Malaria parasites remodel their host erythrocytes to gain nutrients and avoid the immune system. Host erythrocytes are modified by hundreds of effector proteins exported from the parasite into the host cell. Protein export is mediated by the PTEX translocon comprising five core components of which EXP2 is considered to form the putative pore that spans the vacuole membrane enveloping the parasite within its erythrocyte. To explore the function and importance of EXP2 for parasite survival in the asexual blood stage of Plasmodium falciparum we inducibly knocked down the expression of EXP2. Reduction in EXP2 expression strongly reduced parasite growth proportional to the degree of protein knockdown and tended to stall development about half way through the asexual cell cycle. Once the knockdown inducer was removed and EXP2 expression restored, parasite growth recovered dependent upon the length and degree of knockdown. To establish EXP2 function and hence the basis for growth reduction, the trafficking of an exported protein was monitored following EXP2 knockdown. This resulted in severe attenuation of protein export and is consistent with EXP2, and PTEX in general, being the conduit for export of proteins into the host compartment.
Additional file 5: Table S4. KEGG pathway terms predicted to be significantly enriched in gene sets up and downregulated in C. elegans treated with fraction 5 (F5).
Parasitic roundworms (nematodes) cause substantial morbidity and mortality in livestock animals globally, and considerable productivity losses to farmers. The control of these nematodes has relied largely on the use of a limited number of anthelmintics. However, resistance to many of these these anthelmintics is now widespread, and, therefore, there is a need to find new drugs to ensure sustained and effective treatment and control into the future. Recently, we developed a screening assay to test natural, plant extracts with known inhibitory effects against the free-living worm Caenorhabditis elegans. Using this assay, we assessed here the effects of the extracts on motility and development of parasitic larval stages of Haemonchus contortus, one of the most important nematodes of small ruminants worldwide. The study showed that two of five extracts from Picria fel-terrae Lour. have a significant inhibitory effect (at concentrations of 3–5 mg/ml) on the motility and development of H. contortus larvae. Although the two extracts originated from the same plant, they displayed different levels of inhibition on motility and development, which might relate to the presence of various active constituents in these extracts, or the same constituents at different concentrations in distinct parts of the plant. These results suggest that extracts from P. fel-terrae Lour. have promising anthelmintic activity and that more broadly, plant extracts are a potential rich source of anthelmintics to combat helminthic diseases.
Parasitic nematode diseases cause major morbidity and mortality in humans globally and impact the world economy through extensive infection of crops and livestock. Resistance to anthelmintics is widespread in worm populations and therefore there is a continuous need for the new anthelmintic. Using parasitic worms in screens for new anthelmintics is extremely costly and has a low hit rate. This thesis aimed to overcome those problems by selecting ethnomedicinal plants based on their traditional use by indigenous people, and combine this preselection tool with the most studied nematode Caenorhabditis elegans as a model to screen selected ethnomedicinal plant extracts for their anthelmintic activity.
The first aim of this thesis was to screen traditional medicinal plant extracts for their anthelmintic activity using C. elegans. The plant extracts were selected by indigenous communities from Sarawak, Malaysia, based on their use in de-worming remedies. A 96 well plate based assay was developed to screen the nematocidal activity of the plant extracts. The effective plant extracts were tested against Haemonchus contortus, the most economically important animal parasitic nematode in the world. To further investigate their effect on C. elegans, we took advantage of the vast genetic resource of C. elegans to characterize the stress responses of the effective plant extracts and discovered effective plant extracts elicit unique stress reporter profiles compared to the commercially available anthelmintics. The second aim of this project was designed to illustrate the most effective plant extract and investigate its killing mechanism. We fractionated the most effective plant extract, and tested fractions on C. elegans. The anthelmintic activity was determined to exist in a single fraction which killed 90% of C. elegans adults. The efficacy of this fraction on parasitic nematodes was validated via H. contortus motility assay. Analysis of gene expression of selected genes by qRT-PCR confirmed
iv
the stress reporter strain findings and gave temporal changes in gene expression post exposure to the most effective fraction. Transcriptome-wide analysis of gene expression by next-generation sequencing discovered the effects of compounds on ER, mitochondria and lipid metabolism affecting energy production in C. elegans. Our findings showed that C. elegans is not only a good tool for preliminary screening, but may also be useful for examining the effects of compounds on nematodes and to understand the mechanism of action of the compounds. Moreover, the project demonstrates that selecting plants on the basis of their traditional application is an important approach to identify new anthelmintic compounds.
Additional file 4: Table S3. KEGG BRITE terms predicted to be significantly enriched in gene sets up and downregulated in C. elegans treated with fraction 5 (F5).
γδ T cells play an essential role in the immune response to many pathogens, including Plasmodium. However, long-lasting effects of infection on the γδ T cell population still remain inadequately understood. This study focused on assessing molecular and functional changes that persist in the γδ T cell population following resolution of malaria infection. We investigated transcriptional changes and memory-like functional capacity of malaria pre-exposed γδ T cells using a Plasmodium chabaudi infection model. We show that multiple genes associated with effector function (chemokines, cytokines and cytotoxicity) and antigen-presentation were upregulated in P. chabaudi-exposed γδ T cells compared to γδ T cells from naïve mice. This transcriptional profile was positively correlated with profiles observed in conventional memory CD8+ T cells and was accompanied by enhanced reactivation upon secondary encounter with Plasmodium-infected red blood cells in vitro. Collectively our data demonstrate that Plasmodium exposure result in "memory-like imprints" in the γδ T cell population and also promotes γδ T cells that can support antigen-presentation during subsequent infections.
Anthelmintic resistance is widespread in gastrointestinal nematode populations, such that there is a consistent need to search for new anthelmintics. However, the cost of screening for new compounds is high and has a very low success rate. Using the knowledge of traditional healers from Borneo Rainforests (Sarawak, Malaysia), we have previously shown that some traditional medicinal plants are a rich source of potential new anthelmintic drug candidates. In this study, Picria fel-terrae Lour. plant extract, which has previously shown promising anthelmintic activities, was fractionated via the use of a solid phase extraction cartridge and each isolated fraction was then tested on free-living nematode Caenorhabditis elegans and the parasitic nematode Haemonchus contortus. We found that a single fraction was enriched for nematocidal activity, killing ≥90% of C. elegans adults and inhibiting the motility of exsheathed L3 of H. contortus, while having minimal cytotoxic activity in mammalian cell culture. Metabolic profiling and chemometric analysis of the effective fraction indicated medium chained fatty acids and phenolic acids were highly represented.
Abstract γδ T cells play an essential role in the immune response to malaria infection. However, long-lasting effects of malaria infection on the γδ T cell population still remain inadequately understood. This study investigated transcriptional changes and memory-like functional capacity of malaria pre-exposed γδ T cells using a Plasmodium chabaudi infection model. We show that multiple genes associated with effector function (chemokines, cytokines and cytotoxicity) and antigen-presentation were upregulated in P. chabaudi -exposed γδ T cells compared to γδ T cells from naïve mice. This transcriptional profile was positively correlated with profiles observed in conventional memory CD8 + T cells and was accompanied by enhanced reactivation upon secondary encounter with Plasmodium -infected red blood cells in vitro . Collectively our data demonstrate that Plasmodium exposure result in “memory-like imprints” in the γδ T cell population and also promotes γδ T cells that can support antigen-presentation during subsequent infections.
Additional file 6: Table S5. Gene ontology terms predicted to be significantly enriched in gene sets up and downregulated in C. elegans treated with fraction 5 (F5).
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