Molecular and biochemical parasitology最新文献

Leishmania parasites undergo morphological changes during their infectious life cycle, including developmental transitions within the sandfly vector, culminating in metacyclic stages that are pre-adapted for infection. Upon entering vertebrate host phagocytes, Leishmania differentiate into intracellular amastigotes, the form that is ultimately transmitted back to the vector to complete the life cycle. Although environmental conditions that induce these cellular transitions are well-established, molecular mechanisms governing Leishmania morphologic differentiation in response to these cues remain largely uncharacterized. Previous studies indicate a key role for HSP83 in both promastigote metacyclogenesis and amastigote differentiation. To further elucidate HSP83 functions in the Leishmania lifecycle, we examined the biological impact of experimentally elevating HSP83 gene expression in Leishmania. Significantly, HSP83 overexpression was associated with altered metacyclic morphology, increased protein kinase A (PKA) activity and decreased expression of the Leishmania major surface protease, GP63. Corroborating these findings, overexpression of the L. amazonensis PKA catalytic subunit resulted in a largely similar phenotype. Our findings demonstrate for the first time in Leishmania, a functional link between HSP83 and PKA in the control of Leishmania gene expression, replication and morphogenesis.

Cerebral Malaria (CM) is associated with the complex neurological syndrome, whose pathology is mediated by severe inflammatory processes following infection with Plasmodium falciparum. Coenzyme-Q₁₀ (Co-Q₁₀) is a potent anti-inflammatory, anti-oxidant, and anti-apoptotic agent with numerous clinical applications. The aim of this study was to elucidate the role of oral administration of Co-Q₁₀ on the initiation or regulation of inflammatory immune response during experimental cerebral malaria (ECM). For this purpose, the pre-clinical effect of Co-Q₁₀ was evaluated in C57BL/6 J mice infected with Plasmodium berghei ANKA (PbA). Treatment with Co-Q₁₀ resulted in the reduction of infiltrating parasite load, greatly improved the survival rate of PbA-infected mice that occurred independent of parasitaemia and prevented PbA-induced disruption of the blood-brain barrier (BBB) integrity. Exposure to Co-Q₁₀ resulted in the reduction of infiltration of effector CD8 + T cells in the brain and secretion of cytolytic Granzyme B molecules. Notably, Co-Q₁₀-treated mice had reduced levels of CD8 +T cell chemokines CXCR3, CCR2, and CCR5 in the brain following PbA-infection. Brain tissue analysis showed a reduction in the levels of inflammatory mediators TNF- α, CCL3, and RANTES in Co-Q₁₀ administered mice. In addition, Co-Q₁₀ modulated the differentiation and maturation of both splenic and brain dendritic cells and cross-presentation (CD8α+DCs) during ECM. Remarkably, Co-Q₁₀ was very effective in decreasing levels of CD86, MHC-II, and CD40 in macrophages associated with ECM pathology. Exposure to Co-Q₁₀ resulted in increased expression levels of Arginase-1 and Ym1/chitinase 3–like 3, which is linked to ECM protection. Furthermore, Co-Q₁₀ supplementation prevented PbA-induced depletion of Arginase and CD206 mannose receptor levels. Co-Q₁₀ abrogated PbA-driven elevation in pro-inflammatory cytokines IL-1β, IL-18, and IL-6 levels. In conclusion, the oral supplementation with Co-Q₁₀ decelerates the occurrence of ECM by preventing lethal inflammatory immune responses and dampening genes associated with inflammation and immune-pathology during ECM, and offers an inimitable opening for developing an anti-inflammatory agent against cerebral malaria.

Diclazuril is a classic anticoccidial drug. The key molecules of diclazuril in anticoccidial action allows target screening for the development of anticoccidial drugs. Cyclin-dependent kinases (CDK) are prominent target proteins in apicomplexan parasites. In this study, a diclazuril anticoccidiosis animal model was established, and the transcription and translation levels of the CDK-related kinase 2 of Eimeria tenella (EtCRK2) were detected. mRNA and protein expression levels of EtCRK2 decreased in the infected/diclazuril group compared with those in the infected/control group. In addition, immunofluorescence analysis showed that EtCRK2 was localised in the cytoplasm of the merozoites. The fluorescence intensity of EtCRK2 in the infected/diclazuril group was significantly weaker than that in the infected/control group. The anticoccidial drug diclazuril against E.tenella affects the expression pattern of EtCRK2 molecule, and EtCRK2 is a potential target for new drug development.

