Bond, 21 November 1859

https://egrove.olemiss.edu/aldrichcorr_c/1177/thumbnail.jp

Shining a Light on Dense Granules – A Biochemical, Genetic and Cell Biological Investigation of an Essential but Understudied Compartment of the Malaria Parasite

Malaria is a significant infectious disease of tropical and sub-tropical regions that is caused by six species of Apicomplexan parasites of the genus Plasmodium. With 228 million cases and an estimated 619,000 mortalities worldwide in 2021 (1), this disease is endemic in developing countries in which the transmission vector, species of the Anopheles mosquito, most commonly A. gambiae, is found. Further, malaria is a major cause of mortality in children, with 65% of cases occurring in those under the age of 5 (2). Between 2000 and 2019 rates of malaria fatality decreased steadily, with a 10% increase in mortality in 2020 followed by a slight decline in 2021 (1). However, the disease remains a major global health burden and new insights into the pathogenesis of the disease are needed if new interventions are to be developed. The clinical symptoms of malaria are caused by invasion, replication within, and destruction of host red blood cells by the Plasmodium parasite. Invasion of the host cell by the parasite requires the highly regulated secretion of proteins from three specialised secretory organelles: micronemes, rhoptries, and dense granules (DGs). Microneme and rhoptry proteins function in host cell recognition attachment and invasion and establishment of the parasitophorous vacuole (PV), respectively, in Plasmodium spp. (3,4). DGs are speculated to be required for the erythrocyte remodelling that enables parasite survival and replication within the host cell post invasion, with known proteins functioning in transport of parasite effector proteins into the erythrocyte (5) and alteration of host cell mechanical properties (6). Host erythrocyte remodelling by the parasite is a process that may be exploited for development of drugs capable of inhibiting parasite growth and replication, blocking further cycles of infection. Very little is known about Plasmodium DGs, therefore shedding light on the biogenesis, protein composition and function of DGs in Plasmodium may aid drug development efforts. This project aims to address these questions in Plasmodium falciparum using molecular biological and bioinformatics techniques

Developing Novel Imaging Techniques for Analysis of Non-Fixed Cryptosporidium parvum Oocysts under Physiological Conditions

Cryptosporidium is a waterborne apicomplexan parasite typically infecting the upper gastrointestinal tract of humans and animals. Infection in immunocompetent hosts can cause acute self-limiting diarrhoeal symptoms, although in infants and the immunocompromised, infection can be life threatening. The infective stage of the parasite survives in the environment as a hardy cyst/spore or 'oocyst'. These oocysts have high resistance to disinfectants, enabling them to survive for long periods in various environments whilst remaining infective. The nature and characteristics of these "spores" remain elusive, and further research into oocyst composition is necessary to enable the development of effective water treatment methods and medical prophylaxis, for which options are currently limited. This project describes a novel method for live imaging and peak force quantitative nanomechanical property mapping of Cryptosporidium spp. oocysts using atomic force microscopy (AFM). Applying this method, data on the surface topography and deformation characteristics of Cryptosporidium parvum oocysts has been acquired and analysed to identify physiological characteristics of live oocyst of the species C. parvum, both in air and in a near native liquid environment. Scanning electron microscopy (SEM), field emission scanning electron microscopy (FESEM), and fluorescence microscopy were used for comparison between imaging methods, and previous reports. This work will enable investigations into live Cryptosporidium spp. oocyst structure, composition, and mechanical properties in unprecedented depth. In turn these capacities can be used to inform and advance our understanding of host specificity and excystation control, and to develop new methods for treatment and eradication of the parasite, all areas of vital importance to progress research towards combating this significant disease

Distribution of malaria parasite-derived phosphatidylcholine in the infected erythrocyte

Malaria parasites modify their host erythrocyte in multiple ways, leading to changes in the deformability, adhesiveness, and permeability of the host erythrocyte. Most of these changes are mediated by proteins exported from the parasite to the host erythrocyte, where these proteins interact with the host cell cytoskeleton or form complexes in the plasma membrane of the infected erythrocyte. In addition, malaria parasites induce the formation of membranous compartments-the parasitophorous vacuole, the tubovesicular network (TVN), the Maurer's clefts and small vesicles-within the infected erythrocyte, a cell that is normally devoid of internal membranes. After infection, changes also occur in the composition and asymmetry of the erythrocyte plasma membrane. Although many aspects of the mechanism of export of parasite proteins have become clear, the mechanism by which these membranous compartments are formed and expanded is almost entirely unknown. To determine whether parasite-derived phospholipids play a part in these processes, we applied a metabolic labeling technique that allows phosphatidylcholine to be labeled with a fluorophore. As the host erythrocyte cannot synthesize phospholipids, within infected erythrocytes, only parasite-derived phosphatidylcholine will be labeled with this technique. The results revealed that phosphatidylcholine produced by the parasite is distributed throughout the infected erythrocyte, including the TVN and the erythrocyte plasma membrane, but not Maurer's clefts. Interestingly, labeled phospholipids were also detected in the erythrocyte plasma membrane very soon after invasion of the parasites, indicating that the parasite may add phospholipids to the host erythrocyte during invasion. IMPORTANCE Here, we describe a previously unappreciated way in which the malaria parasite interacts with the host erythrocyte, namely, by the transfer of parasite phospholipids to the erythrocyte plasma membrane. This likely has important consequences for the survival of the parasite in the host cell and the host organism. We show that parasite-derived phospholipids are transferred from the parasite to the host erythrocyte plasma membrane and that other internal membranes that are produced after the parasite has invaded the cell are produced, at least in part, using parasite-derived phospholipids. The one exception to this is the Maurer's cleft, a membranous organelle that is involved in the transport of parasite proteins to the surface of the erythrocyte. This reveals that the Maurer's cleft is produced in a different manner than the other parasite-induced membranes. Overall, these findings provide a platform for the study of a new aspect of the host-parasite interaction

Timing of dense granule biogenesis in asexual malaria parasites.

Malaria is an important infectious disease that continues to claim hundreds of thousands of lives annually. The disease is caused by infection of host erythrocytes by apicomplexan parasites of the genus Plasmodium. The parasite contains three different apical organelles - micronemes, rhoptries and dense granules (DGs) - whose contents are secreted to mediate binding to and invasion of the host cell and the extensive remodelling of the host cell that occurs following invasion. Whereas the roles of micronemes and rhoptries in binding and invasion of the host erythrocyte have been studied in detail, the roles of DGs in Plasmodium parasites are poorly understood. They have been proposed to control host cell remodelling through regulated protein secretion after invasion, but many basic aspects of the biology of DGs remain unknown. Here we describe DG biogenesis timing for the first time, using RESA localization as a proxy for the timing of DG formation. We show that DG formation commences approximately 37 min prior to schizont egress, as measured by the recruitment of the DG marker RESA. Furthermore, using a bioinformatics approach, we aimed to predict additional cargo of the DGs and identified the J-dot protein HSP40 as a DG protein, further supporting the very early role of these organelles in the interaction of the parasite with the host cell