Eimeria bovis infection modulates endothelial host cell cholesterol metabolism for successful replication
© Hamid et al. 2015
Received: 27 March 2015
Accepted: 4 August 2015
Published: 23 September 2015
During first merogony Eimeria bovis forms large macromeronts in endothelial host cells containing >120 000 merozoites I. During multiplication, large amounts of cholesterol are indispensable for the enormous offspring membrane production. Cholesterol auxotrophy was proven for other apicomplexan parasites. Consequently they scavenge cholesterol from their host cell apparently in a parasite-specific manner. We here analyzed the influence of E. bovis infection on endothelial host cell cholesterol metabolism and found considerable differences to other coccidian parasites. Overall, free cholesterol significantly accumulated in E. bovis infected host cells. Furthermore, a striking increase of lipid droplet formation was observed within immature macromeronts. Artificial host cell lipid droplet enrichment significantly improved E. bovis merozoite I production confirming the key role of lipid droplet contents for optimal parasite proliferation. The transcription of several genes being involved in both, cholesterol de novo biosynthesis and low density lipoprotein-(LDL) mediated uptake, was significantly up-regulated at a time in infected cells suggesting a simultaneous exploitation of these two cholesterol acquisition pathways. E. bovis scavenges LDL-derived cholesterol apparently through significantly increased levels of surface LDL receptor abundance and LDL binding to infected cells. Consequently, LDL supplementation significantly improved parasite replication. The up-regulation of the oxidized LDL receptor 1 furthermore identified this scavenger receptor as a key molecule in parasite-triggered LDL uptake. Moreover, cellular cholesterol processing was altered in infected cells as indicated by up-regulation of cholesterol-25-hydroxylase and sterol O-acyltransferase. Overall, these results show that E. bovis considerably exploits the host cell cholesterol metabolism to guarantee its massive intracellular growth and replication.
Eimeria bovis represents one of the most pathogenic Eimeria species causing cattle coccidiosis . During its long-lasting intracellular first merogony (14–18 days of duration) E. bovis forms large macromeronts of up to 400 μm in size containing ≥120 000 merozoites I within a parasitophorous vacuole (PV) in host endothelial cells . Given that the invading sporozoite stage alone cannot provide all components necessary for this nutrient and energy demanding process, the parasite needs to scavenge molecules from the host cell. Especially for the offspring membrane biosynthesis, large amounts of cholesterol are indispensable for a successful replication process.
Coccidian parasites have been described as auxotrophic in their capability to synthesize cholesterol by themselves [3–9]. Thus, cholesterol auxotrophy was reported for Toxoplasma gondii and Cryptosporidium parvum [4,7,9]. In consequence these parasites scavenge cholesterol from their host cell thereby exploiting different cellular pathways. Interestingly, different cholesterol auxotrophic parasites appear to follow different strategies of cholesterol scavenge suggesting the modulation of host cell cholesterol metabolism as a parasite-specific and/or host cell-specific process. Coppens et al.  experimentally proved T. gondii as deficient in cholesterol biosynthesis and showed that this parasite scavenges cholesterol from its host cell. However, different strategies were described in different T. gondii-infected host cell types. Thus, in contrast to macrophages , T. gondii exclusively utilized cholesterol derived from internalized LDL particles in CHO cells  whilst transcriptomic data on infected fibroblasts indicated an up-regulation of molecules being involved in the mevalonate pathway . The pivotal role of cholesterol was also proven by exogenously supplied cholesterol which improved T. gondii replication . Cholesterol is inserted in the parasite plasma membrane, the parasitophorous vacuolar membrane (PVM), sequestered in cholesterol-rich organelles in T. gondii-infected host cells and is furthermore esterified for lipid droplet deposition [4,12–14]. Accordingly, host and parasite esterification activity was shown to be essential for T. gondii intracellular growth . Correspondingly, T. gondii infection leads to enhanced cytoplasmic lipid droplet formation in skeletal muscle cell cultures .
By far less data on cholesterol scavenge is available from other coccidian parasites. Cryptosporidium parvum mainly acquires cholesterol from LDL particles and from micelles being internalized by the infected enterocytes but only to a minor degree from host cellular de novo-synthesis . The non-coccidian but apicomplexan parasite Plasmodium yoelii in principle salvages cholesterol in infected hepatocytes from both host cellular pathways, [6,17] although host cellular cholesterol acquisition does not appear to be essential for its optimal proliferation . The authors interpret these results by a moderate parasitic need of sterols and by an adaptive reaction to cholesterol-restricted conditions in terms of alternative source utilization. Abundant lipid droplet formation was also reported for P. berghei- or P. falciparum-infected host cells [18,19], but no cholesteryl ester formation was detected in Plasmodium-infected cells, implying a lack of lipid storage activity [19–22] which may argue for a continuous cholesterol acquisition from the host cell as hypothesized by Coppens .
To date, no detailed analyses on cholesterol acquisition exist for any Eimeria species. All farm animals kept under conventional farming conditions unavoidably are exposed to Eimeria spp. infections worldwide [1,23] and infection-induced impaired animal performance, mortality and anticoccidial treatment costs generally result in considerable economic losses [1,24]. As an obligate intracellular protozoa residing within a PV, Eimeria spp. require cholesterol not only for PV establishment and host cell (membrane) enlargement (especially in macromeront-forming species) but in particular for massive offspring production (>120 000 merozoites I in case of E. bovis). Consequently, global transcriptomic approaches on infected endothelial host cells have indicated that E. bovis is in need for cholesterol for its successful replication . In addition, recent inhibitor analyses indicated a pivotal role of the host cellular cholesterol de novo synthesis for parasite replication since treatments of E. bovis-infected cells with inhibitors of the mevalonate pathway significantly blocked parasite proliferation . However, detailed analyses on this topic are lacking so far. Therefore we here conducted analyses on E. bovis-induced alterations of the mevalonate pathway, on lipid droplet formation and on LDL-mediated cholesterol internalization and showed that apparently this parasite applies comparable but also different strategies compared to other coccidian species to meet its needs for cholesterol for successful macromeront formation.