Toxoplasmosis is a zoonotic disease that infects most animals, including humans. Pyrimethamine/sulfadiazine is the standard treatment for toxoplasmosis. Although this treatment has been successful, it is often associated with side effects that cannot be tolerated. Therefore, various compounds have been proposed as alternative treatments for toxoplasmosis. Antimicrobial peptides (AMPs) act on various pathogens, from viruses to protozoa.

The purpose of the present study was to evaluate the effects of CM11 on in vitro and in vivo Toxoplasma gondii infection. For in vitro experiments, VERO cells were treated with different concentrations of CM11 (1–128 μg/ml) compared to sulfadiazine (SDZ) (0.78–100 μg/ml). MTT and lactate dehydrogenase (LDH) assays evaluated the cell viability and plasma membrane integrity. Then, the inhibitory concentration (IC₅₀) values were determined for treating tachyzoites of T. gondii before or on cells previously infected. Annexin V-FITC/propidium iodide (PI) staining was used to distinguish viable and apoptotic cells. The effect of CM11, SDZ, and a combination of CM11 and SDZ was evaluated in the BALB/c mouse model of acute toxoplasmosis.

CM11 was effective on tachyzoites of T. gondii and had a time and dose-dependent manner. The results of the MTT assay showed that the CC₅₀ values of CM11 and SDZ were estimated at 17.4 µg/ml and 62.3 µg/ml after 24-h, respectively. The inhibitory concentration (IC₅₀) of CM11 and SDZ on infected cells was estimated at 1.9 µg/ml and 1.4 µg/ml after 24-h, respectively. The highest rate of apoptosis (early and late) in high concentrations of SDZ and CM11 was determined for tachyzoites (2.13 % and 13.88 %), non-infected VERO cells (6.1 % and 19.76 %), and infected VERO cells (7.45 % and 29.9 %), respectively. Treating infected mice with CM11 and a combination of CM11 and SDZ had increased survival time.

Based on the mentioned results, it can be concluded that CM11 has a beneficial effect on tachyzoites of T. gondii in vitro. The result of the mouse model suggests that CM11, either alone or in combination with other chemotherapeutic agents, could be a potential therapeutic for toxoplasmosis. Hence, antimicrobial peptides could be applied as promising anti-toxoplasma agents for treating toxoplasmosis.

Previous work has shown that the TbFUT1 and LmjFUT1 genes encode essential fucosyltransferases located inside the single mitochondria of the protozoan parasites Trypanosoma brucei and Leishmania major, respectively. However, nothing was known about the orthologous gene TcFUT1 or its gene product in Trypanosoma cruzi, aetiological agent of Chagas disease. In this study, we describe the overexpression of TcFUT1 with a C-terminal 6xMyc epitope tag in T. cruzi epimastigote cells. Overexpressed and immunoprecipitated TcFUT1–6xMyc was used to demonstrate enzymatic activity and to explore substrate specificity. This defined TcFUT1 as a GDP-Fuc : βGal α1–2 fucosyltransferase with a strict requirement for acceptor glycans with non-reducing terminal Galβ1–3GlcNAc structures. This differs from the specificity of the T. brucei orthologue TbFUT1, which can also tolerate non-reducing terminal Galβ1–4GlcNAc and Galβ1–4Glc acceptor sites. Immunofluorescence microscopy using α-Myc tag antibodies also showed a mitochondrial location for TcFUT1 in T. cruzi epimastigote cells. Collectively, these results are like those described for TbFUT1 and LmjFUT1 from T. brucei and L. major, suggesting that FUT1 gene products have conserved function for across the trypanosomatids and may share therapeutic target potential.

Trypanosoma theileri maintains a long-term extracellular infection with a low parasitaemia in bovids. The surface of this parasite is predicted to be decorated with several surface molecules including membrane surface proteases (MSPs), trans-sialidases and T. theileri putative surface proteins (TTPSPs). However, there are no experimental data to verify this hypothesis. Here, we have purified and partially characterized the surface glycoconjugates of T. theileri using biochemical and mass spectrometry-based approaches. The glycoconjugates fall into two classes: glycoproteins and glycolipids. Proteomic analysis of the glycoprotein fraction demonstrated the presence of MSPs and abundant mucin-like TTPSPs, with most predicted to be GPI-anchored. Mass spectrometric characterization of the glycolipid fraction showed that these are mannose- and galactose-containing glycoinositolphospholipids (GIPLs) that are larger and more diverse than those of its phylogenetic relative T. cruzi, containing up to 10 hexose residues and carrying either alkylacyl-phosphatidylinositol or inositol-phospho-ceramide (IPC) lipid components.

