were detected in aqueous phases of all three T. orientalis genotypes. These proteins facilitate protein folding by isomerisation of proline residues. One enzyme from this family (PIN1) is known to be involved in induction of a cancer-like state in the transforming theilerias [63]. While the T. orientalis homolog of this protein lacks the signal peptide found in the transforming theilerias, buparvaquone interacts directly with the T. annulata PIN1, inhibiting the isomerase activity of this enzyme. Buparvaquone is also effective against Theileria orientalis infections, but it is unclear whether this is due to activity against the PIN1 orthologue as this drug also targets cytochrome b [64].
A homolog of Plasmodium hemolysin III was identified in the detergent phase of the Ikeda genotype. In P. falciparum, hemolysin III is a pore-forming protein expressed during the blood phase of the parasite. This protein is capable of erythrocyte lysis which may contribute to malaria-associated anaemia [65]. As anaemia is the major pathogenic effect of T. orientalis infection in cattle, further functional characterisation of this protein in Theileria spp. may be warranted.
A membrane attack complex (MAC)/perforin domain protein was identified in the Chitose genotype of T. orientalis. Proteins containing these domains are widespread in both prokaryotes and eukaryotes and they are responsible for forming pores in membranes of target cells. In apicomplexans, these proteins play important roles in the progression of the lifecycle, with species reliant on arthropod transmission (such as Plasmodium, Babesia and Theileria spp.) having a larger number of MAC/perforin domain proteins due to the need to traverse a wide variety of vertebrate and invertebrate host cell types [66]. Seven MAC/perforin domain proteins have been identified in T. annulata, with the T. orientalis protein most closely related to TA11680; however, there is little information about the cells targeted by these proteins. Expression of the T. orientalis MAC/perforin protein in the piroplasm phase of the lifecycle suggests that this homolog is most likely involved in traversing erythrocyte membranes.
Glycosylphosphatidylinositol (GPI) anchors are the most common carbohydrate modification in apicomplexan parasites and are critical for interactions with host cells. Many GPI anchored surface proteins have been identified in Toxoplasma, Plasmodium and Cryptosporidium and in the former two organisms the GPI biosynthetic pathway is essential for parasite survival [67]. GPI-anchored proteins also tend to be immunodominant [67,70]. For these reasons, GPI-anchored proteins are common targets for development of apicomplexan vaccines and chemotherapeutics [71,72,73]. A phosphatidylinositol glycan Q (PIG Q) homolog was identified in the detergent phase of the T. orientalis Ikeda genotype. PIG Q initiates the GPI biosynthetic pathway, suggesting that the GPI anchors are important in the erythrocyte phase of the T. orientalis lifecycle. Identification of GPI anchored proteins may assist in developing therapeutics for this species.
One such protein that is predicted to be GPI-anchored in T. parva is the 104 kDa microneme-rhoptry protein. Most apicomplexans produce micronemes, secretory organelles located at the apical end of sporozoites (and merozoites) that release proteins responsible for gliding motility and mediating cell entry [74]. However, Theileria sporozoites and merozoites are round, lacking an apical structure, are non-motile and lack discernible micronemes; although rhoptries (club-shaped secretory organelles usually associated with micronemes) are produced. P104 is the only known protein of the apical microneme-rhoptry complex found in Theileria spp. and is found on the sporozoite [75] and the schizont surface [76], where it is believed to interact with host cell microtubules. Sporozoite neutralising antisera react with p104 indicating that it might be a suitable vaccine candidate [73]. Here, we confirmed a prior study demonstrating that a homolog of p104 in T. orientalis (denoted ToMRP) [28] is also found in the red blood cell phase of the parasite, although interestingly this protein was sequestered in the aqueous phase rather than the detergent phase of the TX114 extraction, contrary to findings from proteomic analysis of the T. annulata schizont [24]. Prior studies of this protein indicate that it is expressed in the early and late erythrocytic stages and may be involved in invasion or egress from bovine red blood cells [28].
The MPSP and another piroplasm surface protein, P23, were amongst the most abundant detergent phase proteins identified in this study across all genotypes. These proteins are immunodominant [35,36] and the MPSP in particular has been the target for subunit vaccine development [41,42]. Furthermore, the MPSP is homologous to the Theileria annulata merozoite surface protein (Tams-1). Tams-1 is also highly expressed and immunodominant during the T. annulata merozoite phase and is considered a promising target for vaccine development [68]. Functional studies on these proteins are lacking but the T. orientalis MPSP has been implicated in binding to both heparin and bovine erythrocytes [43], suggesting that this protein may be involved in gaining erythrocyte entry. P23 also has a homolog in T. annulata, although the function has not yet been characterised. However, in T. orientalis, like the MPSP, P23 has been identified as a heparin-binding protein [35]. Indeed, as for P. falciparum, heparin has been shown to inhibit T. orientalis invasion of erythrocytes [77] and heparin analogues are considered promising chemotherapeutics for blood stage apicomplexan infections [78].

