Transcriptional Memory-Like Imprints and Enhanced Functional Activity in γδ T Cells Following Resolution of Malaria Infection.

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 Plasmodiumchabaudi 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.

A 4-cyano-3-methylisoquinoline inhibitor of Plasmodium falciparum growth targets the sodium efflux pump PfATP4.

We developed a novel series of antimalarial compounds based on a 4-cyano-3-methylisoquinoline. Our lead compound MB14 achieved modest inhibition of the growth in vitro of the human malaria parasite, Plasmodium falciparum. To identify its biological target we selected for parasites resistant to MB14. Genome sequencing revealed that all resistant parasites bore a single point S374R mutation in the sodium (Na+) efflux transporter PfATP4. There are many compounds known to inhibit PfATP4 and some are under preclinical development. MB14 was shown to inhibit Na+ dependent ATPase activity in parasite membranes, consistent with the compound targeting PfATP4 directly. PfATP4 inhibitors cause swelling and lysis of infected erythrocytes, attributed to the accumulation of Na+ inside the intracellular parasites and the resultant parasite swelling. We show here that inhibitor-induced lysis of infected erythrocytes is dependent upon the parasite protein RhopH2, a component of the new permeability pathways that are induced by the parasite in the erythrocyte membrane. These pathways mediate the influx of Na+ into the infected erythrocyte and their suppression via RhopH2 knockdown limits the accumulation of Na+ within the parasite hence protecting the infected erythrocyte from lysis. This study reveals a role for the parasite-induced new permeability pathways in the mechanism of action of PfATP4 inhibitors.

Human Antibodies that Slow Erythrocyte Invasion Potentiate Malaria-Neutralizing Antibodies.

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However, little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional, or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite, thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria.

Transcriptional alterations in Caenorhabditis elegans following exposure to an anthelmintic fraction of the plant Picria fel-terrae Lour.

BACKGROUND: Natural compounds from plants are known to provide a source of anthelmintic molecules. In previous studies, we have shown that plant extracts from the plant Picria fel-terrae Lour. and particular fractions thereof have activity against the free-living nematode Caenorhabditis elegans, causing quite pronounced stress responses in this nematode. We have also shown that a fraction, designated Pf-fraction 5, derived from this plant has a substantial adverse effect on this worm; however, nothing is known about the molecular processes affected in the worm. In the present study, we explored this aspect. RESULTS: Key biological processes linked to upregulated genes (n = 214) included 'response to endoplasmic reticulum stress' and 'lipid metabolism', and processes representing downregulated genes (n = 357) included 'DNA-conformation change' and 'cellular lipid metabolism'. CONCLUSIONS: Exposure of C. elegans to Pf-fraction 5 induces significant changes in the transcriptome. Gene ontology analysis suggests that Pf-fraction 5 induces endoplasmic reticulum and mitochondrial stress, and the changes in gene expression are either a direct or indirect consequence of this. Further work is required to assess specific responses to sub-fractions of Pf-fraction 5 in time-course experiments in C. elegans, to define the chemical(s) with potent anthelmintic properties, to attempt to unravel their mode(s) of action and to assess their selectivity against nematodes.

Knockdown of the translocon protein EXP2 in Plasmodium falciparum reduces growth and protein export.

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.

Metabolic profiling and in vitro assessment of anthelmintic fractions of Picria fel-terrae Lour.

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.

Anthelmintic activity of selected ethno-medicinal plant extracts on parasitic stages of Haemonchus contortus.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.