Serine protease activity in developmental stages of Eimeria tenella

A number of complex processes are involved in Eimeria spp. survival, including control of sporulation, intracellular invasion, evasion of host immune responses, successful reproduction, and nutrition. Proteases have been implicated in many of these processes, but the occurrence and functions of serine proteases have not been characterized. Bioinformatic analysis suggests that the Eimeria tenella genome contains several serine proteases that lack homology to trypsin. Using RT-PCR, a gene encoding a subtilisin-like and a rhomboid protease-like serine protease was shown to be developmentally regulated, both being poorly expressed in sporozoites (SZ) and merozoites (MZ). Casein substrate gel electrophoresis of oocyst extracts during sporulation demonstrated bands of proteolytic activity with relative molecular weights (Mr) of 18, 25, and 45 kDa that were eliminated by coincubation with serine protease inhibitors. A protease with Mr of 25 kDa was purified from extracts of unsporulated oocysts by a combination of affinity and anion exchange chromatography. Extracts of SZ contained only a single band of inhibitor-sensitive proteolytic activity at 25 kDa, while the pattern of proteases from extracts of MZ was similar to that of oocysts except for the occurrence of a 90 kDa protease, resistant to protease inhibitors. Excretory-secretory products (ESP) from MZ contained AEBSF (4-[2-Aminoethyl] benzenesulphonyl fluoride)-sensitive protease activity with a specific activity about 10 times greater than that observed in MZ extracts. No protease activity was observed in the ESP from SZ. Pretreatment of SZ with AEBSF significantly reduced SZ invasion and the release of the microneme protein, MIC2. The current results suggest that serine proteases are present in all the developmental stages examined.     

Hybridoma antibody characterization of stage-specific and stage-cross-reactive antigens of Eimeria tenella

Hybridoma antibodies (HAb) have been raised against the sporozoite stage of 3 species of avian coccidia. These HAb were utilized in Western blot analysis, resulting in the immunoenzymatic detection of sporozoite and merozoite antigens of 1 species, Eimeria tenella. The 5 HAb specific for the sporozoite stage showed either single bands at 22 and 28 kDa or a large diffuse band in the 7-10-kDa range. The 4 HAb that cross-reacted with both asexual stages recognized either a single sporozoite or merozoite antigen of 90 kDa, or multiple antigens (47-69 kDa) for both stages. The 9 HAb demonstrated 5 different immunofluorescent antibody (IFA) patterns, and the 4 cross-reactive HAb showed similar IFA patterns with both asexual stages of E. tenella. The sporozoite-specific HAb which identified the 22, 7-10, and 7-8 kDa antigens showed surface, surface-internal, or internal IFA patterns. The other sporozoite-specific HAb, which labeled the 28-kDa antigen, stained the refractile body. The IFA of the 4 stage-cross-reactive HAb, which recognized the 45-60-kDa and the 90- or 47-69-kDa antigens, localized these antigens to the surface and tip, respectively. Rabbit anti-sporozoite serum appeared to recognize all of the sporozoite and merozoite antigens identified by the HAb as well as a variety of additional stage-cross-reactive antigens.     

Proteins and antigens of merozoites and sporozoites of Eimeria bovis (Apicomplexa)

Proteins and antigens of first-generation merozoites and sporozoites of Eimeria bovis were examined using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and lactoperoxidase iodination procedures. SDS-PAGE gels revealed both common and unique protein bands in merozoite and sporozoite extracts, ranging in molecular weight (Mr) from 15,000 to 215,000. Nitrocellulose immunoblots of separated proteins, when probed with sera obtained from immunized calves, revealed numerous IgG-binding antigens of Mr 18,000 to 180,000 in merozoites and Mr 28,000 to approximately 118,000 in sporozoites. Although merozoite and sporozoite preparations each contained antigens of different molecular weights, 4 antigens had the same migratory distance in both preparations (Mr 58,000, 70,000, 83,000, 98,000). Of 3 types of immune sera used to probe immunoblots, serum taken from a calf that had been inoculated with oocysts of E. bovis and boosted 10 wk later by subcutaneous injection with 2 X 10(7) live merozoites emulsified in Freund's complete adjuvant consistently identified and reacted more intensely with more antigens of merozoites and sporozoites than the other immune sera tested. Autoradiographic analysis of radioiodinated parasites revealed major surface proteins on merozoites of between 15,000 and 18,000 Mr and 3 surface proteins on sporozoites of Mr 28,000, 77,000, and 183,000. All but the 183,000 protein elicited an IgG antibody response in the host.     

Characterization of Eimeria tenella unsporulated oocyst-specific cDNA clones.