3.6. Future Directions

One of the limitations of this study was the relatively low number of proteins identified from each individual T. orientalis genotype, which precluded meaningful comparison of the protein expression pattern across pathogenic and non-pathogenic strains. A more in-depth proteomic characterisation between genotypes is warranted as this may improve understanding of the drivers of pathogenicity in the Ikeda vs. apathogenic types. Furthermore, characterisation of the expressed T. orientalis proteome during lifecycle phases occurring in the tick host, such as the sporozoite phase, would be useful direction for future research to elucidate differences in the biological processes occurring in the arthropod versus mammalian host.

4. Conclusions

This study provides the first global proteomic study of T. orientalis and the expressed proteins identified in the piroplasm stage of the lifecycle represent approximately one quarter of the total predicted proteome. While the number of expressed proteins identified did not allow for meaningful cross comparison of expression patterns between T. orientalis genotypes, this study provides clues to the metabolic and pathogenic processes used by the parasite during the intraerythrocytic lifecycle stage and thereby provides a basis for the development of chemotherapeutics or vaccines against key expressed proteins.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pathogens11101135/s1. Figure S1: aqueous and detergent phase protein fractions from TX-114 extraction of T. orientalis. Figure S2: Alignment of phosphofructokinase sequences from representative apicomplexans. Figure S3: Alignment of uncharacterised aqueous phase proteins found to be highly abundant in the T. orientalis Ikeda. Table S1: Number of T. orientalis proteins identified by functional group. Table S2: All proteins identified from the aqueous and detergent phases of T. orientalis Ikeda, Chitose and Buffeli, and their relative abundances. Table S3: Highly abundant uncharacterised protein orthologs identified in the T. orientalis Ikeda aqueous phase.

Author Contributions

Conceptualisation, C.J.; methodology, C.J., D.R.B., M.P.P. and M.L.M.; formal analysis, M.P.P., D.R.B. and C.J.; investigation, M.L.M., M.P.P., D.R.B. and C.J.; resources, M.P.P. and C.J.; data curation, M.P.P., C.J. and D.R.B.; writing—original draft preparation, C.J; writing—review and editing, C.J., M.L.M., M.P.P. and D.R.B.; supervision, C.J.; project administration, C.J.; funding acquisition, C.J. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by a Meat and Livestock Australia (Project B.AHE.0213) grant to CJ and by the McGarvie Smith Trust.

Institutional Review Board Statement

This research was carried out in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes at the Tick Fever Centre, Wacol, QLD, Australia and was approved by the Department of Agriculture and Fisheries Animal Ethics Committee (Approval SA 2013/09/443).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article or Supplementary Materials. The data presented in this study are available in Table S2.

