Toxoplasma gondii glutathione S-transferase 2 plays an important role in partial secretory protein transport.

Toxoplasma gondii is an apicomplexan parasite, which has three unique secretory organelles: micronemes, rhoptries, and dense granules. Almost all the secreted proteins are transported through the endoplasmic reticulum (ER) and Golgi system to function in their respective destination by accurate targeting and packaging. Glutathione S-transferase (GST) is a supergene family enzyme that has multiple functions, which include regulation of cell proliferation and death signaling pathways, and participation in transportation and metabolism in mammal cells. However, the role of GST in Toxoplasma gondii has not been explained. In this study, we identified three GST proteins in T gondii, of which GST2 acts as a membrane protein that localizes to the Golgi-endosomal system and colocalizes with proteins involved in vesicle transport as well, including synaptobrevin, putative sortilin (VPS10), Rab5 and Rab6, which function as vesicle transport factors. Moreover, the loss of TgGST2 leads to Rab5 and Rab6 distribution of discrete puncta, and incorrect localization and decreased expression of several secretory proteins, and to significantly reduced invasion capacity and virulence to mice. Consistent with its relation to vesicle transport proteins, the distribution of TgGST2 relies on post-Golgi trafficking. Overall, our findings demonstrated that TgGST2 contributes to vesicle trafficking and plays a critical role in parasite lytic cycle.

Functional characterization of acyl-CoA binding protein in Neospora caninum.

BACKGROUND: Lipid metabolism is pivotal for the growth of apicomplexan parasites. Lipid synthesis requires bulk carbon skeleton acyl-CoAs, the transport of which depends on the acyl-CoA binding protein (ACBP). In Neospora caninum, the causative agent of neosporosis, the FASII pathway is required for growth and pathogenicity. However, little is known about the fatty acid transport mechanism in N. caninum. METHODS: We have identified a cytosolic acyl-CoA binding protein, with highly conserved amino acid residues and a typical acyl-CoA binding domain in N. caninum. The recombinant NcACBP protein was expressed to verify the binding activities of NcACBP in vitro, and the heterologous expression of NcACBP in Δacbp yeast in vivo. Lipid extraction from ΔNcACBP or the wild-type of N. caninum was analyzed by GC-MS or TLC. Furthermore, transcriptome analysis was performed to compare the gene expression in different strains. RESULTS: The NcACBP recombinant protein was able to specifically bind acyl-CoA esters in vitro. A yeast complementation assay showed that heterologous expression of NcACBP rescued the phenotypic defects in Δacbp yeast, indicating of the binding activity of NcACBP in vivo. The disruption of NcACBP did not perturb the parasite's growth but enhanced its pathogenicity in mice. The lipidomic analysis showed that disruption of NcACBP caused no obvious changes in the overall abundance and turnover of fatty acids while knockout resulted in the accumulation of triacylglycerol. Transcriptional analysis of ACBP-deficient parasites revealed differentially expressed genes involved in a wide range of biological processes such as lipid metabolism, posttranslational modification, and membrane biogenesis. CONCLUSIONS: Our study demonstrated that genetic ablation of NcACBP did not impair the survival and growth phenotype of N. caninum but enhanced its pathogenicity in mice. This deletion did not affect the overall fatty acid composition but modified the abundance of TAG. The loss of NcACBP resulted in global changes in the expression of multiple genes. This study provides a foundation for elucidating the molecular mechanism of lipid metabolism in N. caninum.

Triclosan inhibits the growth of Neospora caninum in vitro and in vivo.

