Host reticulocytes provide metabolic reservoirs that can be exploited by malaria parasites

Human malaria parasites proliferate in different erythroid cell types during infection. Whilst Plasmodium vivax exhibits a strong preference for immature reticulocytes, the more pathogenic P. falciparum primarily infects mature erythrocytes. In order to assess if these two cell types offer different growth conditions and relate them to parasite preference, we compared the metabolomes of human and rodent reticulocytes with those of their mature erythrocyte counterparts. Reticulocytes were found to have a more complex, enriched metabolic profile than mature erythrocytes and a higher level of metabolic overlap between reticulocyte resident parasite stages and their host cell. This redundancy was assessed by generating a panel of mutants of the rodent malaria parasite P. berghei with defects in intermediary carbon metabolism (ICM) and pyrimidine biosynthesis known to be important for P. falciparum growth and survival in vitro in mature erythrocytes. P. berghei ICM mutants (pbpepc-, phosphoenolpyruvate carboxylase and pbmdh-, malate dehydrogenase) multiplied in reticulocytes and committed to sexual development like wild type parasites. However, P. berghei pyrimidine biosynthesis mutants (pboprt-, orotate phosphoribosyltransferase and pbompdc-, orotidine 5′-monophosphate decarboxylase) were restricted to growth in the youngest forms of reticulocytes and had a severe slow growth phenotype in part resulting from reduced merozoite production. The pbpepc-, pboprt- and pbompdc- mutants retained virulence in mice implying that malaria parasites can partially salvage pyrimidines but failed to complete differentiation to various stages in mosquitoes. These findings suggest that species-specific differences in Plasmodium host cell tropism result in marked differences in the necessity for parasite intrinsic metabolism. These data have implications for drug design when targeting mature erythrocyte or reticulocyte resident parasites

Malaria parasites do respond to heat

The capacity of malaria parasites to respond to changes in their environment at the transcriptional level has been the subject of debate, but recent evidence has unambiguously demonstrated that Plasmodium spp. can produce adaptive transcriptional responses when exposed to some specific types of stress. These include metabolic conditions and febrile temperature. The Plasmodium falciparum protective response to thermal stress is similar to the response in other organisms, but it is regulated by a transcription factor evolutionarily unrelated to the conserved transcription factor that drives the heat shock (HS) response in most eukaryotes. Of the many genes that change expression during HS, only a subset constitutes an authentic response that contributes to parasite survival. © 2022 Elsevier Lt

How merozoite surface antigen-specific antibodies inhibit Plasmodium falciparum growth in vitro

In hyperendemic malarious regions adults develop protective immunity to Plasmodium falciparum infection. In order for this immunity to develop the host immune system must be able to recognise the parasite. One stage at which this occurs is prior to red blood cell invasion when the extracellular form of the parasite, the merozoite, presents the host immune system with a number of potential immunogens termed merozoite surface antigens. Antibodies to merozoite surface antigens are able to inhibit the growth and development of the parasite in vitro. This thesis explores the mechanisms by which merozoite surface antigen -specific antibodies exert this inhibition.The affinity, fine specificity and Fc- mediated effects of antibodies may affect their functional activity. Immortalised B cell lines producing merozoite surface antigen - specific human monoclonal antibodies were generated in order to investigate the effect of these factors on their growth inhibitory activity in vitro. The preliminary characterisation of these mAbs is described in chapter 3. However, sufficient quantities of these mAbs could not be generated for their functional activity to be investigated in vitro.Current dogma holds that the primary function of antibodies is to provide a molecular link between antigen recognition and pathogen destruction. However, all Abs have the ability to catalyse a reaction between singlet oxygen and water to generate hydrogen peroxide. This thesis explored the hypothesis that this antibody-catalysed water-oxidation pathway is responsible for the intraerythrocytic growth inhibition exerted by MSP-1-₁₉-specific Abs. An in vitro ACWO assay was developed to test this hypothesis and data suggest that ACWO may occur in infected RBCs associated with an anti- MSP-1-₁₉ monoclonal antibody.Antibodies specific to an intrinsically unstructured region from the C- terminal half of merozoite surface protein 3.3, designated MSP3.3C, are highly effective at inhibiting iii the in vitro growth of P. falciparum. This thesis explored the mechanisms responsible for this inhibition. This inhibition is caused by inhibition of the intraerythrocytic development of the parasite and not by inhibition of merozoite invasion. MSP3.3C specific Abs can access the intraerythrocytic parasite post invasion and completely arrest parasite development by inducing parasite death.The findings presented in this thesis expand current knowledge of the mechanisms by which MSA- specific Abs inhibit the growth of P. falciparum in vitro. This may prove informative both in terms of our understanding of naturally acquired antibody mediated immunity to P. falciparum asexual stages and in furthering effective vaccine design against this deadly pathogen

Changes in metabolic phenotypes of Plasmodium falciparum in vitro cultures during gametocyte development.