Acknowledgments

We appreciate the assistance of Phillip Carter and Susan Robinson (Tick Fever Centre, Wacol, QLD, Australia) who assisted with the original piroplasm purifications that formed the basis of this study.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Izzo, M.M.; Poe, I.; Horadagoda, N.; De Vos, A.J.; House, J.K. Haemolytic anaemia in cattle in NSW associated with Theileria infections. Aust. Vet. J. 2010, 88, 45–51. [Google Scholar] [CrossRef] [PubMed]
  2. Eamens, G.J.; Gonsalves, J.R.; Jenkins, C.; Collins, D.; Bailey, G. Theileria orientalis MPSP types in Australian cattle herds associated with outbreaks of clinical disease and their association with clinical pathology findings. Vet. Parasitol. 2013, 191, 209–217. [Google Scholar] [CrossRef] [PubMed]
  3. Uilenberg, G.; Perié, N.M.; Spanjer, A.A.; Franssen, F.F. Theileria orientalis, a cosmopolitan blood parasite of cattle: Demonstration of the schizont stage. Res. Vet. Sci. 1985, 38, 352. [Google Scholar] [CrossRef]
  4. Kawamoto, S.; Takahashi, K.; Onuma, M.; Kubota, S.; Nejo, H.O.; Kurosawa, T.; Sonoda, M. Rebound phenomenon of parasitemia in splenectomized calves primarily infected with Theileria sergenti. J. Vet. Med. Sci. 1991, 53, 127–128. [Google Scholar] [CrossRef]
  5. Eamens, G.J.; Bailey, G.; Jenkins, C.; Gonsalves, J.R. Significance of Theileria orientalis types in individual affected beef herds in New South Wales based on clinical, smear and PCR findings. Vet. Parasitol. 2013, 196, 96–105. [Google Scholar] [CrossRef]
  6. Pulford, D.; McFadden, A.; Hamilton, J.; Donald, J. Investigation of the index case herd and identification of the genotypes of Theileria orientalis associated with outbreaks of bovine anaemia in New Zealand in 2012. New Zealand Vet. J. 2016, 64, 21–28. [Google Scholar] [CrossRef]
  7. Gebrekidan, H.; Nelson, L.; Smith, G.; Gasser, R.B.; Jabbar, A. An outbreak of oriental theileriosis in dairy cattle imported to Vietnam from Australia. Parasitology 2017, 144, 738–746. [Google Scholar] [CrossRef]
  8. Kakati, P.; Sarmah, P.C.; Ray, D.; Bhattacharjee, K.; Sharma, R.K.; Barkalita, L.M.; Sarma, D.K.; Baishya, B.C.; Borah, P.; Stanley, B. Emergence of oriental theileriosis in cattle and its transmission through Rhipicephalus (Boophilus) microplus in Assam, India. Vet. World 2015, 8, 1099–1104. [Google Scholar] [CrossRef]
  9. Oakes, V.J.; Yabsley, M.J.; Schwartz, D.; LeRoith, T.; Bissett, C.; Broaddus, C.; Schlater, J.L.; Todd, S.M.; Boes, K.M.; Brookhart, M.; et al. Theileria orientalis Ikeda Genotype in Cattle, Virginia, USA. Emerg. Infect. Dis. 2019, 25, 1653–1659. [Google Scholar] [CrossRef]
  10. Jenkins, C.; Bogema, D.R. Factors associated with seroconversion to the major piroplasm surface protein of the bovine haemoparasite Theileria orientalis. Parasites Vectors 2016, 9, 106. [Google Scholar] [CrossRef] [Green Version]
  11. Emery, D.L. Approaches to Integrated Parasite Management (IPM) for Theileria orientalis with an Emphasis on Immunity. Pathogens 2021, 10, 1153. [Google Scholar] [CrossRef]
  12. Lawrence, K.; Gedye, K.; McFadden, A.; Pulford, D.; Heath, A.; Pomroy, W. Review of the New Zealand Theileria orientalis Ikeda Type Epidemic and Epidemiological Research since 2012. Pathogens 2021, 10, 1346. [Google Scholar] [CrossRef]
  13. Swilks, E.; Fell, S.A.; Hammer, J.F.; Sales, N.; Krebs, G.L.; Jenkins, C. Transplacental transmission of Theileria orientalis occurs at a low rate in field-affected cattle: Infection in utero does not appear to be a major cause of abortion. Parasites Vectors 2017, 10, 227. [Google Scholar] [CrossRef]
  14. Bailey, G. Buparvaquone Tissue Residue Study; Meat & Livestock Australia: North Sydney, Australia, 2013. [Google Scholar]
  15. Mehlhorn, H. (Ed.) Theileriosis. In Encyclopedic Reference of Parasitology: Diseases, Treatment, Therapy; Springer Berlin Heidelberg: Berlin/Heidelberg, Germany, 2001; pp. 568–570. [Google Scholar]
  16. Jenkins, C.; Micallef, M.; Alex, S.M.; Collins, D.; Djordjevic, S.P.; Bogema, D.R. Temporal dynamics and subpopulation analysis of Theileria orientalis genotypes in cattle. Infect. Genet. Evol. 2015, 32, 199–207. [Google Scholar] [CrossRef]