A cDNA library was constructed with poly(A)+ RNA from unsporulated oocysts of Eimeria tenella in pUC18. After screening, 4 cDNA clones that hybridized to RNA of unsporulated and sporulating oocysts but not to RNA of either sporulated oocysts or second generation merozoites were isolated and characterized. Each of the cDNA clones is unique. The loci for 2 of the clones are on E. tenella chromosome 7, the site of the third is located on chromosome 6 and the last clone hybridizes, for the most part, to chromosome 5 but also to other E. tenella chromosomes. The cognate RNAs for each of the cDNA clones show differential patterns of hybridization during oocyst sporulation with the levels of RNA being low at the start of sporulation (0 hr), increasing to peak levels between 6.5 and 23 hr after the onset of sporulation and, in each case, decreasing to low hybridization levels at 48 hr after initiation of sporulation. These results establish that specific mRNA levels are differentially regulated during sporulation.     

Molecular interactions of cultured turkey kidney cells with specific antigens of Eimeria adenoeides sporozoites

Earlier studies suggested that specific communication between the parasite and the host cell may play a role in cellular invasion by sporozoites of species of avian Eimeria. In this study, quantification of cellular invasion and modified Western blot analysis were used to explore the possibility that parasite receptors for interaction with the host cell might be involved in the sporozoite-host cell communication. Invasion in cultured cells treated with a homogenate of Eimeria adenoeides sporozoites was approximately 50% lower than that in untreated cultures. When the sporozoite homogenate was solubilized in sodium dodecyl sulfate and electrophoretically separated, components of the cultured host cells bound consistently to sporozoite bands having Mr of 23 and 40 kDa. Biotinylation of intact sporozoites revealed at least 14 biotin-labeled bands, including bands at 23 and 40 kDa, that were considered to be surface molecules. If the sporozoites were incubated in trypsin after they were biotinylated, only two biotinylated bands at 18 and 23 kDa remained; the 40-kDa biotinylated band was not detected. Despite the removal of the majority of the surface molecules, the cell homogenate still bound to the trypsin-treated sporozoites; the intensity of the label was similar to that resulting from the binding of cell homogenate to untreated sporozoites. The data show specific interactions between 23- and 40-kDa sporozoite bands and host cell components, and provide evidence that the 23-kDa molecule may be located on the sporozoite surface and the 40-kDa molecule located intracellularly.     

Changes of Nucleic Acids and Proteins in the Oocysts of Eimeria tenella during Sporulation

SYNOPSIS. The total content of DNA in Eimeria tenella , estimated at 5.8 × 10⁻¹² gm/oocyst, varies little during sporulation. Its buoyant density is 1.682 gm/cm³, reflecting a G + C content of ∼41%. Thymidine is not incorporated into any TCA insoluble fraction of sporulating oocysts, but radioactivity from [³H]uridine and [³H]deoxyuridine are incorporated into RNA at a linear rate during the first 5 hr of sporulation. The labeled RNA, found mainly in the paranuclear bodies of newly formed sporozoites, contains ∼0.15 nmole [³H]uridine/10⁶ oocysts at the completion of sporulation. One nmole of leucine is incorporated into the hot TCA insoluble fraction of 10⁶ oocysts during the first 7 hr of sporulation after an initial lag. The incorporated amino acid is mainly in the cytoplasm of the sporozoites, and an analysis by SDS-gel electrophoresis reveals most of the radioactivity in a narrow band with a molecular weight of ∼50,000 daltons. Incorporation of uridine and leucine, however, can be totally suppressed by respiratory inhibition. Further analysis of the proteins in the oocysts reveals that the total protein content remains relatively unchanged at 2.64 × 10⁻¹⁶ gm/oocyst during sporulation, but there is a shift of 13–14% of total protein from the soluble cytoplasm to the 15,000 g pellets. By polyacrylamide gel electrophoresis, a major protein band. possibly a glycoprotein, is shown in the soluble cytoplasm of unsporulated oocysts. This band disappears during sporulation.     

Isolation of amylopectin granules and identification of amylopectin phosphorylase in the oocysts of Eimeria tenella.

Amylopectin granules were purified from Eimeria tenella oocysts following digestion with sodium dodecyl sulfate and pronase. The oval granules had a uniform size of 0.5 X 0.7 mum, and consisted of only glucose polymers. alpha-Amylase treatment yielded 235 nmoles of maltose from the granules from 10(6) unsporulated oocysts and 93 nmoles maltose from those from 10(6) sporulated oocysts. Amylopectin phosphorylase activity was detected in the cytoplasm of unsporulated oocysts of E. tenella. It had a specific activity of 13 U/mg protein in crude extracts, and a pH optimum of 6.0. The Km values determined were 9.1 mM for glucose-1-phosphate and 5.6 mM for glucose end groups in potato amylopectin. Enzyme activity declined at a linear rate during sporulation, sporulated oocysts containing less than 8% of the activity of unsporulated oocysts. No amylase-type activity was found in the parasite.     