BACKGROUND: Gametocytes are the Plasmodium life stage that is solely responsible for malaria transmission. Despite their important role in perpetuating malaria, gametocyte differentiation and development is poorly understood. METHODS: To shed light on the biochemical changes that occur during asexual and gametocyte development, metabolic characterization of media from in vitro intra-erythrocytic Plasmodium falciparum cultures was performed throughout gametocyte development by applying 1H nuclear magnetic spectroscopy, and using sham erythrocyte cultures as controls. Spectral differences between parasite and sham cultures were assessed via principal component analyses and partial-least squares analyses, and univariate statistical methods. RESULTS: Clear parasite-associated changes in metabolism were observed throughout the culture period, revealing differences between asexual parasites and gametocyte stages. With culture progression and development of gametocytes, parasitic release of the glycolytic end products lactate, pyruvate, alanine, and glycerol, were found to be dramatically reduced whilst acetate release was greatly increased. Also, uptake of lipid moieties CH(2), CH(3), and CH = CH-CH(2)-CH(2) increased throughout gametocyte development, peaking with maturity. CONCLUSIONS: This study uniquely presents an initial characterization of the metabolic exchange between parasite and culture medium during in vitro P. falciparum gametocyte culture. Results suggest that energy metabolism and lipid utilization between the asexual stages and gametocytes is different. This study provides new insights for gametocyte-specific nutritional requirements to aid future optimization and standardization of in vitro gametocyte cultivation, and highlights areas of novel gametocyte cell biology that deserve to be studied in greater detail and may yield new targets for transmission-blocking drugs

An Exported Malaria Protein Regulates Glucose Uptake During Intraerythrocytic Infection

Malaria is the world\u27s second biggest infectious killer after tuberculosis. It accounts for 219 million cases each year, with an estimated 660,000 deaths. The majority of these deaths occur in sub-Saharan Africa, in children under 5 years old. In addition to Africa, malaria is endemic to Asia, Central and South America, the Caribbean and the Middle East. Plasmodium falciparum (P. falciparum) is the protozoan parasite that is responsible for the deadliest form of human malaria. Plasmodia are carried by the female Anopheles mosquito and infected into humans during a blood meal. The parasites invade liver cells and form merozoites which erupt from liver cells to invade red blood cells. The intraerythrocytic cycle of infection is responsible for the clinical manifestations of malaria, namely fever and chills. The intraerythrocytic cycle is also the stage of disease that is most studied and targeted for treatment. Although treatment for malaria is available, drug-resistant forms of the parasite are increasingly rampant. For this reason, new, more effective treatments for malaria are necessary. To develop these treatments, we must have a better understanding of the biological processes that the parasite employs to survive in the host to cause disease. In 1996, an international effort was launched to sequence the genome of P. falciparum with the expectation that the genome sequence could be exploited in the search for new drugs and vaccines to fight malaria. In 2002, the genome sequence was published with gaps in some chromosomes. Approximately 5,300 protein-encoding genes were identified; of these about 60% were labeled as hypothetical proteins. Our studies focus on determining the function of one hypothetical protein, PFB0923c, that we now call Glucose Uptake Restoration Protein (GURP). We show that GURP localizes to novel double membrane vesicles in the RBC cytosol and is essential during P. falciparum intraerythrocytic infection. GURP interacts with and sequesters the host protein stomatin, which is known to depress glucose uptake in mammalian cells. Knockdown of GURP decreases glucose uptake and impairs parasite growth in RBCs. This phenotype can be rescued with antioxidants, suggesting that hexose monophosphate/pentose phosphate pathway impairment is lethal in the knockdown parasites. GURP C183 is essential to parasite viability and trafficking of GURP vesicles to the RBC cytosol. Together, these data demonstrate that GURP is essential to P. falciparum viability and glucose uptake during infection of red blood cells

Malaria Pathophysiology as a Syndrome: Focus on Glucose Homeostasis in Severe Malaria and Phytotherapeutics Management of the Disease