Neospora caninum is an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide; thus, there is an urgent need to develop novel therapeutic agents to control the neosporosis. Enoyl acyl carrier protein reductase (ENR) is a key enzyme of the type II fatty acid synthesis pathway (FAS II), which is essential for apicomplexan parasite survival. The antimicrobial agent triclosan has been shown to be a very potent inhibitor of ENR. In this study, we identified an E. coli ENR-like protein in N. caninum. Multiple sequence alignment showed all the requisite features of ENR existed in this protein, so we named this protein NcENR. Swiss-Model analysis showed NcENR interacts with triclosan. We observed that ENR is localized in the apicoplast, a plastid-like organelle. Similar to the potent inhibition of triclosan on other apicomplexa parasites, this compound markedly inhibits the growth of N. caninum at low concentrations. Further research showed that triclosan attenuated the invasion ability and proliferation ability of N. caninum at low concentrations. The results from in vivo studies in the mouse showed that triclosan attenuated the virulence of N. caninum in mice mildly and reduced the parasite burden in the brain significantly. Taken together, triclosan inhibits the growth of N. caninum both in vitro and in vivo at low concentrations.

Melatonin promotes secondary hair follicle development of early postnatal cashmere goat and improves cashmere quantity and quality by enhancing antioxidant capacity and suppressing apoptosis.

Development of secondary hair follicles in early postnatal cashmere goats may be adversely affected by reactive oxygen species which cause oxidative stress. Because melatonin is a potent antioxidant and scavenger of free radicals, this study explored the effects of melatonin on secondary hair follicle development and subsequent cashmere production. It was found that the initiation of new secondary follicles in early postnatal Inner Mongolian cashmere goats of both melatonin-treated and control goats occurred in the first 10 weeks of age. Melatonin promoted the initiation and maturation of secondary follicles and increased their population. Importantly, the beneficial effect of melatonin on secondary follicle population remained throughout life. As a result, melatonin increased cashmere production and improved its quality in terms of reduced fiber diameter. The mechanisms underlying the role of melatonin on secondary follicle development included the enhancement of activities of antioxidant enzymes, for example, superoxide dismutase and glutathione peroxidase (GSH-Px), elevated total antioxidant capacity, and upregulated anti-apoptotic Bcl-2 expression and downregulated expression of the pro-apoptotic proteins, Bax and caspase-3. These results reveal that melatonin serves to promote secondary hair follicle development in early postnatal cashmere goats and expands our understanding of melatonin application in cashmere production. Melatonin treatment led to an increase in both the quantity and quality of cashmere fiber. This increased the textile value of the fibers and provided economic benefit.

An efficient rearing system rapidly producing large quantities of poultry red mites, Dermanyssus gallinae (Acari: Dermanyssidae), under laboratory conditions.

The poultry red mite, Dermanyssus gallinae, is one of the most economically deleterious ectoparasites affecting egg-laying hens in many parts of the world. New approaches to control D. gallinae often require the maintenance of colonies of D. gallinae under laboratory conditions. In the present study, we present an efficient rearing system for D. gallinae, consisting of a metal cage, a plastic storage box and a tray filled with water. Chicks were raised in the cage as host animals. A novel trap was developed to monitor the dynamic changes of mite populations, made with a plastic centrifuge tube and a disposable breathing mask with folds. Mite parameters were analyzed, including number of mites and eggs, survival and feeding rates, oviposition, hatchability and the proportion of D. gallinae at different life stages. The results show that the rearing system had a 53.5-fold increase in the number of mites over a period of six weeks after the introduction of mites. The survival rates of mites were above 94%, and the mean feeding rates ranged from 22.57% to 37.30%. The mean number of eggs per female ranged from 3.42 to 3.50, with the hatchability of eggs above 97%. Nymphs made up most of the population, ranging from 71.46% to 81.37%, while the population of larvae was minor and ranging from 7.54% to 13.04%. The mask trap used in this study was an effective and convenient device to shelter D. gallinae and monitor the dynamic changes of the mite population. The rearing system proved very effective in maintaining and reproducing colonies of D. gallinae, with great potential for the evaluation of the efficacy of vaccines or compounds against D. gallinae under laboratory conditions. It would be a useful tool for close observations in studies on the biology, acology and physiology of poultry red mites.

