Biochemical and Biophysical Parasitology

Niaid Extramural Activities

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Abstract

This year saw the continuation of some on-going projects, conclusion and publication of the results of some projects and the initiation of new projects.I.Lipid membrane domain dynamicsWe quantified heterogeneous membrane domain formation in synthetic lipid membranes by near-field scanning optical microscopy to improve our understanding of the basic behavior and physical chemistry of lipid membranes. We found that both nanometer- and micrometer-scale membrane domains co-existed; the domain sizes were modulated by cholesterol. This is the first quantitative near-field scanning optical microscope study of dynamic changes in raft-like membrane domains at a level of resolution below the diffraction limit of a conventional light microscope. II.Quantum dot-based immunochemistry of P. falciparum infected erythrocytes We used quantum dots in an immunocytochemical approach to quantify band 3 modifications in P. falciparum-infected hemoglobin C erythrocytes. This work, carried out in collaboration with the Wellems lab, was directed to an elucidation of the innate protection mechanisms against severe malaria present in hemoglobin C individuals. The high photostability of quantum dots permitted 3D erythrocyte image reconstruction and the demonstration of anomalies in CC erythrocyte shape resulting from a P. falciparum infection. Power spectra and autocorrelations analyses were used to quantify band 3 cluster size. We found that all infected erythrocytes irrespective of hemoglobin genotype (normal AA and a mutant CC erythrocytes) have increased band 3 clustering, but the phenomenon was more profound in CC erythrocytes. In addition, band 3 clustering in uninfected CC erythrocytes is similar to infected AA cells with a mean cluster size of ~ 500 nm. However, infected CC erythrocytes have a mean cluster size of ~ 1?m. This increased band 3 cluster size in CC erythrocytes may enhance autoantibody recognition of abnormal erythrocytes play a major role in the innate protection mechanism exhibited by hemoglobin C individuals.III. Quantitative AFM study of Plasmodium falciparum-infected hemoglobin C erythrocytes. There is a well established clinical association between hemoglobin genotype and innate protection against P. falciparum malaria. In contrast to normal hemoglobin AA, mutant hemoglobin C (HbC) is associated with substantial reductions in the risk of severe malaria in both heterozygous AC (HbC-trait) and homozygous CC individuals. Irrespective of hemoglobin genotype, parasites may induce knob-like projections on the erythrocyte surface. The knobs play a major role in the adherence of P. falciparum-infected erythrocytes to microvascular endothelia. Within AA erythrocytes, the knobs are uniformly distributed over the erythrocyte surface and the total number of knobs per unit area increases as the parasite matures. To evaluate the influence of hemoglobin genotype on knob formation, we studied AA, AC and CC erythrocytes using a combination of atomic force and light microscopy for concomitant topographic and wide-field fluorescence imaging. A detailed analysis of knob width showed a major peak with a width of ~70 nm in all infected erythrocytes. In parasitized AC and CC erythrocytes, however, a second larger knob population with a peak of ~120 nm was found. The large knob population size increased as the parasites matured. Furthermore, spatial knob distribution analyses demonstrated that knobs on CC erythrocytes are more [original contained 7,347 characters. NIDB was suppose to allow 7,900 characters but truncated my Summary w/o explanation!!]