Materials and methods
The E. bovis strain H used in the present study was initially isolated from the field in Northern Germany  and maintained by passages in parasite-free male Holstein Friesian calves . All animal procedures were performed according to the Justus Liebig University Animal Care Committee guidelines, approved by the Ethic Commission for Experimental Animal Studies of the State of Hesse (Regierungspräsidium Giessen, GI 18/10 No A37/2011, JLU-No 494) and in accordance to the current German Animal Protection Laws.
For oocysts production, calves were infected orally at the age of 10 weeks with 3 × 105 sporulated E. bovis oocysts. The oocysts were isolated from the faeces beginning 18 days post infection (pi) according to the method of Jackson . The sporulation of oocysts was achieved by incubation in a 2% (w/v) potassium dichromate (Merck) solution at room temperature (RT). Sporulated oocysts were stored in 2% (w/v) potassium dichromate solution at 4 °C until further use. Sporozoites were excysted from sporulated oocysts as previously described . Free sporozoites were washed three times in phosphate buffered-solution (PBS) and counted in a Neubauer chamber.
Host cells and Eimeria bovis host cell infections
Primary bovine umbilical vein endothelial cells (BUVEC) were isolated according to Jaffe et al. . Umbilical cords were collected under aseptic conditions from animals born by sectio caesaria and kept at 4 °C in 0.9% HBSS-HEPES buffer (pH 7.4, Gibco) supplemented with 1% penicillin (500 U/mL, Sigma-Aldrich) and streptomycin (50 μg/mL, Sigma-Aldrich) until use. For the isolation of endothelial cells, 0.025% collagenase type II (Worthington Biochemical Corporation) suspended in Pucks solution (Gibco) was infused into the lumen of the ligated umbilical vein and incubated for 20 min at 37 °C in 5% CO2 atmosphere. After gently massaging the umbilical vein, the cell suspension was collected and supplemented with 1 mL fetal calf serum (FCS, Gibco) in order to inactivate the collagenase. After two washings (400 × g, 10 min, 4 °C), cells were resuspended in complete endothelial cell growth medium (ECGM, PromoCell), plated in 25 cm2 tissue plastic culture flasks (Greiner) and kept at 37 °C in 5% CO2 atmosphere. BUVEC were cultured in modified ECGM medium (EGCM, PromoCell, diluted 0.3× in M199 medium, Sigma-Aldrich) with medium changes every 2–3 days. BUVEC cell layers were used for infection after 1–2 passages in vitro.
BUVEC layers in 24-well or 25 cm2-flask formats were infected at 80–90% confluency with 2 × 104 or 1.5 × 106 E. bovis sporozoites, respectively. Culture medium was changed 24 h after parasite infection and thereafter every third day. Merozoites I were collected from cell culture supernatants from days 17–20 pi onwards and washed three times in PBS (600 × g, 15 min) before further use.
Staining of cholesterol and lipid droplets
For staining of intracellular stages (1, 8, 14 and 17 days pi), BUVEC were grown on cover slips and infected, whereas invasive stages (sporozoites and merozoites I) were directly dropped onto poly-L-lysine coated coverslips. Specimens were washed with PBS, fixed in 4% paraformaldehyde (10 min), washed three times in PBS and incubated in 1% glycine PBS (10 min) to quench non-specific signals, followed by three washings in PBS. To detect free cholesterol, the samples were stained by filipin (0.05 mg/mL in PBS containing 10% FCS, Sigma-Aldrich, 2 h, in the dark, RT). For neutral lipid/lipid droplet visualization the cells were stained in Nile Red (1:1000, Cayman Chemical, 15 min, RT, in the dark) or with Bodipy 493/503 (1 μg/mL, Life Technologies; 10 min, RT, in the dark). All samples were washed in PBS, mounted in Prolong antifading mounting medium (Life Technologies) and analyzed using an inverted fluorescence microscope or confocal microscope applying the UV filter set for filipin (340–380 nm excitation, 430 nm pass filter) or FITC filter settings for Nile red and Bodipy 493/503. For an alternative LD staining, osmium tetroxide staining  was performed with modification. Therefore, cells were washed four times in PBS, fixed (2% paraformaldehyde containing 0.1% glutaraldehyde, EM grade, Sigma-Aldrich, RT, 30 min) and osmium tetroxide-stained (0.1%, Sigma-Aldrich, RT, 30 min). The samples were washed three times in PBS, mounted in PBS and analyzed under bright field conditions.
Total cholesterol measurement
Total lipid extractions from E. bovis-infected (4, 8 and 17 days pi) and control BUVEC (n = 7) were performed in hexane:isopropanol according to Hara and Radin . The cells were washed twice in ice-cold PBS, trypsinized and washed again (400 × g, 10 min). The total cell numbers were counted using a Neubauer chamber. Hexane:isopropanol (3:2, v/v) was added to the cell pellet. Cells were disrupted for 10 min in a Tissue Lyser (Qiagen, Hilden, Germany) using stainless steel beads. The samples were centrifuged (8000 × g, 1 min) and the supernatants were collected. The extraction was repeated once for each sample. The supernatants were then combined and dried manually under gentle liquid nitrogen stream. The total lipid extracts were reconstituted in 500 μL isopropanol:NP40 (9:1, v/v) followed by sonication in a waterbath (RT, 30 min). 5 μL of each sample were pre-treated with catalase [(5 μL of 0.5 mg/mL) in 40 μL of 1X reaction buffer (37 °C, 15 min)] in 96-well black clear-bottom plates (Greiner Bio-One) to reduce background fluorescence of peroxides in the solvents  before the enzyme cocktail of the Amplex-red kit (Life Technologies) was added. Cholesterol standards (Sigma-Aldrich; 10, 5, 2.5, 1.25, 0.625 and 0.325 μM) and blanks (solvent only) were included in each experiment. Fifty microliters of enzyme mixture (0.1 M potassium phosphate buffer, pH 7.4; 0.25 M NaCl, 5 mM cholic acid, 0.1% Triton X-100, 0.3 U/mL cholesterol oxidase, cholesterol esterase, 1.3 U/mL HRP, and 0.4 mM ADHP) were added and incubated (37 °C, 15 min). Resorufin formation was measured by fluorescence intensities (excitation wavelength of 530 nm, emission wavelength of 580 nm) in the Varioskan Flash Multimode Reader (Thermo Scientific). Total cholesterol of the samples was extrapolated to the values of the cholesterol standard. The total cholesterol content of each sample was normalized to its total cell number counts.
