Eimeria tenella pyrroline -5-carboxylate reductase is a secreted protein and involved in host cell invasion.

Chicken coccidiosis, which caused by Eimeria spp, is a parasitic protozoal disease. At present, control measures of this disease depend mainly on anticoccidial drugs and live vaccines. But these control strategies have drawbacks such as drug resistance and limitations in live vaccines production. Therefore, novel control approaches are urgently need to study to control this disease effectively. In this study, the function and characteristics of the pyrroline-5-carboxylate reductase of Eimeria tenella (EtPYCR) protein were preliminary analyzed. The transcription and translation level were analyzed by using qPCR and Western blot. The results showed that the mRNA transcription and translation levels of EtPYCR were higher in unsporulated oocysts (UO) and second generation merozoites (Mrz) than that in sporulated oocysts (SO) and sporozoites. Enzyme activity showed that the enzyme activity of EtPYCR was also higher in the UO and Mrz than that in the SO and sporozoites. Immunofluorescence localization showed EtPYCR was mainly located on the top of sporozoites and the whole cytoplasm and surface of Mrz. The secretion assay indicated that EtPYCR was secretion protein, but not from micronemes. Invasion inhibition assay showed that rabbit anti-rEtPYCR polyclonal antibodies can effectively inhibit sporozoite invasion of DF-1 cells. These results showed that EtPYCR possess several important roles that separate and distinct from its conversion 1-pyrroline-5-carboxylate (P5C) into proline and maybe involved in the host cell invasion and development of parasites in host cells.

Proteomic Analysis of Fractionated Eimeria tenella Sporulated Oocysts Reveals Involvement in Oocyst Wall Formation.

Eimeria tenella is the most pathogenic intracellular protozoan parasite of the Eimeria species. Eimeria oocyst wall biogenesis appears to play a central role in oocyst transmission. Proteome profiling offers insights into the mechanisms governing the molecular basis of oocyst wall formation and identifies targets for blocking parasite transmission. Tandem mass tags (TMT)-labeled quantitative proteomics was used to analyze the oocyst wall and sporocysts of E. tenella. A combined total of 2865 E. tenella proteins were identified in the oocyst wall and sporocyst fractions; among these, 401 DEPs were identified, of which 211 were upregulated and 190 were downregulated. The 211 up-regulated DEPs were involved in various biological processes, including DNA replication, fatty acid metabolism and biosynthesis, glutathione metabolism, and propanoate metabolism. Among these proteins, several are of interest for their likely role in oocyst wall formation, including two tyrosine-rich gametocyte proteins (EtGAM56, EtSWP1) and two cysteine-rich proteins (EtOWP2, EtOWP6). Concurrently, 96 uncharacterized proteins may also participate in oocyst wall formation. The present study significantly expands our knowledge of the proteome of the oocyst wall of E. tenella, thereby providing a theoretical basis for further understanding of the biosynthesis and resilience of the E. tenella oocyst wall.

Molecular characterization and immune protection by cystathionine ß-synthase from Eimeria tenella.

Eimeria tenella is an obligate intracellular apicomplexan parasite that causes avian coccidiosis and leads to severe economic losses in the global poultry industry. Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CGL) act together to generate H2S in the reverse transsulfuration pathway. In this study, E. tenella Cystathionine ß-synthase (EtCBS) was cloned using rapid amplification of cDNA 5'-ends (5'RACE) and characterized, and its immunoprotective effects were evaluated. The recombinant EtCBS protein (rEtCBS) was expressed and successfully recognized by anti-sporozoites (Spz) protein rabbit serum. EtCBS mRNA levels were highest in Spz by qPCR, and the protein expression levels were higher in unsporulated oocysts (UO) than in other stages by Western blot. Indirect immunofluorescence showed that EtCBS protein was found on the surface of Spz and second-generation merozoites (Mrz). The invasion inhibition assays showed that rabbit anti-rEtCBS polyclonal antibodies effectively inhibited parasite invasion host cells. Chickens immunized with rEtCBS protein showed prominently increased weight gains and decreased oocyst output compared to nonimmunized and infected control group. The results suggest that EtCBS could be a potential vaccine candidate against E. tenella.

Molecular characterization of glyceraldehyde-3-phosphate dehydrogenase from Eimeria tenella.