Identification and partial purification of a lectin on the surface of the sporozoite of Cryptosporidium parvum.

A human-derived isolate of Cryptosporidium parvum from a symptomatic patient with the acquired immunodeficiency syndrome was expanded in vivo by infecting a neonatal calf with 10(8) oocysts. Sporozoites were isolated from 4 x 10(10) oocysts harvested from this single infection, and the characteristics of mixed hemagglutination (HA) with rabbit erythrocytes were determined. Sporozoite HA was inhibited by bovine submaxillary mucin (BSM), hog gastric mucin, and orosomucoid, but not by simple sugars, including sialic acid. Carbohydrate-inhibitable HA (lectin) activity increased with sporozoite lysis and was associated with the sporozoite membrane fractions. The ability of intact sporozoites to form rosettes around erythrocytes indicates that the HA (lectin) is, at least in part, present on the parasite surface. Hemagglutination (lectin) activity was partially purified from sporozoite lysates by affinity chromatography with BSM coupled to Sepharose-4B. Best elution was obtained with ethylene glycol and NaCl, which resulted in enrichment of 6 bands compared to the crude starting lysate (Mr = 60, 24, 22, 20, and 15 kDa and a 40-kDa doublet). Our results indicate that an HA (lectin) activity is present on the surface of intact sporozoites where it could play a role in cell-to-cell interactions with eukaryotic targets.     

Detection of Cryptosporidium parvum oocysts by dot-blotting using monoclonal antibodies to Cryptosporidium parvum virus 40-kDa capsid protein

Monoclonal antibodies (MAb) were prepared against the 40-kDa capsid protein of Cryptosporidium parvum virus (CPV) by immunizing mice with purified recombinant CPV40 protein. In immunoblotting analysis, MAbCPV40-1 bound to a 40-kDa protein in extracts of C. parvum oocysts. This 40-kDa protein was localized in the sporozoite cytoplasm by immunofluorescence (IFA) staining with MAbCPV40-1. In a dot-blot assay, MAbCPV40-1 was capable of detecting 10(2) non-bleach-treated and 10(2)-10(3) bleach-treated C. parvum oocysts. MAbCPV40-1 was capable of detecting CPV40 antigen in both soluble and total C. parvum oocyst protein extracts, indicating a potential use for detecting this parasite in environmental samples.     

Eimeria tenella: hsp70 expression during sporogony.

There is an increasing interest in identifying the parasite components involved in the maturation, development, and infectivity of intracellular protozoan parasites. In the present study, a heat shock protein (hsp) of the family of 70 kDa hsp (hsp70), which play important roles in the stage conversion and virulence of these parasites, was examined. Whereas hsp70 expression has been examined in Eimeria tenella within host tissues, in the present study, oocysts of E. tenella were used to investigate the expression of hsp70 during sporulation without interference from the host; hsp70 expression during excystation was induced by incubating sporulated oocysts under various experimental conditions to produce the stimuli necessary for sporozoites to become active and to excyst in vitro. Hsp70 was detected by immunohistochemical techniques; quantitative flow cytometric analysis was also been carried out using specific monoclonal antibodies against hsp70. Hsp70 was expressed during sporulation but was not found in sporulated oocysts after the completion of sporulation. Oocysts re-expressed hsp70 when excystation was induced. The presence of hsp70 prior to infection may preadapt the parasite for additional stress in the host and may be involved in the formation of sporozoites.     

Isolation of Amylopectin Granules and Identification of Amylopectin Phosphorylase in the Oocysts of Eimeria tenella

SYNOPSIS. Amylopectin granules were purified from Eimeria tenella oocysts following digestion with sodium dodecyl sulfate and pronase. The oval granules had a uniform size of 0.5 × 0.7 μm, and consisted of only glucose polymers. α-Amylase treatment yielded 235 nmoles of maltose from the granules from 10⁶ unsporulated oocysts and 93 nmoles maltose from those from 10⁶ sporulated oocysts.
Amylopectin phosphorylase activity was detected in the cytoplasm of unsporulated oocysts of E. tenella. It had a specific activity of 13 U/mg protein in crude extracts, and a pH optimum of 6.0. The K m values determined were 9.1 mM for glucose-1-phosphate and 5.6 mM for glucose end groups in potato amylopectin. Enzyme activity declined at a linear rate during sporulation, sporulated oocysts containing less than 8% of the activity of unsporulated oocysts. No amylase-type activity was found in the parasite.     