Quantitative analysis of lipid droplet accumulation in infected BUVEC
E. bovis-infected and control BUVEC (n = 3) were trypsinized at days 8, 17 and 21 pi and pelleted in PBS (400 × g, 3 min, 4 °C). The resuspended cells were stained with Bodipy 493/503 (10 min, on ice) and washed twice with 1 ml PBS (400 × g, 3 min, 4 °C). The cells were transferred to 5-mL FACS tubes containing 200 μL of PBS and were processed in a FACS Calibur flow cytometer (BD Biosciences, Heidelberg, Germany) by laser excitation at 488 nm (FL1-H channel). Flow cytometry data were acquired by the BD CellQuest Pro software.
Cholesterol, LDL and lipid droplet enrichment
For cholesterol enrichment, both, cholesterol and desmosterol (both Sigma-Aldrich) dissolved in ethanol were continuously added to BUVEC cultures (n = 5) at 5 μM final concentration. Although not being physiological the administration of cholesterol at low concentration (in ethanol) according to Xu et al.  was chosen to reduce its toxicity in long-term application. Supplementation of cholesterol complexed to cyclodextrin was not applicable to long-term culture in our hands owing to its high cytotoxicity.
For LDL enrichment, LDL (Sigma-Aldrich, 10 mg/mL final concentration) was supplemented to E. bovis-infected BUVEC (n = 5) from 10 days pi onwards. To artificially enhance lipid droplet formation in host cells, oleic acid (Sigma-Aldrich) was supplemented in BSA formulation complexes  to the cell culture medium. Direct conjugation was performed by mixing oleic acid-free BSA (fraction V, Roth) with oleic acid at the molar ratio of 6:1 (oleic acid:BSA). BUVEC (n = 3) were treated with 5 μM oleic acid/BSA complexes for an induction period of 1 h, and thereafter continuously with 2.5 μM to prevent toxicity. Treatments were repeated every second day from 8 days pi onwards.
Low density lipoprotein (LDL) binding assay
The binding of LDL to receptors on the surface of infected cells and non-infected controls was estimated using Bodipy-labelled LDL (Life Technologies). Therefore, E. bovis-infected BUVEC (15 days pi) were cultivated in FCS-free endothelial cell basal medium (PromoCell) supplemented with 10% lipoprotein deficient-serum (Sigma-Aldrich) for 36–48 h prior to medium supplementation with Bodipy-labelled LDL (10 μg/mL). The cells were incubated for 1 h at 4 °C followed by 4 h at 37 °C and also 3 h, 5 h for fluorescence microscopy. For qualitative analyses the cells were washed in PBS, fixed (4% paraformaldehyde, 10 min) and mounted in Prolong antifading mounting medium prior to fluorescence microscopy. For quantitative analyses, BUVEC were treated with 3% NaN3 (5 min, RT) to inhibit receptor recycling and washed with ice-cold PBS. Monolayers were detached by Accutase (Sigma-Aldrich) treatment (5 min, 37 °C), washed in PBS (400 × g, 5 min) and transferred to 5-mL FACS tubes containing 200 μL of PBS. The cells were processed in a FACS Calibur flow cytometer using the FL1-H channel.
Real-time quantitative PCR assay for the detection of cholesterol metabolism-associated molecules
Sequences of primers and probes used in real-time qPCR
Probe (reporter 5’-3’ quencher)
Acetyl-CoA acetyltransferase 1
Acetyl-CoA acetyltransferase 2
3-hydroxy-3-methylglutaryl-CoA synthase 1
Sterol O-acyltransferase 1 (SOAT1)
Low density lipoprotein receptor
Oxidized low density lipoprotein/(lectin-like) receptor 1
E. bovis-infected and control BUVEC (n = 3) were processed for total RNA isolation at different time points of macromeront maturation (days 12, 14, 17 and 20 pi). Total RNA isolation was performed using the RNeasy kit (Qiagen) according to manufacturer’s protocol. Therefore BUVEC were lysed within the cell culture flasks with RLT lysis buffer (600 μL/25 cm2 flask) and processed as proposed by the manufacturer. An on-column DNase treatment (Qiagen) according to manufacturer’s instruction was included in the total RNA isolation procedure. Total RNAs were stored at −20 °C until further use. The quality of total RNA samples was controlled via Agilent 2100 bioanalyzer (Agilent Technologies, USA). In order to remove any DNA leftover, a second genomic DNA digestion step was performed. Therefore, 1 μg of total RNA was treated with 10 U DNase I in 1x DNase reaction buffer (all Thermo Scientific, 37 °C, 1 h). DNase was inactivated by heating the sample (65 °C, 10 min). The efficiency of genomic DNA digestion was controlled by including no-RT-controls in each real-time PCR experiment.
cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System according to manufacturer’s protocol with slight modifications.1 μg of DNase-treated total RNA was added to 1 μL of 50 μM oligo d(T), 1 μL of 50 ng/μL random hexamer primer, 1 μL of 10 mM dNTP mix and DEPC-treated water was adjusted to 10 μL total volume. The sample was incubated at 65 °C for 5 min and then immediately cooled on ice. For first strand cDNA synthesis, 2 μL of 10x RT buffer, 1 μL 25 mM MgCl2, 1 μL 0.1 M DTT, 1 μL RNaseOUT (40 U/μL) and 1 μL SuperScript III enzyme (200 U/μL) were added and the sample was incubated at 50 °C for 60 min followed by a 85 °C-inactivation step for 15 min.