Chicken coccidiosis is an extremely common and lethally epidemic disease caused by Eimeria spp. The control measures of coccidiosis depend mainly on drugs. However, the ensuing drug resistance problem has brought considerable economic loss to the poultry industry. In our previous study, comparative transcriptome analyses of a drug-sensitive (DS) strain and two drug-resistant strains (diclazuril-resistant (DZR) and maduramicin-resistant (MRR) strains) of Eimeria tenella were carried out by transcriptome sequencing. The expression of glyceraldehyde-3-phosphate dehydrogenase of E. tenella (EtGAPDH) was upregulated in the two resistant strains. In this study, we cloned and characterized EtGAPDH. Indirect immunofluorescence localization was used to observe the distribution of EtGAPDH in E. tenella. The results showed that the protein was distributed mainly on the surface of sporozoites and merozoites, and in the cytoplasm of merozoites. qPCR was performed to detect the transcription level of EtGAPDH in the different developmental stages of the E. tenella DS strain. The transcription level of EtGAPDH was significantly higher in second-generation merozoites than in the other three stages. The transcription level of EtGAPDH in the different drug-resistant strains and DS strain of E. tenella was also analyzed by qPCR. The results showed that the transcription level was significantly higher in the two drug-resistant strains (MRR and DZR) than in the DS strain. As the concentration of diclazuril and maduramicin increased, the transcription levels also increased. Western blot results showed that EtGAPDH protein was upregulated in the DZR and MRR strains. Enzyme activity showed that the enzyme activity of EtGAPDH was higher in the two resistant strains than in the DS strain. These results showed that EtGAPDH possess several roles that separate and distinct from its glycolytic function and maybe involved in the development of E. tenella resistance to anticoccidial drugs.

Development of Novel Immunoprophylactic Agents against Multidrug-Resistant Gram-Negative Bacterial Infections.

The widespread emergence of antibiotic resistance, including multidrug resistance in Gram-negative (G-) bacterial pathogens, poses a critical challenge to the current antimicrobial armamentarium. Antibody-drug conjugates (ADCs), primarily used in anticancer therapy, offer a promising treatment alternative due to their ability to deliver a therapeutic molecule while simultaneously activating the host immune response. The Cloudbreak platform is being used to develop ADCs to treat infectious diseases, composed of a therapeutic targeting moiety (TM) attached via a noncleavable linker to an effector moiety (EM) to treat infectious diseases. In this proof-of-concept study, 21 novel dimeric peptidic molecules (TMs) were evaluated for activity against a screening panel of G- pathogens. The activities of the TMs were not impacted by existing drug resistance. Potent TMs were conjugated to the Fc fragment of human IgG1 (EM), resulting in 4 novel ADCs. These ADCs were evaluated for immunoprophylactic efficacy in a neutropenic mouse model of deep thigh infection. In colistin-sensitive infections, 3 of the 4 ADCs offered protection similar to that of therapeutically dosed colistin, while CTC-171 offered enhanced protection. The efficacy of these ADCs was unchanged in colistin-resistant infections. Together, these results indicate that the ADCs used here are capable of potent binding to G- pathogens regardless of lipopolysaccharide (LPS) modifications that otherwise lead to antibiotic resistance and support further exploration of ADCs in the treatment of infections caused by drug-resistant G- bacteria.

Identification and Characterization of a Novel Apical Membrane Antigen 3 in Eimeria tenella.

Eimeria tenella is an obligate intracellular parasite in the phylum Apicomplexa. As described for other members of Apicomplexa, apical membrane antigen 1 (AMA1) has been shown to be critical for sporozoite invasion of host cells by E. tenella. Recently, an E. tenella paralogue of AMA1 (EtAMA1), dubbed sporoAMA1 (EtAMA3), was identified in proteomic and transcriptomic analyses of E. tenella, but not further characterized. Here, we show that EtAMA3 is a type I integral membrane protein that has 24% -38% identity with other EtAMAs. EtAMA3 has the same pattern of Cys residues in domains I and II of AMA1 orthologs from apicomplexan parasites, but high variance in domain III, with all six invariant Cys residues absent. EtAMA3 expression was developmentally regulated at the mRNA and protein levels. EtAMA3 protein was detected in sporulated oocysts and sporozoites, but not in the unsporulated oocysts or second-generation merozoites. EtAMA3 is secreted by micronemes and is primarily localized to the apical end of sporozoites during host-cell invasion. Additionally, pretreatment of sporozoites with rEtAMA3-specific antibodies substantially impeded their invasion into host cells. These results suggest EtAMA3 is a sporozoite-specific protein that is involved in host-cell sporozoite invasion.