Severe malaria presents with varied pathophysiological manifestations to include derangement in glucose homeostasis. The changes in glucose management by the infected human host emanate from both Plasmodium parasitic and host factors and/or influences which are aimed at creating a proliferative advantage to the parasite. This also includes morphological changes that that take place to both infected and uninfected cells as structural alterations occur on the cell membranes to allow for increased nutrients (glucose) transportation into the cells. Without the availability, effective and efficient intervention there is a high cost incurred by the human host. Hyperglycaemia, hypoglycaemia and hyperinsulinemia are critical aspects displayed in severe malaria. Conventional treatment to malaria renders itself hostile to the host with negative glucose metabolism changes experiences in the young, pregnant women and malaria naïve individuals. In malaria, therefore, host effects, parasite imperatives and treatment regimens play a pivotal role in the return to wellness of the patient. Phytotherapeutics are emerging as treatment alternatives that ameliorate glucose homeostasis alternations as well as combat malaria parasitaemia. The phytochemicals e.g. triterpenes, have been shown to alleviate the “disease” and “parasitic” aspects of malaria pointing at key aspects in ameliorating malaria glucose homeostasis fallings-out that are experienced in malaria

Experimental and computational applications of microarray technology for malaria eradication in Africa

Various mutation assisted drug resistance evolved in Plasmodium falciparum strains and insecticide resistance to female Anopheles mosquito account for major biomedical catastrophes standing against all efforts to eradicate malaria in Sub-Saharan Africa. Malaria is endemic in more than 100 countries and by far the most costly disease in terms of human health causing major losses among many African nations including Nigeria. The fight against malaria is failing and DNA microarray analysis need to keep up the pace in order to unravel the evolving parasite’s gene expression profile which is a pointer to monitoring the genes involved in malaria’s infective metabolic pathway. Huge data is generated and biologists have the challenge of extracting useful information from volumes of microarray data. Expression levels for tens of thousands of genes can be simultaneously measured in a single hybridization experiment and are collectively called a “gene expression profile”. Gene expression profiles can also be used in studying various state of malaria development in which expression profiles of different disease states at different time points are collected and compared to each other to establish a classifying scheme for purposes such as diagnosis and treatments with adequate drugs. This paper examines microarray technology and its application as supported by appropriate software tools from experimental set-up to the level of data analysis. An assessment of the level of microarray technology in Africa, its availability and techniques required for malaria eradication and effective healthcare in Nigeria and Africa in general were also underscored

Analysis of slow (theta) oscillations as a potential temporal reference frame for information coding in sensory cortices

While sensory neurons carry behaviorally relevant information in responses that often extend over hundreds of milliseconds, the key units of neural information likely consist of much shorter and temporally precise spike patterns. The mechanisms and temporal reference frames by which sensory networks partition responses into these shorter units of information remain unknown. One hypothesis holds that slow oscillations provide a network-intrinsic reference to temporally partitioned spike trains without exploiting the millisecond-precise alignment of spikes to sensory stimuli. We tested this hypothesis on neural responses recorded in visual and auditory cortices of macaque monkeys in response to natural stimuli. Comparing different schemes for response partitioning revealed that theta band oscillations provide a temporal reference that permits extracting significantly more information than can be obtained from spike counts, and sometimes almost as much information as obtained by partitioning spike trains using precisely stimulus-locked time bins. We further tested the robustness of these partitioning schemes to temporal uncertainty in the decoding process and to noise in the sensory input. This revealed that partitioning using an oscillatory reference provides greater robustness than partitioning using precisely stimulus-locked time bins. Overall, these results provide a computational proof of concept for the hypothesis that slow rhythmic network activity may serve as internal reference frame for information coding in sensory cortices and they foster the notion that slow oscillations serve as key elements for the computations underlying perception

Systems analysis of host-parasite interactions.

Parasitic diseases caused by protozoan pathogens lead to hundreds of thousands of deaths per year in addition to substantial suffering and socioeconomic decline for millions of people worldwide. The lack of effective vaccines coupled with the widespread emergence of drug-resistant parasites necessitates that the research community take an active role in understanding host-parasite infection biology in order to develop improved therapeutics. Recent advances in next-generation sequencing and the rapid development of publicly accessible genomic databases for many human pathogens have facilitated the application of systems biology to the study of host-parasite interactions. Over the past decade, these technologies have led to the discovery of many important biological processes governing parasitic disease. The integration and interpretation of high-throughput -omic data will undoubtedly generate extraordinary insight into host-parasite interaction networks essential to navigate the intricacies of these complex systems. As systems analysis continues to build the foundation for our understanding of host-parasite biology, this will provide the framework necessary to drive drug discovery research forward and accelerate the development of new antiparasitic therapies