Real-time PCR was performed in duplicates in a 10 μL total volume containing 400 nM forward and reverse primers, 200 nM probe, 10 ng cDNA and 5 μL 2× PerfeCTa qPCR FastMix (Quanta Biosciences, USA). The reaction conditions for all systems were as follows: 95 °C for 10 min, 40 cycles at 95 °C for 10 s, 60 °C for 15 s and 72 °C for 30 s. PCRs were performed on a Rotor-GeneQ cycler (Qiagen, Hilden, Germany). No-template controls (NTC) and no-RT reactions were included in each experiment. As a reference gene we chose with GAPDH a gene that was previously used in other publications reporting on E. bovis-infected BUVEC [24,38]. To prove that the GAPDH gene is a suitable reference gene in this experimental set-up, we have previously performed experiments on the stability of GAPDH gene transcription in E. bovis-infected BUVEC. Therefore, four different BUVEC isolates (=4 biological replicates) were infected with E. bovis and tested for GAPDH gene transcription (in duplicates) throughout infection (at days 4, 8, 12, 14 and 17 pi). Considering all samples of all isolates at Ct level we showed that GAPDH gene transcription was not significantly influenced by the infection (mean ± standard deviation of the Cts of all samples: 21.31 ± 0.84) but proved stabile within the kinetics, since the variation of the data accounted to less than 4%. Analyses of the qPCR data used the comparative Ct method (ΔΔC T method) and reported as n-fold differences comparing E. bovis-infected BUVEC with non-infected ones after normalizing the samples with the GAPDH reference gene.
Merozoites I quantification via Eimeria bovis microneme protein 4 (EbMIC4)-based real-time PCR
For merozoites I quantification we used a real-timePCR system based on the detection of the single copy gene of E. bovis microneme protein 4 (EbMIC4, ) applying the following primers and probe: forward primer: 5’-CACAGAAAGCAAAAGACA-3’, reverse primer: 5’-GACCATTCTCCAAATTCC-3’ and probe: 5’-FAM-CGCAGTCAGTCTTCTCCTTCC-BHQ1-3’ . Therefore, cell culture supernatants containing merozoites I were collected at indicated time points pi. At the end of the culture period the cells were trypsinized and collected together with the supernatant followed by centrifugation (600 × g, 15 min). The cell pellet was treated with 200 μL of cell lysis buffer containing 0.32 M Sucrose, 1% Triton X-100, 0.01 M Tris-HCl (pH 7.5), 5 mM MgCl2 and incubated in 100 μL 1X PCR buffer (Quanta, USA) containing 20 μL proteinase K (20 mg/mL; Qiagen) at 56 °C for 1 h. Proteinase K was heat-inactivated (95 °C, 10 min) and the DNA-containing samples were frozen at − 20 °C until further use. All samples were processed as triplicates.
Real-time PCR was performed in a total volume of 20 μL containing 5 μl DNA of merozoites I samples, 400 nM of each primer, 200 nM probe and 10 μL PerfeCTa MasterMix (Quanta, USA) at 95 °C for 10 min; 40 cycles at 95 °C for 10 s, 60 °C for 15 s and 72 °C for 30 s. In each PCR experiment serial dilutions of merozoites I covering 6 magnitude orders of 10-fold (106-10) were included allowing for absolute quantification of merozoites I counts in the samples.
Real-time PCR data analysis was performed by the ΔΔC T method, normalized to GAPDH and expressed as relative fold change compared to control [40,41]. For statistics data were analyzed by Student’s t-tests comparing control and treated groups. GraphPad Prism 6.02 was used to visualize graphs.
Free cholesterol and neutral lipid/lipid droplet distribution in E. bovis invasive stages
Cholesterol and lipid droplets accumulate in E. bovis-infected cells during macromeront formation
Given that the fixation mode may alter lipid droplet integrity leading to dimmed LD appearance , we additionally used osmium tetroxide staining which is known to better preserve lipid body structures. However, osmium tetroxide treatments led to similar results to those of Bodipy 493/503 staining thus confirming the significant increase of lipid droplet abundance (visible as small black dots) in E. bovis macromeront-carrying host cells (Figure 3F).
Extracellular sterol enrichment and enhanced cellular lipid droplet disposability boost parasite replication
E. bovis infection enhances surface LDL receptor abundance on infected cells and exogenously supplied LDL improves merozoite I production
To estimate whether the increase in LDL-binding originated from enhanced surface LDL receptor (LDLR) abundance, a flow cytometry-based assay using an LDLR-specific antibody was established and tested on E. bovis-infected BUVEC at 17 days pi. Overall, the data revealed a significant infection-induced increase of LDLR surface expression (1.92 fold) in macromeront-carrying host cells (infected vs. non-infected cells: p < 0.0001; Figures 7C and 7D). These data are in agreement with transcriptional profiles of LDLR in E. bovis-infected cells, as described later (Figure 9).
E. bovis infection induces up-regulation of the gene transcription of molecules involved in the mevalonate pathway and in cholesterol hydroxylation and esterification
Following de novo biosynthesis, cholesterol may either be recycled to the membranes or modified via further enzymatic steps promoting hydroxylation via CH25H or esterification via SOAT1 [45–47]. Analyses on E. bovis-infected BUVEC revealed that both pathways of cholesterol processing were significantly up-regulated at times of macromeront formation. Thus, SOAT1 gene transcripts were found significantly increased for 3.3- and 10.2-fold at 14 and 17 days pi (both: p ≤ 0.01), respectively, when compared to non-infected controls (Figure 9). Given that cholesterylesters are stored in lipid droplets, these results indirectly confirmed the data on enhanced lipid droplet formation in E. bovis-infected host cells. The overall strongest up-regulation of all gene transcripts tested was measured for CH25H (Figure 9). Hence, significant enhancements of gene transcription were detected at days 14, 17 (both: p ≤ 0.001) and 20 (p ≤ 0.01) pi, suggesting a crucial role of 25-hydroxycholesterol synthesis in the development of E. bovis macromeronts.