  17. Bogema, D.R.; Deutscher, A.T.; Fell, S.; Collins, D.; Eamens, G.J.; Jenkins, C. Development and validation of a quantitative PCR assay using multiplexed hydrolysis probes for detection and quantification of Theileria orientalis isolates and differentiation of clinically relevant subtypes. J. Clin. Microbiol. 2015, 53, 941–950. [Google Scholar] [CrossRef]
  18. Bogema, D.R.; Micallef, M.L.; Liu, M.; Padula, M.P.; Djordjevic, S.P.; Darling, A.E.; Jenkins, C. Analysis of Theileria orientalis draft genome sequences reveals potential species-level divergence of the Ikeda, Chitose and Buffeli genotypes. BMC Genom. 2018, 19, 298. [Google Scholar] [CrossRef]
  19. Shimizu, S.; Suzuki, K.; Nakamura, K.; Kadota, K.; Fujisaki, K.; Ito, S.; Minami, T. Isolation of Theileria sergenti piroplasms from infected erythrocytes and development of an enzyme-linked immunosorbent assay for serodiagnosis of T sergenti infections. Res. Vet. Sci. 1988, 45, 206–212. [Google Scholar] [CrossRef]
  20. Tacchi, J.L.; Raymond, B.B.A.; Haynes, P.A.; Berry, I.J.; Widjaja, M.; Bogema, D.R.; Woolley, L.K.; Jenkins, C.; Minion, F.C.; Padula, M.P.; et al. Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae. Open Biol. 2016, 6, 150210. [Google Scholar] [CrossRef]
  21. Ishihama, Y.; Oda, Y.; Tabata, T.; Sato, T.; Nagasu, T.; Rappsilber, J.; Mann, M. Exponentially Modified Protein Abundance Index (emPAI) for Estimation of Absolute Protein Amount in Proteomics by the Number of Sequenced Peptides per Protein. Mol. Cell. Proteom. 2005, 4, 1265–1272. [Google Scholar] [CrossRef]
  22. Yam, J.; Bogema, D.R.; Micallef, M.L.; Djordjevic, S.P.; Jenkins, C. Complete Genomes of Theileria orientalis Chitose and Buffeli Genotypes Reveal within Species Translocations and Differences in ABC Transporter Content. Pathogens 2022, 11, 801. [Google Scholar] [CrossRef]
  23. Hayashida, K.; Hara, Y.; Abe, T.; Yamasaki, C.; Toyoda, A.; Kosuge, T.; Suzuki, Y.; Sato, Y.; Kawashima, S.; Katayama, T.; et al. Comparative Genome Analysis of Three Eukaryotic Parasites with Differing Abilities To Transform Leukocytes Reveals Key Mediators of Theileria-Induced Leukocyte Transformation. mBio 2012, 3, e00204–e00212. [Google Scholar] [CrossRef]
  24. Witschi, M.; Xia, D.; Sanderson, S.; Baumgartner, M.; Wastling, J.M.; Dobbelaere, D.A. Proteomic analysis of the Theileria annulata schizont. Int. J. Parasitol. 2013, 43, 173–180. [Google Scholar] [CrossRef]
  25. Shonhai, A. Plasmodial heat shock proteins: Targets for chemotherapy. FEMS Immunol. Med. Microbiol. 2010, 58, 61–74. [Google Scholar] [CrossRef]
  26. Suthar, A.; Maji, C.; Gopalkrishnan, A.; Raval, S.H.; Kumar, R.; Kumar, S. Anti-piroplasmic activity of novobiocin as heat shock protein 90 inhibitor against in vitro cultured Theileria equi and Babesia caballi parasites. Ticks Tick Borne Dis. 2021, 12, 101696. [Google Scholar] [CrossRef]
  27. Abdo, J.; Liu, Z.; Yin, H.; Kullmann, B.; Ahmed, J.S.; Seitzer, U. Identification of clone-9 antigenic protein of Theileria uilenbergi and evaluation of its application for serodiagnosis. Parasitol. Res. 2010, 107, 517–524. [Google Scholar] [CrossRef]
  28. Kim, J.-Y.; Yokoyama, N.; Kumar, S.; Inoue, N.; Inaba, M.; Fujisaki, K.; Sugimoto, C. Identification of a piroplasm protein of Theileria orientalis that binds to bovine erythrocyte band 3. Mol. Biochem. Parasitol. 2004, 137, 193–200. [Google Scholar] [CrossRef]
  29. Kandeel, M.; Kitade, Y. Substrate specificity and nucleotides binding properties of NM23H2/nucleoside diphosphate kinase homolog from Plasmodium falciparum. J. Bioenerg. Biomembr. 2010, 42, 361–369. [Google Scholar] [CrossRef]
  30. Nguyen, C.; Kasinathan, G.; Leal-Cortijo, I.; Musso-Buendia, A.; Kaiser, M.; Brun, R.; Ruiz-Pérez, L.M.; Johansson, N.G.; González-Pacanowska, D.; Gilbert, I.H. Deoxyuridine triphosphate nucleotidohydrolase as a potential antiparasitic drug target. J. Med. Chem. 2005, 48, 5942–5954. [Google Scholar] [CrossRef]