Toxoplasma gondii rhoptry protein38 (TgROP38) affects parasite invasion, egress, and induces IL-18 secretion during early infection.

Toxoplasma rhoptry protein 38 (TgROP38) is a new active kinase that modulates host cell signal transduction and TgROP38 expression shows strain-specificity and stage-specificity in different isolates. In the present study, we overexpressed ROP38 in the RH and prugniaud (PRU) strain (RH+rop38II and PRU+rop38II), disrupted ROP38 (PRUΔROP38) in the PRU strain, complemented the ROP38 (PRUΔROP38comp+) in the PRUΔROP38 strain, and compared phenotypes of gene-edited and parental strains. We found that knockout of ROP38 led to increased proliferation (P < 0.01) and invasion (P < 0.01) ability of the parasite. However, intraperitoneal infection with 1000 tachyzoites, PRUΔROP38 showed almost no virulent to mice compared with PRU (P < 0.01). Mice infected with low dose of PRU parasites produced higher levels of IL-18 and IL-1ß compared with those infected with the PRUΔROP38 parasites during early days (P < 0.01). IL-18 produced by the PRU-infected group was significantly higher than that of the PRUΔROP38-infected group in vitro (P < 0.01). These phenomena may be related to the involvement of TgROP38 in the regulation of TgProfilin (TgPRF) protein, which could be recognized by host Toll-like receptor 11 and 12 (TLR11 and TLR12), an activation of host immune response. We also found that TgPRF expression was obviously decreased in PRUΔROP38, which was related to the cytokines production in mice model. These findings reveal an intriguing biological function of ROP38 in the RH and PRU toxoplasma, which may provide us with some clues of the existence of this protein in other isolates.

Toxoplasma gondii Infection Induces High Mobility Group Box 1 Released from Mouse Macrophages.

High mobility group box 1 (HMGB1) is abundantly expressed in intracellular engaged DNA binding ability. However, more importantly, it is a weapon against infection through proinflammatory response and immune regulation while released to extracellular. Toxoplasma gondii causes inflammatory pathological changes including ileitis and encephalitis in chronic infection. To investigate whether HMGB1 contributes to the toxoplasmosis lesions, we examined HMGB1 changes during T. gondii infection. The results showed that HMGB1 transcription was down-regulated in the murine macrophage ANA1 cell line and mouse peritoneal macrophages (PMΦs) after T. gondii inoculation, but up-regulated in the IFN-γ treated macrophages and the intraperitoneal exudate cells from the T. gondii infected mice. The content of intracellular HMGB1 are basically consistent with the transcription levels in ANA1 assay, while there were no obvious changes in the mouse PMΦs. Both ANA1 and mouse PMΦs released HMGB1 after parasites infection, and no obvious HMGB1 aggregation in cytoplasm compare to the IFN-γ treatment group. Furthermore, we demonstrated that T. gondii invasion led to HMGB1 release, which was dependent on the Caspase 1 activity. These finding should promote to further investigate the functions of extracellular HMGB1 in the toxoplasmosis.

A new microneme protein of Neospora caninum, NcMIC8 is involved in host cell invasion.

Microneme proteins play an important role in the invasion process of Apicomplexan parasites through adhesion to host cells. We discovered a new N. caninum protein, NcMIC8, which is highly identical to TgMIC8. The NcMIC8 sequence has 2049 bp and no intron in the open reading fragment. It has a molecular weight of 73.8 kDa and contains a signal peptide, a transmembrane region, a low complexity region and 10 epidermal growth factor (EGF) domains. Immuno-fluorescence assay showed that NcMIC8 is located in the microneme. NcMIC8 was secreted to culture medium under stimulation of 1% ethanol, and cleaved to form the mature body of 40 kDa before transporting to microneme or during secretion. Blocking NcMIC8 using anti-NcMIC8 serum effectively inhibited host cell invasion by tachyzoites in vitro. NcMIC8 in the form of mature body interacts with NcMIC3, and the two microneme proteins form a complex probably during transportation. NcMIC8 is a new microneme protein of N. caninum and could be an attractive target for the control of neosporosis.