The transcriptional profiling of LDLR and OLR1 in E. bovis-infected host cells additionally showed significantly enhanced mRNA levels for both molecules. Compared to non-infected controls, significant reactions were detected for LDLR at 12 and 17 days pi (both: p ≤ 0.01) reaching an up to 9.49-fold increase (Figure 9). OLR1 gene transcripts were significantly enhanced during the entire period of investigation (days 12, 17 and 20 pi: p ≤ 0.01, day 14 pi: p ≤ 0.05, Figure 9) reaching an average of up to 7-fold increase in E. bovis infected cells.
During first merogony E. bovis forms the PV, enlarges the endothelial host cell and produces thousands of merozoites I and, consequently, is in considerable need for cholesterol. We here show that the parasite significantly utilizes different host cellular cholesterol acquisition pathways and demonstrate that E. bovis-induced modulation of the host cell cholesterol metabolism differs from other apicomplexan parasites for several reasons: i) the massive induction of lipid droplets within the immature meront I stage, ii) the simultaneous interference with both, the host cellular de novo synthesis and LDL-mediated cholesterol up-take, iii) the involvement of OLR1 and iv) the induction of CH25H in host cellular cholesterol modification.
In contrast to other apicomplexan parasites, such as T. gondii, the monoxenous protozoa E. bovis represents a strict host- and host cell-specific parasite. Thus, first merogony exclusively occurs in bovine endothelial cells and lasts for up to three weeks. Filipin staining experiments ascertained that E. bovis stages indeed do contain free cholesterol molecules. This is in line with tachyzoites of T. gondii, [4,47–49] but in contrast to P. yoelii free sporozoites which lack filipin staining . Early after infection and in cells carrying developing macromeronts strong filipin reaction were observed indicating high cholesterol contents in infected cells. Quantitative analyses revealed an almost doubling of total cholesterol content in times of E. bovis macromeront maturation. Furthermore and in accordance to studies on T. gondii , excess exogenous cholesterol resulted in significantly enhanced offspring production confirming the key role of high cholesterol abundance for successful E. bovis development.
Neutral lipid staining in E. bovis free stages revealed a differential distribution of free cholesterol and neutral lipids in sporozoites and merozoites I. Thus, neutral lipids exclusively occurred in the refractile bodies of sporozoites and in cytosolic lipid droplet-like structures of both stages. This is in line with observations on E. tenella sporozoites [50,51] and T. gondii tachyzoites [4,12,15].
The most striking feature in E. bovis-infected host cells was the massive induction of lipid droplets within the immature meront I (note that the cytoplasm did not show any lipid droplets) which finds no correlate in any other coccidian infection. Although a stage-dependent increase of lipid droplets was also reported in P. falciparum-infected erythrocytes [19–21], these organelles were mainly localized in the host cell cytoplasm and not within the parasitic stages and were of much less numbers. Interestingly, the maximum lipid droplet generation was observed in late immature macromeronts, i.e. at times when merozoites I are about to be formed. In contrast, in mature macromeronts carrying fully developed merozoites I, structurally defined lipid droplets were hardly observed, suggesting that lipid droplet contents were almost totally consumed during offspring formation. Confocal microscopy analyses revealed considerable variations of lipid droplet sizes in macromeront-carrying host cells, ranging from submicrometer diameters to large sizes of ≥5 μm. Accordingly, “gigantic” lipid droplets (being situated within the host cell cytoplasm) were previously described in Trypanosomacruzi-infected host cells  but not in any other coccidian parasite, so far. The general phenomenon of enhanced lipid droplet formation in infected host cells was also reported for other protozoan parasites such as T. cruzi, T. gondii, P. berghei or P. falciparum [16,18,19,52–54]. However, it has to be stressed that the localization and the degree of lipid droplet formation in the latter cases was not at all comparable to E. bovis infections and most reports referred to host cell cytosolic lipid droplets and not parasitic ones. Whilst in T. cruzi-, T. gondii- or P. falciparum-induced host cell lipid droplets never exceeded a number of ~20 [16,19,55], the numbers of these organelles was 100-1000-fold higher in immature E. bovis meronts (suspected numbers since the true numbers are uncountable owing to the thickness of the specimens). The key role of lipid droplets was furthermore proven by oleic acid treatments which significantly improved parasite proliferative capacities. Given that excess cellular cholesterol is stored in the esterified form in lipid droplets, we additionally analyzed the gene transcription of SOAT1, i.e., the enzyme promoting cholesterol esterification, and demonstrated a significant increase of respective transcripts in E. bovis-infected cells. In line with other apicomplexan parasites [14,15] the key role of cholesterol esterification for optimal E. bovis proliferation cells was recently confirmed by chemical SOAT inhibition leading to almost total blockage of E. bovis replication in endothelial host cells .
Besides esterification, hydroxylation of cholesterol is also performed to detoxify this molecule in mammalian cells. Transcriptional profiling and protein expression (P. Hamid, personal communication) analyses of E. bovis-infected BUVEC showed a significant up-regulation of CH25H which promotes cholesterol hydroxylation. It is noteworthy, that the overall highest up-regulation of gene transcripts and proteins concerned CH25H suggesting enhanced 25-OH-cholesterol (25-OHC) synthesis to occur in E. bovis-infected host cells. So far, no comparable phenomenon was reported in any other coccidian parasite infection. It has to be mentioned that levels of gene transcription and protein expression do not necessarily reflect cellular enzyme activities. However, preliminary results on biochemical 25-OHC measurements showed enhanced concentrations of this molecule in E. bovis-infected host cells (A. Taubert, unpublished data) indicating a new strategy of cholesterol processing in coccidian-infected host cells.