  31. Matthews, K.; Kalanon, M.; Chisholm, S.A.; Sturm, A.; Goodman, C.D.; Dixon, M.W.A.; Sanders, P.R.; Nebl, T.; Fraser, F.; Haase, S.; et al. The Plasmodium translocon of exported proteins (PTEX) component thioredoxin-2 is important for maintaining normal blood-stage growth. Mol. Microbiol. 2013, 89, 1167–1186. [Google Scholar] [CrossRef]
  32. Nyagwange, J.; Tijhaar, E.; Ternette, N.; Mobegi, F.; Tretina, K.; Silva, J.C.; Pelle, R.; Nene, V. Characterization of the Theileria parva sporozoite proteome. Int. J. Parasitol. 2018, 48, 265–273. [Google Scholar] [CrossRef]
  33. Chaudhary, K.; Darling, J.A.; Fohl, L.M.; Sullivan, W.J.; Donald, R.G.K.; Pfefferkorn, E.R.; Ullman, B.; Roos, D.S. Purine Salvage Pathways in the Apicomplexan Parasite Toxoplasma gondii. J. Biol. Chem. 2004, 279, 31221–31227. [Google Scholar] [CrossRef]
  34. Tanaka, M.; Sakurai, T.; Yokoyama, N.; Inoue, N.; Kawazu, S.-I. Cloning and characterization of peroxiredoxin in Babesia bovis. Parasitol. Res. 2009, 105, 1473. [Google Scholar] [CrossRef] [PubMed]
  35. Takemae, H.; Sugi, T.; Kobayashi, K.; Murakoshi, F.; Recuenco, F.C.; Ishiwa, A.; Inomata, A.; Horimoto, T.; Yokoyama, N.; Kato, K.; et al. Interaction between Theileria orientalis 23-kDa piroplasm membrane protein and heparin. Jpn. J. Vet. Res. 2014, 62, 17–24. [Google Scholar] [CrossRef] [PubMed]
  36. Kawazu, S.; Sugimoto, C.; Kamio, T.; Fujisaki, K. Analysis of the genes encoding immunodominant piroplasm surface proteins of Theileria sergenti and Theileria buffeli by nucleotide sequencing and polymerase chain reaction. Mol. Biochem. Parasitol. 1992, 56, 169–175. [Google Scholar] [CrossRef]
  37. Sako, Y.; Sugimoto, C.; Onuma, M. Expression of a Major Piroplasm Surface Protein of Theileria sergenti in Sporozoite Stage. J. Vet. Med. Sci. 1999, 61, 275–277. [Google Scholar] [CrossRef] [PubMed]
  38. Liendo, A.; Stedman, T.T.; Ngô, H.M.; Chaturvedi, S.; Hoppe, H.C.; Joiner, K.A. Toxoplasma gondii ADP-ribosylation Factor 1 Mediates Enhanced Release of Constitutively Secreted Dense Granule Proteins. J. Biol. Chem. 2001, 276, 18272–18281. [Google Scholar] [CrossRef] [PubMed]
  39. Woods, K.; Perry, C.; Brühlmann, F.; Olias, P. Theileria’s Strategies and Effector Mechanisms for Host Cell Transformation: From Invasion to Immortalization. Front. Cell Dev. Biol. 2021, 9, 662805. [Google Scholar] [CrossRef] [PubMed]
  40. Shaw, M.K.; Tilney, L.G. The entry of Theileria parva merozoites into bovine erythrocytes occurs by a process similar to sporozoite invasion of lymphocytes. Parasitology 1995, 111 Pt 4, 455–461. [Google Scholar] [CrossRef]
  41. Onuma, M.; Kubota, S.; Kakuda, T.; Sako, Y.; Asada, M.; Kabeya, H.; Sugimoto, C. Control of Theileria sergenti infection by vaccination. Trop. Anim. Health Prod. 1997, 29, 119s–123s. [Google Scholar] [CrossRef]
  42. Tanaka, M.; Ohgitani, T.; Okabe, T.; Kawamoto, S.; Takahashi, K.; Onuma, M.; Kawakami, Y.; Sasaki, N. Protective Effect against Intraerythrocytic Merozoites of Theileria-Sergenti Infection in Calves by Passive Transfer of Monoclonal-Antibody. Jpn. J. Vet. Sci. 1990, 52, 631–633. [Google Scholar] [CrossRef] [Green Version]
  43. Takemae, H.; Sugi, T.; Kobayashi, K.; Murakoshi, F.; Recuenco, F.C.; Ishiwa, A.; Inomata, A.; Horimoto, T.; Yokoyama, N.; Kato, K. Analyses of the binding between Theileria orientalis major piroplasm surface proteins and bovine red blood cells. Vet. Rec. 2014, 175, 149. [Google Scholar] [CrossRef]
  44. Gardner, M.J.; Bishop, R.; Shah, T.; de Villiers, E.P.; Carlton, J.M.; Hall, N.; Ren, Q.; Paulsen, I.T.; Pain, A.; Berriman, M.; et al. Genome Sequence of Theileria parva, a Bovine Pathogen That Transforms Lymphocytes. Science 2005, 309, 134–137. [Google Scholar] [CrossRef]
  45. He, W.; Ohashi, K.; Sugimoto, C.; Onuma, M. Theileria orientalis: Cloning a cDNA encoding a protein similar to thiol protease with haemoglobin-binding activity. Exp. Parasitol. 2005, 111, 143–153. [Google Scholar] [CrossRef]
  46. Yang, X.; Yin, X.; Liu, J.; Niu, Z.; Yang, J.; Shen, B. Essential role of pyrophosphate homeostasis mediated by the pyrophosphate-dependent phosphofructokinase in Toxoplasma gondii. PLOS Pathog. 2022, 18, e1010293. [Google Scholar] [CrossRef]