Plasmodium sporozoites can block apoptotic pathways within host hepatocytes, ensuring the survival of the parasite. However, attenuated plasmodial sporozoites are unable to prevent apoptosis, which provides many parasite antigens to immune cells. This exposure leads to protection against Malaria in both human and animal models. If these hosts are later inoculated with infectious sporozoites, apoptosis of infected hepatocytes will occur, preventing parasite development. Considering that hydrogen peroxide can induce apoptosis, it is plausible that it plays a role in the mechanisms associated with the protection mediated by attenuated plasmodial sporozoites. Based on published results that describe the relationship between Plasmodium, hydrogen peroxide, and apoptosis, a rational explanation can be provided for this hypothesis.

Acanthamoeba are known to cause a vision threatening eye infection typically due to contact lens wear, and an infection of the central nervous system. The ability of these amoebae to switch phenotypes, from an active trophozoite to a resistant cyst form is not well understood; the cyst stage is often resistant to chemotherapy, which is of concern given the rise of contact lens use and the ineffective disinfectants available, versus the cyst stage. Herein, for the first time, a range of raloxifene sulfonate/sulfamate derivatives which target nucleotide pyrophosphatase/phosphodiesterase enzymes, were assessed using amoebicidal and excystation tests versus the trophozoite and cyst stage of Acanthamoeba. Moreover, the potential for cytopathogenicity inhibition in amoebae was assessed. Each of the derivatives showed considerable anti-amoebic activity as well as the ability to suppress phenotypic switching (except for compound 1a). Selected raloxifene derivatives reduced Acanthamoeba-mediated host cell damage using lactate dehydrogenase assay. These findings suggest that pyrophosphatase/phosphodiesterase enzymes may be valuable targets against Acanthamoeba infections.

Glutathione transferase enzymes (GSTs) are believed to be a major detoxification system in helminth parasites and have been associated with immunomodulation of the host response. Echinococcus granulosus sensu lato (s.l.) is a cestode parasite known to express at least five different GSTs, but no Omega-class enzymes have been reported in this parasite or in any other cestode. Herein we report the identification of a new member of the GST superfamily in E. granulosus s.l., which is phylogenetically related to the Omega-class: EgrGSTO. Through mass spectrometry, we showed that the 237 amino acids protein EgrGSTO is expressed by the parasite. Moreover, we identified homologues of EgrGSTO in other eight members of the Taeniidae family, including E. canadensis, E. multilocularis, E. oligarthrus, Hydatigera taeniaeformis, Taenia asiatica, T. multiceps, T. saginata and T. solium. A manual sequence inspection and rational modification yielded eight Taeniidae’s GSTO sequences, each one encoding for a 237 aa polypeptide showing 80.2% overall identity. To the best of our knowledge, this is the first description of genes encoding for Omega-class GSTs in worms belonging to the Taeniidae family -that at least in E. granulosus s.l. is expressed as a protein- suggesting the gene encodes for a functional protein.

Interleukin 9 (IL-9) is an effective cytokine secreted by newly defined Th9 cells, which is involved in allergic and infectious diseases. In this study, lymphocytes were isolated from mesenteric lymph node (MLN), spleen, liver, lung, and Peyer’s patches (PP) of C57BL/6 mice 5–6 weeks after S. japonicum infection, intracellular cytokine staining was done to detect the percentage of IL-9-producing CD4⁺ T cells. The qPCR and ELISA were used to verify the content of IL-9 in MLN. The population of IL-9-producing lymphocyte subset was identified by FACS. In addition, the dynamic changes and cytokine profiles of Th9 cells in the MLN of infected mice were detected by FACS. ELISA was used to detect IL-9 induced by soluble egg antigen (SEA) from isolated lymphocytes in mouse MLN. The results showed that the percentage of IL-9-secreting Th9 cells in the MLN of the infected mouse was higher than that in the spleen, liver, lung, or PP. Though CD8⁺ Tc cells, NKT cells, and γδT cells could secrete IL-9, CD4⁺ Th cells were the main source of IL-9 in S. japonicum-infected C57BL/6 mice (P < 0.05). The percentage of Th9 cells in MLN of infected mouse increased from week 3–4, and reached a peak at week 5–6, then began to decrease from week 7–8 (P < 0.05). Moreover, Th9 cells could also secrete a small amount of IL-4, IFN-γ, IL-5, and IL-10. Our results suggested a higher percentage of Th9 cells was induced in the MLN of S. japonicum-infected mice, which might play an important role in the early stage of S. japonicum-induced disease.