Transcriptional profiling of E. bovis-infected host cells showed that all molecules tested being involved in the mevalonate pathway or in early substrate synthesis were found up-regulated in times of merozoite I formation. An enhancement of ACAT1 was also confirmed on the protein level (P. Hamid, personal communication). These data are in line with recent data on chemical HMGCS1 and SQLE inhibition leading to a significant blockage of E. bovis proliferation . Related data in T. gondii infections are somewhat conflicting: whilst Coppens et al.  denied any involvement of this pathway in T. gondii-infected host cells, other studies indicated that de novo synthesis indeed contributes to T. gondii growth [10,11,56].
Besides de novo biosynthesis, exogenous cholesterol is acquired via cellular lipoprotein internalization. LDL has been reported as major exogenous cholesterol source for T. gondii, C. parvum and Plasmodium spp. [4,6,7]. Accordingly, we here report on enhanced binding and up-take of LDL at times of E. bovis macromeront formation. Consistent with recent microarray data , LDLR gene transcripts were found significantly up-regulated throughout E. bovis macromeront development. Furthermore, a significant increase of surface LDLR abundance was detected on infected host cells via FACS analyses. These results are in line with data on T. gondii-infected CHO cells , but contrast to the fact that reduced LDLR expression did not affect the liver stage burden in the case of Plasmodium spp. . In accordance to T. gondii-infected CHO cells , LDL supplementation had beneficial effects on E. bovis merozoite I production confirming the pivotal role of LDL for optimal parasite replication. In contrast, LDL enrichment had no stimulatory effects on Plasmodium spp. and C. parvum proliferation [6,7] or on T. gondii growth in macrophages  indicating parasite- and cell type-specific mechanisms.
The current transcriptional data additionally revealed the scavenger receptor OLR1 (syn. LOX-1) to be involved in E. bovis-triggered LDL-uptake since a strong up-regulation of this receptor was detected in times of macromeront formation. These data are in agreement with recent microarray data  and were confirmed on protein level (P. Hamid, personal observation.). OLR1 is considered as the major receptor for oxidized LDL (oxLDL) internalization in vascular endothelial cells [57–59]. To our knowledge, this represents the first report indicating an involvement of OLR1/oxLDL in coccidian host cell infections.
Overall, the current data indicate that E. bovis has the capacity to scavenge cholesterol from several cellular sources at a time, which clearly contrasts to T. gondii . Given that cellular cholesterol synthesis is tightly regulated by a complex network of cellular mechanisms, more research is needed to understand how E. bovis outwits this regulatory network.
The authors thank A. Wehrend (Clinic for Obstetrics, Gynaecology and Andrology of Large and Small Animals, Justus Liebig University, Giessen, Germany) and K. Herzog (Clinic for Cattle, University of Veterinary Medicine Hannover, Germany for their continuous supply of bovine umbilical cords. PH was supported by a DIKTI (Direktorat Jenderal Pendidikan Tinggi Indonesia) PhD scholarship. Several experiments were supported and financed by the German Research Foundation (DFG; grant number: TA 291/10-1).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Daugschies A, Najdrowski M (2005) Eimeriosis in cattle: current understanding. J Vet Med B Infect Dis Vet Public Health 52:417–427View ArticlePubMedGoogle Scholar
- Hammond DM, Bowman GW, Davis LR, Simms BT (1946) The endogenous phase of the life cycle of Eimeria bovis. J Parasitolo 32:409–427View ArticleGoogle Scholar
- Furlong ST (1989) Sterols of parasitic protozoa and helminths. Exp Parasitol 68:482–485View ArticlePubMedGoogle Scholar
- Coppens I, Sinai AP, Joiner KA (2000) Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition. J Cell Biol 149:167–180PubMed CentralView ArticlePubMedGoogle Scholar
- Bano N, Romano JD, Jayabalasingham B, Coppens I (2007) Cellular interactions of Plasmodium liver stage with its host mammalian cell. Int J Parasitol 37:1329–1341View ArticlePubMedGoogle Scholar
- Labaied M, Jayabalasingham B, Bano N, Cha SJ, Sandoval J, Guan G, Coppens I (2011) Plasmodium salvages cholesterol internalized by LDL and synthesized de novo in the liver. Cell Microbiol 13:569–586View ArticlePubMedGoogle Scholar
- Ehrenman K, Wanyiri JW, Bhat N, Ward HD, Coppens I (2013) Cryptosporidium parvum scavenges LDL-derived cholesterol and micellar cholesterol internalized into enterocytes. Cell Microbiol 15:1182–1197PubMed CentralView ArticlePubMedGoogle Scholar
- Bansal D, Bhatti HS, Sehgal R (2005) Role of cholesterol in parasitic infections. Lipids Health Dis 4:10PubMed CentralView ArticlePubMedGoogle Scholar
- Coppens I (2013) Targeting lipid biosynthesis and salvage in apicomplexan parasites for improved chemotherapies. Nat Rev Microbiol 11:823–835View ArticlePubMedGoogle Scholar
- Nishikawa Y, Ibrahim HM, Kameyama K, Shiga I, Hiasa J, Xuan X (2011) Host cholesterol synthesis contributes to growth of intracellular Toxoplasma gondii in macrophages. J Vet Med Sci 73:633–639View ArticlePubMedGoogle Scholar