  47. Srivastava, A.; Philip, N.; Hughes, K.R.; Georgiou, K.; MacRae, J.I.; Barrett, M.P.; Creek, D.J.; McConville, M.J.; Waters, A.P. Stage-Specific Changes in Plasmodium Metabolism Required for Differentiation and Adaptation to Different Host and Vector Environments. PLoS Pathog. 2016, 12, e1006094. [Google Scholar] [CrossRef]
  48. Jacot, D.; Waller, R.F.; Soldati-Favre, D.; MacPherson, D.A.; MacRae, J.I. Apicomplexan Energy Metabolism: Carbon Source Promiscuity and the Quiescence Hyperbole. Trends Parasitol. 2016, 32, 56–70. [Google Scholar] [CrossRef]
  49. Ralph, S.A. Strange organelles–Plasmodium mitochondria lack a pyruvate dehydrogenase complex. Mol. Microbiol. 2005, 55, 1–4. [Google Scholar] [CrossRef]
  50. Shaw, M.K. Cell invasion by Theileria sporozoites. Trends Parasitol. 2003, 19, 2–6. [Google Scholar] [CrossRef]
  51. Piro, F.; Focaia, R.; Dou, Z.; Masci, S.; Smith, D.; Di Cristina, M. An Uninvited Seat at the Dinner Table: How Apicomplexan Parasites Scavenge Nutrients from the Host. Microorganisms 2021, 9, 2592. [Google Scholar] [CrossRef]
  52. Krishnan, A.; Soldati-Favre, D. Amino Acid Metabolism in Apicomplexan Parasites. Metabolites 2021, 11, 61. [Google Scholar] [CrossRef]
  53. Conrad, P.A.; Kelly, B.G.; Brown, C.G. Intraerythrocytic schizogony of Theileria annulata. Parasitology 1985, 91, 67–82. [Google Scholar] [CrossRef]
  54. Pishchany, G.; Skaar, E.P. Taste for Blood: Hemoglobin as a Nutrient Source for Pathogens. PLoS Pathog. 2012, 8, e1002535. [Google Scholar] [CrossRef]
  55. Murata, C.E.; Goldberg, D.E. Plasmodium falciparum falcilysin: A metalloprotease with dual specificity. J. Biol. Chem. 2003, 278, 38022–38028. [Google Scholar] [CrossRef]
  56. Klemba, M. On the location of the aminopeptidase N homolog PfA-M1 in Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 2009, 106, E55. [Google Scholar] [CrossRef]
  57. Na, B.K.; Shenai, B.R.; Sijwali, P.S.; Choe, Y.; Pandey, K.C.; Singh, A.; Craik, C.S.; Rosenthal, P.J. Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax. Biochem. J. 2004, 378, 529–538. [Google Scholar] [CrossRef]
  58. Rosenthal, P.J.; Sijwali, P.S.; Singh, A.; Shenai, B.R. Cysteine proteases of malaria parasites: Targets for chemotherapy. Curr. Pharm. Des. 2002, 8, 1659–1672. [Google Scholar] [CrossRef]
  59. Kahlon, G.; Lira, R.; Masvlov, N.; Pompa, E.; Brar, N.; Eagon, S.; Anderson, M.O.; Andaya, A.; Chance, J.P.; Fejzic, H.; et al. Structure guided development of potent piperazine-derived hydroxamic acid inhibitors targeting falcilysin. Bioorganic Med. Chem. Lett. 2021, 32, 127683. [Google Scholar] [CrossRef]
  60. Allary, M.; Schrevel, J.; Florent, I. Properties, stage-dependent expression and localization of Plasmodium falciparum M1 family zinc-aminopeptidase. Parasitology 2002, 125, 1–10. [Google Scholar] [CrossRef]
  61. Sopitthummakhun, K.; Thongpanchang, C.; Vilaivan, T.; Yuthavong, Y.; Chaiyen, P.; Leartsakulpanich, U. Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: Inhibition studies using improved methods for enzyme production and assay. Malar. J. 2012, 11, 194. [Google Scholar] [CrossRef]
  62. Wrenger, C.; Müller, I.B.; Schifferdecker, A.J.; Jain, R.; Jordanova, R.; Groves, M.R. Specific inhibition of the aspartate aminotransferase of Plasmodium falciparum. J. Mol. Biol. 2011, 405, 956–971. [Google Scholar] [CrossRef]
  63. Marsolier, J.; Perichon, M.; DeBarry, J.D.; Villoutreix, B.O.; Chluba, J.; Lopez, T.; Garrido, C.; Zhou, X.Z.; Lu, K.P.; Fritsch, L.; et al. Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation. Nature 2015, 520, 378–382. [Google Scholar] [CrossRef]
  64. Mhadhbi, M.; Chaouch, M.; Ajroud, K.; Darghouth, M.A.; BenAbderrazak, S. Sequence Polymorphism of Cytochrome b Gene in Theileria annulata Tunisian Isolates and Its Association with Buparvaquone Treatment Failure. PLoS ONE 2015, 10, e0129678. [Google Scholar] [CrossRef]
  65. Moonah, S.; Sanders, N.G.; Persichetti, J.K.; Sullivan, D.J. Erythrocyte Lysis and Xenopus laevis Oocyte Rupture by Recombinant Plasmodium falciparum Hemolysin III. Eukaryot. Cell 2014, 13, 1337–1345. [Google Scholar] [CrossRef]