Molecular characterization and functional analysis of Eimeria tenella citrate synthase.

Chicken coccidiosis, caused by an obligate intracellular protozoan parasite of the genus Eimeria, is a major parasitic disease in the intensively reared poultry industry. Due to the widespread use of anticoccidial drugs, resistance has become an inevitable problem. In our previous study, Eimeria tenella citrate synthase (EtCS) was found to be up-expressed in two drug-resistant strains (diclazuril-resistant and maduramycin-resistant strains) compared to drug-sensitive strain by RNA sequence. In this study, we cloned and expressed EtCS and obtain its polyclonal antibodies. Quantitative real-time polymerase chain (qPCR) reactions and Western blots were used to analyze the transcription and translation levels of EtCS in sensitive and three drug-resistant strains. Compared with the sensitive strain, the transcription of EtCS was both significantly upregulated in diclazuril-resistant and maduramycin-resistant strains, but was not significantly different in salinomycin-resistant strain. No significant difference was seen in translation level in the three drug-resistant strains. Indirect immunofluorescence indicated that EtCS was mainly located in the cytoplasm of sporozoites except for posterior refractile bodies and in the cytoplasm and surface of merozoites. Anti-rEtCS antibody has inhibitory effects on E. tenella sporozoite invasion of DF-1 cells and the inhibition rate is more than 83%. Binding of the protein to chicken macrophage (HD11) cells was confirmed by immunofluorescence assays. When macrophages were treated with rEtCS, secretion of nitric oxide and cell proliferation of the macrophages were substantially reduced. These results showed that EtCS may be related to host cell invasion of E. tenella and involve in the development of E.tenella resistance to some drugs.

Comparative Transcriptome Analyses of Drug-sensitive and Drug-resistant Strains of Eimeria tenella by RNA-sequencing.

Avian coccidiosis is a widespread and economically significant disease in poultry. At present, treatment of coccidiosis mainly relies on drugs. Anticoccidial drugs can be divided into two categories: ionophorous compounds and synthetic drugs. However, the emergence of drug-resistant strains has become a challenge for coccidiosis control with anticoccidial drugs. To gain insights into the molecular mechanism governing the drug resistance of Eimeria tenella, two drug-resistant strains of E. tenella, one maduramicin-resistant (MRR) strain and one diclazuril-resistant (DZR) strain, were generated. We carried out comparative transcriptome analyses of a drug-sensitive strain (DS) and two drug-resistant MRR and DZR strains of E. tenella using RNA-sequencing. A total of 1,070 differentially expressed genes (DEGs), 672 upregulated and 398 downregulated, were identified in MRR vs. DS, and 379 DEGs, 330 upregulated and 49 downregulated, were detected in DZR vs. DS. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed to better understand the functions of these DEGs. In the comparison of DZR vs. DS, some DEGs were involved in peroxisome, biosynthesis of unsaturated fatty acids, and fatty acid metabolism. In the comparison of MRR vs. DS, some DEGs were involved in glycolysis/gluconeogenesis, regulation of actin cytoskeleton, and DNA replication. In addition, some DEGs coded for surface antigens that were downregulated in two drug-resistant strains involved invasion, pathogenesis, and host-parasite interactions. These results provided suggestions for further research toward unraveling the molecular mechanisms of drug resistance in Eimeria species and contribute to developing rapid molecular methods to detect resistance to these drugs in Eimeria species in poultry.

Molecular characterization of serine/threonine protein phosphatase of Eimeria tenella.

Avian coccidiosis is a widespread and economically significant poultry disease caused by several Eimeria species, including Eimeria tenella. Previously, E. tenella serine/threonine protein phosphatase (EtSTP) was found to be differentially expressed in drug-sensitive (DS) and drug-resistant strains using RNA-seq. In the present study, we found that transcription and translation levels of EtSTP were higher in diclazuril-resistant (DZR) strains and maduramicin-resistant (MRR) strains than in DS strains using quantitative real-time PCR (qPCR) and Western blotting. Enzyme activity results indicated that the catalytic activity of EtSTP was higher in the two drug-resistant strains than in DS strains. Western blot and qPCR analysis also showed that expression levels of EtSTP were higher in unsporulated oocysts (UO) and second-generation merozoites (SM). Indirect immunofluorescence localization showed that EtSTP was located in most areas of the parasite with the exception of refractile bodies, and fluorescence intensity was enhanced during development. In vitro inhibition experiments showed that the ability of sporozoites (SZ) to invade cells was significantly decreased after treatment with anti-rEtSTP antibody. These results indicated that EtSTP acted mainly during the developmental and reproductive stages of the parasite and may be related to the resistance of coccidia to external drug pressure.