- Blader IJ, Manger ID, Boothroyd JC (2001) Microarray analysis reveals previously unknown changes in Toxoplasma gondii-infected human cells. J Biol Chem 276:24223–24231View ArticlePubMedGoogle Scholar
- Charron AJ, Sibley LD (2002) Host cells: mobilizable lipid resources for the intracellular parasite Toxoplasma gondii. J Cell Sci 115:3049–3059PubMedGoogle Scholar
- Sehgal A, Bettiol S, Pypaert M, Wenk MR, Kaasch A, Blader IJ, Joiner KA, Coppens I (2005) Peculiarities of host cholesterol transport to the unique intracellular vacuole containing Toxoplasma. Traffic 6:1125–1141View ArticlePubMedGoogle Scholar
- Nishikawa Y, Quittnat F, Stedman TT, Voelker DR, Choi JY, Zahn M, Yang M, Pypaert M, Joiner KA, Coppens I (2005) Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma. Cell Microbiol 7:849–867View ArticlePubMedGoogle Scholar
- Sonda S, Ting LM, Novak S, Kim K, Maher JJ, Farese RV Jr, Ernst JD (2001) Cholesterol esterification by host and parasite is essential for optimal proliferation of Toxoplasma gondii. J Biol Chem 276:34434–34440View ArticlePubMedGoogle Scholar
- Gomes AF, Magalhaes KG, Rodrigues RM, de Carvalho L, Molinaro R, Bozza PT, Barbosa HS (2014) Toxoplasma gondii-skeletal muscle cells interaction increases lipid droplet biogenesis and positively modulates the production of IL-12, IFN-g and PGE2. Parasit Vectors 7:47PubMed CentralView ArticlePubMedGoogle Scholar
- Grellier P, Valentin A, Millerioux V, Schrevel J, Rigomier D (1994) 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors lovastatin and simvastatin inhibit in vitro development of Plasmodium falciparum and Babesia divergens in human erythrocytes. Antimicrob Agents Chemother 38:1144–1148PubMed CentralView ArticlePubMedGoogle Scholar
- Rodriguez-Acosta A, Finol HJ, Pulido-Mendez M, Marquez A, Andrade G, Gonzalez N, Aguilar I, Giron ME, Pinto A (1998) Liver ultrastructural pathology in mice infected with Plasmodium berghei. J Submicrosc Cytol Pathol 30:299–307PubMedGoogle Scholar
- Vielemeyer O, McIntosh MT, Joiner KA, Coppens I (2004) Neutral lipid synthesis and storage in the intraerythrocytic stages of Plasmodium falciparum. Mol Biochem Parasitol 135:197–209View ArticlePubMedGoogle Scholar
- Nawabi P, Lykidis A, Ji D, Haldar K (2003) Neutral-lipid analysis reveals elevation of acylglycerols and lack of cholesterol esters in Plasmodium falciparum-infected erythrocytes. Eukaryot Cell 2:1128–1131PubMed CentralView ArticlePubMedGoogle Scholar
- Palacpac NM, Hiramine Y, Mi-ichi F, Torii M, Kita K, Hiramatsu R, Horii T, Mitamura T (2004) Developmental-stage-specific triacylglycerol biosynthesis, degradation and trafficking as lipid bodies in Plasmodium falciparum-infected erythrocytes. J Cell Sci 117:1469–1480View ArticlePubMedGoogle Scholar
- Coppens I, Vielemeyer O (2005) Insights into unique physiological features of neutral lipids in Apicomplexa: from storage to potential mediation in parasite metabolic activities. Int J Parasitol 35:597–615View ArticlePubMedGoogle Scholar
- Faber JE, Kollmann D, Heise A, Bauer C, Failing K, Burger HJ, Zahner H (2002) Eimeria infections in cows in the periparturient phase and their calves: oocyst excretion and levels of specific serum and colostrum antibodies. Vet Parasitol 104:1–17View ArticlePubMedGoogle Scholar
- Hermosilla C, Zahner H, Taubert A (2006) Eimeria bovis modulates adhesion molecule gene transcription in and PMN adhesion to infected bovine endothelial cells. Int J Parasitol 36:423–431View ArticlePubMedGoogle Scholar
- Taubert A, Wimmers K, Ponsuksili S, Jimenez CA, Zahner H, Hermosilla C (2010) Microarray-based transcriptional profiling of Eimeria bovis-infected bovine endothelial host cells. Vet Res 41:70PubMed CentralView ArticlePubMedGoogle Scholar
- Hamid PH, Hirzmann J, Hermosilla C, Taubert A (2014) Differential inhibition of host cell cholesterol de novo biosynthesis and processing abrogates Eimeria bovis intracellular development. Parasitol Res 113:4165–4176View ArticlePubMedGoogle Scholar
- Fiege N, Klatte D, Kollmann D, Zahner H, Burger HJ (1992) Eimeria bovis in cattle: colostral transfer of antibodies and immune response to experimental infections. Parasitol Res 78:32–38View ArticlePubMedGoogle Scholar
- Hermosilla C, Burger HJ, Zahner H (1999) T cell responses in calves to a primary Eimeria bovis infection: phenotypical and functional changes. Vet Parasitol 84:49–64View ArticlePubMedGoogle Scholar
- Jackson AR (1964) The isolation of viable coccidial sporozoites. Parasitology 54:87–93View ArticlePubMedGoogle Scholar
- Hermosilla C, Barbisch B, Heise A, Kowalik S, Zahner H (2002) Development of Eimeria bovis in vitro: suitability of several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal, Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells. Parasitol Res 88:301–307View ArticlePubMedGoogle Scholar
- Jaffe EA, Nachman RL, Becker CG, Minick CR (1973) Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52:2745–2756PubMed CentralView ArticlePubMedGoogle Scholar
- Melo RC, D’Avila H, Wan HC, Bozza PT, Dvorak AM, Weller PF (2011) Lipid bodies in inflammatory cells: structure, function, and current imaging techniques. J Histochem Cytochem 59:540–556PubMed CentralView ArticlePubMedGoogle Scholar