  66. Sassmannshausen, J.; Pradel, G.; Bennink, S. Perforin-Like Proteins of Apicomplexan Parasites. Front. Cell Infect. Microbiol. 2020, 10, 578883. [Google Scholar] [CrossRef]
  67. Debierre-Grockiego, F.; Schwarz, R.T. Immunological reactions in response to apicomplexan glycosylphosphatidylinositols. Glycobiology 2010, 20, 801–811. [Google Scholar] [CrossRef]
  68. Boulter, N.R.; Brown, C.G.; Kirvar, E.; Glass, E.; Campbell, J.; Morzaria, S.; Nene, V.; Musoke, A.; D’Oliveira, C.; Gubbels, M.J.; et al. Different vaccine strategies used to protect against Theileria annulata. Ann. N. Y. Acad. Sci. 1998, 849, 234–246. [Google Scholar] [CrossRef]
  69. Pain, A.; Renauld, H.; Berriman, M.; Murphy, L.; Yeats, C.A.; Weir, W.; Kerhornou, A.; Aslett, M.; Bishop, R.; Bouchier, C.; et al. Genome of the host-cell transforming parasite Theileria annulata compared with T. parva. Science 2005, 309, 131–133. [Google Scholar] [CrossRef]
  70. Abbasnia, T.; Asoodeh, A.; Habibi, G.; Haghparast, A. Isolation and purification of glycosylphosphatidylinositols (GPIs) in the schizont stage of Theileria annulata and determination of antibody response to GPI anchors in vaccinated and infected animals. Parasites Vectors 2018, 11, 82. [Google Scholar] [CrossRef]
  71. Fenollar, À.; Ros-Lucas, A.; Pía Alberione, M.; Martínez-Peinado, N.; Ramírez, M.; Ángel Rosales-Motos, M.; Lee, L.Y.; Alonso-Padilla, J.; Izquierdo, L. Compounds targeting GPI biosynthesis or N-glycosylation are active against Plasmodium falciparum. Comput. Struct. Biotechnol. J. 2022, 20, 850–863. [Google Scholar] [CrossRef]
  72. Nathaly Wieser, S.; Schnittger, L.; Florin-Christensen, M.; Delbecq, S.; Schetters, T. Vaccination against babesiosis using recombinant GPI-anchored proteins. Int. J. Parasitol. 2019, 49, 175–181. [Google Scholar] [CrossRef]
  73. Nyagwange, J.; Nene, V.; Mwalimu, S.; Henson, S.; Steinaa, L.; Nzau, B.; Tijhaar, E.; Pelle, R. Antibodies to in silico selected GPI-anchored Theileria parva proteins neutralize sporozoite infection in vitro. Vet. Immunol. Immunopathol. 2018, 199, 8–14. [Google Scholar] [CrossRef]
  74. Gubbels, M.J.; Duraisingh, M.T. Evolution of apicomplexan secretory organelles. Int. J. Parasitol. 2012, 42, 1071–1081. [Google Scholar] [CrossRef]
  75. Iams, K.P.; Young, J.R.; Nene, V.; Desai, J.; Webster, P.; Ole-MoiYoi, O.K.; Musoke, A.J. Characterisation of the gene encoding a 104-kilodalton micronemerhoptry protein of Theileria parva. Mol. Biochem. Parasitol. 1990, 39, 47–60. [Google Scholar] [CrossRef]
  76. Huber, S.; Theiler, R.; Quervain, D.d.; Wiens, O.; Karangenc, T.; Heussler, V.; Dobbelaere, D.; Woods, K. The Microtubule-Stabilizing Protein CLASP1 Associates with the Theileria annulata Schizont Surface via Its Kinetochore-Binding Domain. mSphere 2017, 2, e00215–e00217. [Google Scholar] [CrossRef]
  77. Hagiwara, K.; Takahashi, M.; Ichikawa, T.; Tsuji, M.; Ikuta, K.; Ishihara, C. Inhibitory effect of heparin on red blood cell invasion by Theileria sergenti merozoites. Int. J. Parasitol. 1997, 27, 535–539. [Google Scholar] [CrossRef]
  78. Burns, A.L.; Dans, M.G.; Balbin, J.M.; de Koning-Ward, T.F.; Gilson, P.R.; Beeson, J.G.; Boyle, M.J.; Wilson, D.W. Targeting malaria parasite invasion of red blood cells as an antimalarial strategy. FEMS Microbiol. Rev. 2019, 43, 223–238. [Google Scholar] [CrossRef]
Pathogens 11 01135 g001 550
Figure 1. Proportion of T. orientalis proteins identified by functional group.
Figure 1. Proportion of T. orientalis proteins identified by functional group.
Pathogens 11 01135 g001
Pathogens 11 01135 g002 550
Figure 2. Metabolic pathways of glycolysis, glycerol catabolism and the TCA cycle with enzymes identified in T. orientalis piroplasms marked in blue, enzymes not identified in the piroplasm proteome in green and enzymes missing from both the piroplasm proteomes and the T. orientalis genomes in red. Genotypes in which each enzyme was identified are shown in brackets (I = Ikeda, B = Buffeli, C = Chitose).