Molecular characterization of 60S ribosomal protein L12 of E. tenella.

This study analyzed the large-subunit (60S) ribosomal protein L12 of Eimeria tenella (Et60s-RPL12). A full-length cDNA was cloned, and the recombinant protein was expressed in E. coli BL21 and inoculated in rabbits to produce the polyclonal antibody. Quantitative real-time polymerase chain reaction and western blotting were used to analyze the transcription levels of Et60s-RPL12 and translation levels in different developmental stages of E. tenella. The results showed that the mRNA transcription level of Et60s-RPL12 was highest in second-generation merozoites, whereas the translation level was highest in unsporulated oocysts. Indirect immunofluorescence showed that Et60s-RPL12 was localized to the anterior region and surface of sporozoites, except for the two refractile bodies. As the invasion of DF-1 cells progressed, fluorescence intensity was increased, and Et60s-RPL12 was localized to the parasitophorous vacuole membrane (PVM). The secretion assay results using staurosporine indicated that this protein was secreted, but not from micronemes. The role of Et60s-RPL12 in invasion was evaluated in vitro. The results of the invasion assay showed that polyclonal antibody inhibited host cell invasion by the parasite, which reached about 12%. However, the rate of invasion was not correlated with the concentration of IgG.

Molecular characterization and immune protection of an AN1-like zinc finger protein of Eimeria tenella.

Coccidiosis is caused by multiple species of the apicomplexan protozoa Eimeria. Among them, Eimeria tenella is frequently considered to be the most pathogenic. Zinc finger proteins (ZnFPs) are a type of protein containing zinc finger domains. In the present study, a putative Eimeria tenella AN1-like ZnFP (E. tenella AN1-like zinc finger domain-containing protein, putative partial mRNA, EtAN1-ZnFP) was cloned and characterized, and its immune protective effects were evaluated. The 798-bp ORF sequence of EtAN1-ZnFP that encoded a protein of approximately 27.0 kDa was obtained. The recombinant EtAN1-ZnFP protein (rEtAN1-ZnFP) was expressed in Escherichia coli. Western blot analysis showed that the recombinant protein was recognized by the anti-GST monoclonal antibody and anti-sporozoite protein rabbit serum. qPCR analysis revealed that EtAN1-ZnFP was highly expressed in unsporulated oocysts and sporozoites. Immunostaining with an anti-rEtAN1-ZnFP antibody indicated that EtAN1-ZnFP was uniformly distributed in the cytoplasm of sporozoites, except for the refractive body; furthermore, this protein was evenly distributed in the cytoplasm of immature schizonts but seldom distributed in mature schizonts. The results of the in vitro invasion inhibition assay indicated that the antibodies against rEtAN1-ZnFP efficiently reduced the ability of E. tenella sporozoites to invade host cells. Animal challenge experiments demonstrated that the chickens immunized with rEtAN1-ZnFP protein significantly decreased mean lesion scores and fecal oocyst output compared with challenged control group. The results suggest that EtAN1-ZnFP can induce partial immune protection against infection with E. tenella and could be an effective candidate for the development of new vaccines.

Preliminary study of the mechanism of action of ethanamizuril against Eimeria tenella.

Ethanamizuril (EZL) is a novel triazine compound with excellent anticoccidial activity. We carried out a preliminary investigation of the effects of EZL on the different life cycle stages of Eimeria tenella. EZL mainly acted on the schizogony stage, with peak activity during the second-generation merozoite stage. We also studied the possible target of EZL by identifying the majorly differentially expressed gene affected by EZL in second-generation merozoites using real-time polymerase chain reaction, and screening for surface antigen proteins (SAGs). The relative expression levels of SAGs were compared by Western blot analysis showing that expression levels of surface antigen family member (SAGfm) and SAG19 were significantly downregulated by EZL. Immunofluorescence analysis indicated that SAGfm and SAG19 were localized on the surface of second-generation merozoites. In addition, fluorescence signals were significantly stronger in second-generation merozoites of infected non-medicated control (INC) group compared with that of the EZL group. Therefore, it was speculated that SAGs might be a potential target of EZL action. The inhibitory effects of anticoccidial drugs on SAG levels in coccidia thus warrant further research.