- Hara A, Radin NS (1978) Lipid extraction of tissues with a low-toxicity solvent. Anal Biochem 90:420–426View ArticlePubMedGoogle Scholar
- Robinet P, Wang Z, Hazen SL, Smith JD (2010) A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells. J Lipid Res 51:3364–3369PubMed CentralView ArticlePubMedGoogle Scholar
- Xu F, Rychnovsky SD, Belani JD, Hobbs HH, Cohen JC, Rawson RB (2005) Dual roles for cholesterol in mammalian cells. Proc Natl Acad Sci U S A 102:14551–14556PubMed CentralView ArticlePubMedGoogle Scholar
- Martin S, Parton RG (2011) Characterization of Rab18, A lipid droplet-associated small GTPAse. In: Conn PM Methods of Enzymology. Academic Press Elsevier, CaliforniaGoogle Scholar
- Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622View ArticlePubMedGoogle Scholar
- Taubert A, Zahner H, Hermosilla C (2006) Dynamics of transcription of immunomodulatory genes in endothelial cells infected with different coccidian parasites. Vet Parasitol 142:214–222View ArticlePubMedGoogle Scholar
- Lutz K (2008) Charakterisierung des Eimeria bovis mikronemenproteins 4 (EbMIC4) und erste studien zur modulation des wirtszell-proteoms durch Eimeria bovis. PhD Thesis, Justus Liebig University, Institute of ParasitologyGoogle Scholar
- Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408View ArticlePubMedGoogle Scholar
- Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108View ArticlePubMedGoogle Scholar
- DiDonato D, Brasaemle DL (2003) Fixation methods for the study of lipid droplets by immunofluorescence microscopy. J Histochem Cytochem 51:773–780View ArticlePubMedGoogle Scholar
- Rodriguez-Acebes S, de la Cueva P, Fernandez-Hernando C, Ferruelo AJ, Lasuncion MA, Rawson RB, Martinez-Botas J, Gomez-Coronado D (2009) Desmosterol can replace cholesterol in sustaining cell proliferation and regulating the SREBP pathway in a sterol-Delta24-reductase-deficient cell line. Biochem J 420:305–315PubMed CentralView ArticlePubMedGoogle Scholar
- Goldstein JL, DeBose-Boyd RA, Brown MS (2006) Protein sensors for membrane sterols. Cell 124:35–46View ArticlePubMedGoogle Scholar
- Ikonen E (2008) Cellular cholesterol trafficking and compartmentalization. Nat Rev Mol Cell Biol 9:125–138View ArticlePubMedGoogle Scholar
- Chang TY, Chang CC, Ohgami N, Yamauchi Y (2006) Cholesterol sensing, trafficking, and esterification. Annu Rev Cell Dev Biol 22:129–157View ArticlePubMedGoogle Scholar
- Foussard F, Leriche MA, Dubremetz JF (1991) Characterization of the lipid content of Toxoplasma gondii rhoptries. Parasitology 102:367–370View ArticlePubMedGoogle Scholar
- Foussard F, Gallois Y, Girault A, Menez JF (1991) Lipids and fatty acids of tachyzoites and purified pellicles of Toxoplasma gondii. Parasitol Res 77:475–477View ArticlePubMedGoogle Scholar
- Coppens I, Joiner KA (2003) Host but not parasite cholesterol controls Toxoplasma cell entry by modulating organelle discharge. Mol Biol Cell 14:3804–3820PubMed CentralView ArticlePubMedGoogle Scholar
- Lemgruber L, Lupetti P (2002) Crystalloid body, refractile body and virus-like particles in Apicomplexa: what is in there? Parasitology 139:285–293View ArticleGoogle Scholar
- de Venevelles P, Francois Chich J, Faigle W, Lombard B, Loew D, Pery P, Labbe M (2006) Study of proteins associated with the Eimeria tenella refractile body by a proteomic approach. Int J Parasitol 36:1399–1407View ArticlePubMedGoogle Scholar
- Melo RC, Fabrino DL, Dias FF, Parreira GG (2006) Lipid bodies: Structural markers of inflammatory macrophages in innate immunity. Inflamm Res 55:342–348View ArticlePubMedGoogle Scholar
- Jackson KE, Klonis N, Ferguson DJ, Adisa A, Dogovski C, Tilley L (2004) Food vacuole-associated lipid bodies and heterogeneous lipid environments in the malaria parasite, Plasmodium falciparum. Mol Microbiol 54:109–122View ArticlePubMedGoogle Scholar
- Coppens I (2006) Contribution of host lipids to Toxoplasma pathogenesis. Cell Microbiol 8:1–9View ArticlePubMedGoogle Scholar
- Melo RC, D’Avila H, Fabrino DL, Almeida PE, Bozza PT (2003) Macrophage lipid body induction by Chagas disease in vivo: putative intracellular domains for eicosanoid formation during infection. Tissue Cell 35:59–67View ArticlePubMedGoogle Scholar
- Martins-Duarte ES, Urbina JA, de Souza W, Vommaro RC (2006) Antiproliferative activities of two novel quinuclidine inhibitors against Toxoplasma gondii tachyzoites in vitro. J Antimicrob Chemother 58:59–65View ArticlePubMedGoogle Scholar
- Sawamura T, Kume N, Aoyama T, Moriwaki H, Hoshikawa H, Aiba Y, Tanaka T, Miwa S, Katsura Y, Kita T, Masaki T (1997) An endothelial receptor for oxidized low-density lipoprotein. Nature 386:73–77View ArticlePubMedGoogle Scholar
- Moriwaki H, Kume N, Sawamura T, Aoyama T, Hoshikawa H, Ochi H, Nishi E, Masaki T, Kita T (1998) Ligand specificity of LOX-1, a novel endothelial receptor for oxidized low density lipoprotein. Arterioscler Thromb Vasc Biol 18:1541–1547View ArticlePubMedGoogle Scholar
- Kume N, Murase T, Moriwaki H, Aoyama T, Sawamura T, Masaki T, Kita T (1998) Inducible expression of lectin-like oxidized LDL receptor-1 in vascular endothelial cells. Circ Res 83:322–327View ArticlePubMedGoogle Scholar