Figure 2. Metabolic pathways of glycolysis, glycerol catabolism and the TCA cycle with enzymes identified in T. orientalis piroplasms marked in blue, enzymes not identified in the piroplasm proteome in green and enzymes missing from both the piroplasm proteomes and the T. orientalis genomes in red. Genotypes in which each enzyme was identified are shown in brackets (I = Ikeda, B = Buffeli, C = Chitose).
Pathogens 11 01135 g002
Table
Table 1. Aqueous and detergent phase proteins identified from individual genotypes of T. orientalis.
Table 1. Aqueous and detergent phase proteins identified from individual genotypes of T. orientalis.
T. orientalis IkedaT. orientalis ChitoseT. orientalis BuffeliAll Genotypes
Aqueous phase272179378829
Detergent phase1303864232
Total proteins *4022174421061
* Numbers reflect target proteins only. Host and contaminating proteins not included.
Table
Table 3. Most abundant (emPAI > 0.5) detergent phase proteins by T. orientalis genotype.
Table 3. Most abundant (emPAI > 0.5) detergent phase proteins by T. orientalis genotype.
GenotypeProtein NameAccessionemPAIFunctionReference
Ikeda Major piroplasm surface protein32402152845.59Host cell interaction[41,42,43]
Piroplasm surface protein P232253202456.56Host cell interaction[35]
Uncharacterised proteinJ4C3E80.8Unknown[23]
ADP-ribosylation factorJ4C8070.58Transcription, regulation and signalling[38]
Uncharacterised proteinJ4DQ050.57Unknown[23]
Sodium/glutamate transporterJ4DA940.52Cellular organisation and transport[24,44]
Tocp1 variantJ4CDK50.52Host cell interaction[45]
Buffeli Major piroplasm surface protein7471566098.86Host cell interaction[41,42,43]
Piroplasm surface protein P232253202373.38Host cell interaction[35]
Genomic DNA chromosome 3 (putative porin)Q4UBJ90.59Cellular organisation and transport[22]
Chitose Major piroplasm surface protein2908830163.03Host cell interaction[41,42,43]
Uncharacterised proteinJ4CDD81.32Unknown[22]
Piroplasm surface protein P23B2LUF80.83Host cell interaction[35]
Table
Table 4. Proteins expressed in T. orientalis piroplasms with potential roles in haemoglobin utilisation.
Table 4. Proteins expressed in T. orientalis piroplasms with potential roles in haemoglobin utilisation.
AccessionAnnotationFunctionReference
J4CDK5Tocp 1Binds haemoglobin[28]
XP_001020177Papain-family cysteine proteaseErythrocyte rupture, haemoglobin-hydrolysis[58]
Q4N067Cysteine proteinaseVivapain-like, hydrolyses haemoglobin[57]
J4C810FalcilysinHaemoglobin-degrading metalloprotease[55]
J4DNF9Aminopeptidase NDegrades haemoglobin-derived peptides into amino acids[60]
Table
Table 5. Proteins involved in host cell interactions and their orthologs.
Table 5. Proteins involved in host cell interactions and their orthologs.
AccessionAnnotationOrthologOrganismReference
J7MEL8Peptidyl-prolyl cis-trans isomeraseTA18945T. annulata[63]
J7MF01
J7M8A7		Hemolysin III
MAC/perforin domain protein		PBANKA-1319100
PY05180		P. berghei
P. yoellii		[65]
[66]
J4C901
D0FY42
J4DP87J4DP87		Phosphatidylinositol glycan Class Q
Microneme-rhoptry related protein
Major piroplasm surface protein (MPSP)
Piroplasm membrane protein P23		YYG_01332
TpMuguga_04g00437
TA17050
TA13810		P. vinckeii
T. parva
T. annulata
T. annulata		[67]
[28]
[68]
[69]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Jenkins, C.; Micallef, M.L.; Padula, M.P.; Bogema, D.R. Characterisation of the Theileria orientalis Piroplasm Proteome across Three Common Genotypes. Pathogens 2022, 11, 1135. https://doi.org/10.3390/pathogens11101135

AMA Style

Jenkins C, Micallef ML, Padula MP, Bogema DR. Characterisation of the Theileria orientalis Piroplasm Proteome across Three Common Genotypes. Pathogens. 2022; 11(10):1135. https://doi.org/10.3390/pathogens11101135

Chicago/Turabian Style

Jenkins, Cheryl, Melinda L. Micallef, Matthew P. Padula, and Daniel R. Bogema. 2022. "Characterisation of the Theileria orientalis Piroplasm Proteome across Three Common Genotypes" Pathogens 11, no. 10: 1135. https://doi.org/10.3390/pathogens11101135

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop