Biophysical Membrane Mechanisms of Exocytosis inside Extracellular Vesicles, Membrane Repair Failing in Muscular Dystrophy, and Parasite Replication

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

In this first toxoplasmosis project, we use high-speed, multi-wavelength fluorescence imaging to spatially monitor both host membrane barrier integrity and invasion in real time. The data reveal that at the earliest stages of invasion the parasite creates a transient perforation in the host cell membrane at the point where the parasite’s apical end contacts the host cell. Parasites depleted of any of five proteins known to be required for rhoptry exocytosis are unable to perforate the host cell membrane. These data suggest a model in which perforating agents stored within rhoptries are released onto the host cell at the initiation of invasion to create a conduit for the delivery of rhoptry effector proteins. In the second toxoplasmosis project, by acquiring electrophysiological recordings of host cells at sub-200 µs resolution, we detected and analyzed a transient increase in host cell membrane conductance following parasite exposure. Transients always preceded invasion, but parasites depleted of RON2 generated transients without invading, ruling out an essential role for RON2 in generating this conductance pathway. Time-series analysis developed for transients and applied to the entire transient dataset (910,000 data points) reveals multiple quantal conductance changes in the parasite-induced transient, consistent with a rapid insertion, then slower removal, blocking, or inactivation of potential pore components. Quantal steps for wild-type RH strain parasites have a principal mode with Gaussian mean of 0.26 nS, similar in step size to the pore forming protein EXP2, part of the PTEX translocon of malaria parasites. Without RON2 the quantal mean (0.19 nS) was significantly different. Because we observed no parasite invasion without poration, the term "invasion pore" is proposed to describe this transient breach in host cell membrane barrier integrity during invasion. To test the first malaria project hypothesis, we used light and electron microscopy on a new model we discovered of parasite development on glass that preserves parasite viability. This new system also eliminates interference of RBC hemoglobin with light microscopy. Here, we report for the first time the steps of parasite metamorphosis and reveal both internal and external requirements needed for successful parasite shape change. We showed that the first critical step of metamorphosis is the dismantling of parasite inner membrane complex, parasite cytoskeleton, needed for invasion of RBC. Parasite plasma membrane, released from the structural containment of cytoskeleton, provided the extra surface area for a large amoeboid parasite. Parasites simultaneously flatten and segment their nuclei making it as thin and flexible as parasite itself, thus contributing to the task of preservation of infected RBC elasticity. These parasite transformations, if prevented by drug interventions, will lead to the natural clearance of malaria parasites from the infected person at the very onset of infection by natural endogenous splenic clearance mechanisms. To test the second malaria project hypothesis, we have tested for correlations between the growth of the malaria parasite and elevated red blood cell (RBC) dehydration. We found a lower propensity of dehydrated RBC to replicate malaria parasites. In addition, we found higher cell fragility of dehydrated RBC during experimental cell movement in the specially designed rotating devices of our own design. Currently we are testing blood of donors caring PIEZO1 gain of function mutation (E756del) and Sickle cell mutation (HbS) both of which are leading to RBC dehydration and hemolysis in vivo. Preliminary data analysis of experiments with multiple donors indicates that both mutations increase RBC hemolysis in the experimental conditions of cell movement and proportionally inhibit malaria parasite replication in these cells. This finding supports our primary hypothesis that mutations lead to increased RBC dehydration and fragility in blood circulation are the protective factors from severe malaria claiming many children’s lives in the malaria endemic areas. By establishing this correlation, we provided a new insight into the understanding of the intricacy of parasite-host interaction for the future development of therapeutic approaches to combat malaria, which is currently on the rise due to spreading of parasite resistance to the current antimalarials.