Molecular characterization of surface antigen 10 of Eimeria tenella.

Chicken coccidiosis is caused by the apicomplexan parasite Eimeria spp. At present, drug resistance of Eimeria is common because of the indiscriminate use of anticoccidial drugs. The gene encoding surface antigen 10 of Eimeria tenella (EtSAG10) is differentially expressed between drug-resistant and drug-sensitive strains. RNA-seq analysis indicated that this gene was downregulated in strains resistant to maduramicin and diclazuril compared to susceptible strains. EtSAG10 DNA sequence alignment revealed that they contained one and ten mutations in MRR and DZR, compared with DS, respectively. A full-length EtSAG10 cDNA was successfully cloned and expressed, and the polyclonal antibody was prepared. The transcription and translation levels of EtSAG10 were analyzed by quantitative real-time PCR (qPCR) and Western blotting. The localization of EtSAG10 in Spz, Mrz, and parasites in the first asexual stage was determined by indirect immunofluorescence. The potential association of EtSAG10 with sporozoite invasion of host cells was assessed by invasion inhibition assays. The results showed that EtSAG10 had a predicted transmembrane domain at the C-terminal end and a predicted signal peptide at the N-terminal end. EtSAG10 was downregulated in drug-resistant strains, which is consistent with the RNA-seq results. The EtSAG10 protein was localized to the parasite surface and parasitophorous vacuole membrane. This protein was shown to play a role in the infection of chicken intestine by sporozoites.

Effects of host fatty acid-binding protein 4 on Eimeria tenella sporozoites invasion of cells.

In our previous study, proteomics analyses of host cells infected with Eimeria tenella sporozoites coupled with isobaric tags for relative and absolute quantitation, identified several host proteins related to Eimeria invasion. In this study, A 458-bp Gallus gallus fatty acid-binding protein 4 (FABP4) gene was cloned and subcloned to pET-28c(+) vector to construct the prokaryotic recombinant expression plasmid pET-28c(+)-FABP4. The 18.5 kDa recombinant FABP4 protein (rFABP4) was expressed and identified by western blotting. Expression of FABP4 in E. tenella sporozoite-infected DF-1 cells was downregulated significantly than in non-infected cells detected by western blotting and immunohistochemistry. The antibody inhibition assay showed that antibodies against FABP4 at 50, 100, 200, 300, and 400 µg/mL had no significant effect on sporozoite invasion. BMS-309403 and transforming growth factor-ß3 (TGF-ß3) was used to inhibit and improve the expression of FABP4 in DF-1 cells, respectively, and their effect on the sporozoite invasion of cells was detected by flow cytometry. Sporozoite invasion rate in the BMS-309403-treated group was not significantly affected; however, the invasion rate in the TGF-ß3-treated group declined significantly. These results show that host FABP4 plays a negative role in Eimeria invasion. However, further studies are needed to elucidate the exact mechanism of how FABP4 negatively regulates Eimeria invasion.

Molecular characterization and functional analysis of Eimeria tenella malate dehydrogenase.

Eimeria tenella is a serious intracellular parasite that actively invades cecal epithelial cells of chickens. The widespread use of drugs causes severe resistance to Eimeria tenella. We detected that malate dehydrogenase (MDH), one of the differentially expressed genes, was upregulated in diclazuril-resistant and maduramicin-resistant strains through transcriptome sequencing. In this study, we cloned and expressed MDH of E. tenella (EtMDH). Quantitative real-time polymerase chain reactions (qPCR) and Western blots were used to analyze the expression of EtMDH in resistant and sensitive strains, indicating EtMDH was upregulated in two resistant strains at the messenger RNA and protein levels. Enzyme activity was tested through absorbance measurement and the EtMDH activity increased in two resistant strains. Expression levels of EtMDH in four developmental stages of E. tenella were tested through qPCR and Western blot. Invasion inhibition assays explored if EtMDH was involved in invasion of DF-1 cells by E. tenella sporozoites. Indirect immunofluorescence assays investigated EtMDH distribution during parasite development in DF-1 cells invaded by E. tenella sporozoites. Experimental results showed that EtMDH may be related to drug resistance of E. tenella during its development and invasion. EtMDH may be an effective molecular marker for detection of E. tenella drug resistance.