Functional characterization of a unique cytochrome P450 in Toxoplasma gondii.

The basic metabolic cytochrome P450 (CYP) proteins are essential for the biotransformation of sterols and xenobiotics. By contrast, the Toxoplasma gondii genome contains only one CYP gene, and the role of this enzyme in the physiology and biochemistry of apicomplexan parasites is unknown. Because it is a potential resistance gene, identifying the functionality of P450 in T. gondii is particularly important. Knocking out Tg-P450 had no significant effect on T. gondii survival, but mice infected with parasites overexpressing Tg-P450 exhibited significantly enhanced pathogenicity. Enzyme activity analyses demonstrated that this protein has mammalian CYP2B and CYP3A enzymatic activity. In addition, T. gondii lacking the P450 gene exhibited reduced resistance to quinine, mefloquine and clarithromycin compared with parasites overexpressing Tg-P450. These results suggest that P450 functions in T. gondii metabolism and detoxification is involved in vitally important processes in parasitic organisms, making this enzyme a potential drug target.

Prevalence of Antibodies against Neospora caninum in Père David's Deer ( Elaphurus davidianus ) in Beijing, China.

Neospora caninum is an apicomplexan protozoan and causes neuromuscular disorders in canids and abortions in cattle worldwide. We screened sera samples from 49 free-ranging Père David's deer ( Elaphurus davidianus ) in a nature reserve in Beijing, China, for antibodies against N. caninum using indirect fluorescence antibody tests and western blot tests. Antibodies were found in 27% of the deer. Western blot analysis revealed antibody reactivity against immunodominant N. caninum antigens of 16, 25, and 37 kDa in size together with other visible bands.

Effects of melatonin implantation on cashmere yield, fibre characteristics, duration of cashmere growth as well as growth and reproductive performance of Inner Mongolian cashmere goats.

BACKGROUND: Exogenous melatonin could induce cashmere growth. However, induced growth of cashmere fleece by melatonin implants cannot be combined with the typical growth, resulting in earlier shedding followed by another cycle of cashmere growth. To address this issue, we examine the effects on the cashmere yield, fibre characteristics, and the growth and reproductive performance of cashmere goats of planned administration of melatonin. METHODS: Eighteen half-sib, female goats were assigned to two treatments (n = 9) including a control and a treatment where melatonin (2 mg/kg BW) was implanted at the end of April and end of June. Cashmere growth and shedding were observed for approximately 1 year following implantation. Fibre samples were collected monthly to determine cumulative cashmere length. Initiation and cessation dates for cashmere growth as well as the rate of cashmere growth were calculated. Cashmere yield, weight gain of dam, kidding date, litter size, and birth weight were also recorded. RESULTS: Melatonin implantation increased cashmere yield by 34.5 % (control 553.7 g vs. melatonin 745.0 g; P < 0.01), cashmere length by 21.3 % (control 95.2 mm vs. melatonin 115.4 mm; P < 0.01), and decreased fibre diameter by 4.4 % (control 14.6 µm vs. melatonin 14.0 µm; P < 0.03). In melatonin-treated goats, the average initiation date was earlier than in control goats (May 18, 2013 vs. July 2, 2013; P < 0.01) but there was a similar cessation date (March 22, 2014 vs. March 27, 2014). Consequently, the duration of cashmere growth was longer in melatonin-treated goats than in control goats (307 vs.270 days; P < 0.01). The final BW, average daily gain, kidding date, litter size, and birth weight were not influenced by melatonin implantation. CONCLUSIONS: These data indicate that melatonin implantation (2 mg/kg BW) on two occasions (late April and June) increased cashmere yield by combining the induced growth of cashmere fleece with the typical growth and decreased the fibre diameter without changing dam growth rate or reproductive performance.