Identification and characterization of Eimeria tenella apical membrane antigen-1 (AMA1)

Apical membrane antigen-1 (AMA1) is a micronemal protein of apicomplexan parasites that appears to be essential during the invasion of host cells. In this study, a full-length cDNA of AMA1 was identified from Eimeria tenella (Et) using expressed sequence tag and the rapid amplification of cDNA ends technique. EtAMA1 had an open reading frame of 1608 bp encoding a protein of 535 amino acids. Quantitative real-time PCR analysis revealed that EtAMA1 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second-generation merozoites). The ectodomain sequence was expressed as recombinant EtAMA1 (rEtAMA1) and rabbit polyclonal antibodies raised against the rEtAMA1 recognized a 58-kDa native parasite protein by Western Blotting and had a potent inhibitory effect on parasite invasion, decreasing it by approximately 70%. Immunofluorescence analysis and immunohistochemistry analysis showed EtAMA1 might play an important role in sporozoite invasion and development.     

Plasmodium vivax:A Monoclonal Antibody Recognizes a Circumsporozoite Protein Precursor on the Sporozoite Surface

Gonzalez-Ceron, L., Rodriguez, M. H., Wirtz, R. A., Sina, B. J., Palomeque, O. L., Nettel, J. A., and Tsutsumi, V. 1998.Plasmodium vivax:A monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.Experimental Parasitology90, 203–211. The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells.Plasmodium vivaxCS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with otherPlasmodiumspecies, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with allP. vivaxsporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.     

Biosynthesis and catabolism of mannitol is developmentally regulated in the protozoan parasite Eimeria tenella

The mannitol cycle is a metabolic branch of the glycolytic pathway found in Eimeria tenella. In this paper, we describe the biosynthesis and consumption of mannitol during parasite development. Low micromolar levels of mannitol were detected in all of the asexual stages and mannitol production increased sharply during the sexual phase of the life cycle. Unsporulated oocysts had high mannitol content (300 mM or 25% of the oocyst mass). Mannitol-1-phosphate dehydrogenase (M1PDH), the first committed step of the mannitol cycle, was also elevated in sexual stages and this coincides with mannitol levels. Approximately 90% of the mannitol present in unsporulated oocysts was consumed in the first 15 hr of sporulation, and levels continued to drop until the sporulation process was complete at approximately 35 hr. Thus, mannitol appears to be the "fuel" for sporulation during the vegetative stage of the parasite life cycle. Evaluation of oocyst extracts from 6 additional Eimeria species for mannitol content and the presence of M1PDH indicated that the mannitol cycle was broadly present in this genus. This finding combined with the lack of mannitol metabolism in higher eukaryotes makes this pathway an attractive chemotherapeutic target.     

Identification and characterization of a serpin from Eimeria acervulina

Serpins are serine protease inhibitors that are widely distributed in metazoans but have not been previously characterized in Eimeria spp. A serpin from Eimeria acervulina was cloned, expressed and characterized. Random screening of an E.acervulina sporozoite cDNA library identified a single clone (D14) whose coding region shared high similarity to consensus structure of serpins. Clone D14 contained an entire open reading frame (ORF) consisting of 1,245 nts that encode a peptide 413 amino acids in length with a predicted molecular weight of 45.5 kDa and containing a signal peptide 28 residues in length. By Western blot analysis, polyclonal antiserum to the recombinant serpin (rbSp) recognized a major 55 kDa protein band in unsporulated oocysts and in oocysts sporulated up to 24 hr (fully sporulated). The anti-rbSp detected bands of 55 kDa and 48 kDa in sporozoites (SZ) and merozoites (MZ) respectively. Analysis of MZ secretion products revealed a single protein of 48 kDa which may correspond to secreted serpin. By immuno-staining the serpin was located in granules distributed throughout both the SZ and MZ but granules appeared to be concentrated in the parasite's anterior. Analysis of the structure predicts that the E. acervulina serpin should be an active inhibitor. However, rbSp was without inhibitory activity against common serine proteases. By Western blot analysis the endogenous serpin in MZ extracts did not form the expected high molecular weight complex when coincubated with either trypsin or subtilisin. The results demonstrate that E. acervulina contains a serpin gene and expresses a protein with structural properties similar to an active serine protease inhibitor. Although the function of the E. acervulina serpin remains unknown the results further suggest that serpin is secreted by the parasite where it may be involved in cell invasion and other basic developmental processes.