Characterization and expression analysis of a new small heat shock protein Hsp20.4 from Eimeria tenella.

Small heat shock proteins (sHsps) are ubiquitous and diverse molecular chaperones. Found in almost all organisms, they regulate protein refolding and protect cells from stress. Until now, no sHsp has been characterized in Eimeria tenella. In this study, the novel EtsHsp20.4 gene was cloned from E. tenella by rapid amplification of cDNA ends based on a previously identified expressed sequence tag. The full-length cDNA was 1019bp in length and contained an open reading frame of 558bp that encoded a 185-amino acid polypeptide with a calculated molecular weight of 20.4 kDa. The EtsHsp20.4 protein contained a distinct HSP20/alpha-crystallin domain that is the key determinant of their function as molecular chaperones and belongs to the HSP20 protein family. EtsHsp20.4 mRNA levels were higher in sporulated oocysts than in sporozoites or second-generation merozoites by real-time quantitative PCR, the transcription of EtsHsp20.4 was barely detectable in unsporulated oocysts. Immunolocalization with EtsHsp20.4 antibody showed that EtsHsp20.4 was mainly located on the surface of sporozoites, first-generation merozoites and second-generation merozoites. Following the development of parasites in DF-1 cells, EtsHsp20.4 protein was uniformly dispersed in trophozoites, immature schizonts, and mature schizonts. Malate dehydrogenase thermal aggregation assays indicated that recombinant EtsHsp20.4 had molecular chaperone activity in vitro. These results suggested that EtsHsp20.4 might be involved in sporulation in external environments and intracellular growth of the parasite in the host.

Screening and characterization of apical membrane antigen 1 interacting proteins in Eimeria tenella.

Avian coccidiosis is a widespread and economically significant disease of poultry. It is an enteric disease caused by several protozoan Eimeria species. Eimeria belongs to the phylum Apicomplexa, which exhibits an unusual mechanism of host cell invasion. During invasion of host cells, the protein apical membrane antigen 1 (AMA1) is essential for invasion of Toxoplasma gondii and Plasmodium. Contrary to the roles of AMA1 during host cell invasion in T. gondii and Plasmodium, the precise functions of Eimeria AMA1 (EtAMA1) are unclear. In order to study the functions of EtAMA1, a yeast two-hybrid cDNA library was constructed from E. tenella sporozoites. The EtAMA1 ectodomain was cloned into the pGBKT7 vector to construct the bait plasmid pGBKT7- EtAMA1. Autoactivation and toxicity of the bait protein in yeast cells were tested by comparison with the pGBKT7 empty vector. Expression of the bait protein was detected by western blots. The bait plasmid pGBKT7-EtAMA1 was used to screen yeast two-hybrid cDNA library from E. tenella sporozoites. After multiple screenings with high-screening-rate medium and exclusion of false-positive plasmids, positive preys were sequenced and analyzed using BLAST. We obtained 14 putative EtAMA1-interacting proteins including E. tenella acidic microneme protein2 (EtMIC2), E. tenella putative cystathionine beta-synthase, E. tenella Eimeria-specific protein, four E. tenella conserved hypothetical proteins (one in the serine/threonine protein kinase family) and seven unknown proteins. Gene Ontology analysis indicated that two known proteins were associated with metabolic process, pyridoxal phosphate binding and protein phosphorylation. Functional analysis indicated EtMIC2 was implicated in parasite motility, migration, recognition and invasion of host cells. The data suggested that EtAMA1 may be important during host cell invasion, but also involved in other biological processes.

Molecular characterization and functional analysis of subunit 7 of eukaryotic initiation factor 3 from Eimeria tenella.

The initiation of translation in eukaryotic cells is stimulated by proteins known as initiation factors (eIFs). A structurally complex eIF composed of multiple subunits, eIF3 has been shown to have various functions in translation in a variety of eukaryotes. Until now, little is known about eIF3 in Eimeria tenella. Based on a previously identified expressed sequence tag(EST), we cloned the eIF3 subunit 7 gene (EteIF3s7) from E. tenella by rapid amplification of the cDNA ends(RACE). The 2278-bp full-length complementary DNA of EteIF3s7 contained a 1716-bp open reading frame (ORF) that encoded a 571-amino acid (aa) polypeptide. The EteIF3s7 protein contained the subunit 7 domain that is characteristic of members of the eIF3 zeta superfamily. The levels of EteIF3s7 messenger RNA and protein were higher in second generation merozoites than in sporulated oocysts, unsporulated oocysts, or sporozoites, and the EteIF3s7 protein was barely detectable in unsporulated oocysts. Our immunofluorescence analysis showed that the EteIF3s7 protein was uniformly distributed throughout the cytoplasm of sporozoites. After sporozoites were incubated in complete medium, the EteIF3s7 protein localized to the anterior region of the parasite. Following the first schizogenous division, the protein was uniformly dispersed in trophozoites, immature schizonts, and mature schizonts, and the EteIF3s7 protein was observed to be closely associated with the parasitophorous vacuole membrane. An anti-rEteIF3s7 polyclonal antibody inhibited the ability of E. tenella to invade DF-1 cells, which suggested that EteIF3s7 might be involved in host cell invasion and required for the growth of the parasite in the host.

Mutations in the catalytic domain of human matrix metalloproteinase-1 (MMP-1) that allow for regulated activity through the use of Ca2+.

Conditionally active proteins regulated by a physiological parameter represent a potential new class of protein therapeutics. By systematically creating point mutations in the catalytic and linker domains of human MMP-1, we generated a protein library amenable to physiological parameter-based screening. Mutants screened for temperature-sensitive activity had mutations clustered at or near amino acids critical for metal binding. One mutant, GVSK (Gly(159) to Val, Ser(208) to Lys), contains mutations in regions of the catalytic domain involved in calcium and zinc binding. The in vitro activity of GVSK at 37 °C in high Ca(2+) (10 mm) was comparable with MMP-1 (wild type), but in low Ca(2+) (1 mm), there was an over 10-fold loss in activity despite having similar kinetic parameters. Activity decreased over 50% within 15 min and correlated with the degradation of the activated protein, suggesting that GVSK was unstable in low Ca(2+). Varying the concentration of Zn(2+) had no effect on GVSK activity in vitro. As compared with MMP-1, GVSK degraded soluble collagen I at the high but not the low Ca(2+) concentration. In vivo, MMP-1 and GVSK degraded collagen I when perfused in Zucker rat ventral skin and formed higher molecular weight complexes with α2-macroglobulin, an inhibitor of MMPs. In vitro and in vivo complex formation and subsequent enzyme inactivation occurred faster with GVSK, especially at the low Ca(2+) concentration. These data suggest that the activity of the human MMP-1 mutant GVSK can be regulated by Ca(2+) both in vitro and in vivo and may represent a novel approach to engineering matrix-remodeling enzymes for therapeutic applications.

Molecular cloning and characterization of lactate dehydrogenase gene from Eimeria tenella.

Lactate dehydrogenase (LDH) is a key enzyme in the glycolytic pathway and is crucial for parasite survival. In this study, we cloned and expressed the LDH of Eimeria tenella (EtLDH). Real-time polymerase chain reaction and Western blot analysis revealed that the expression of EtLDH was developmentally regulated at the messenger RNA (mRNA) and protein levels. EtLDH mRNA levels were higher in second-generation merozoites than in other developmental stages (unsporulated oocysts, sporulated oocysts, and sporozoites). EtLDH protein expression levels were most prominent in second-generation merozoites, moderately expressed in unsporulated oocysts and sporulated oocysts, and weakly detected in sporozoites. Immunostaining with anti-recombinant EtLDH (rEtLDH) antibody indicated that EtLDH was mainly located in the anterior region in free sporozoites and became concentrated in the anterior region of intracellular sporozoites except for the apex after invasion into DF-1 cells. Specific staining of EtLDH protein was more intense in trophozoites and immature first-generation schizonts, but decreased in mature first-generation schizonts. Inhibition of EtLDH function using specific antibodies cannot efficiently reduce the ability of E. tenella sporozoites to invade host cells. These results suggest that EtLDH may be involved in glycolysis during the first-generation merogony stage in E. tenella and has little role in host invasion.