Dissecting the mechanism of severe malaria

&nbsp;This study answered the effect of deletion of CISH gene on the susceptibility to malaria. It charted haematological parameters, cytokine profiles, bone marrow and spleen cell populations. The findings pointed that perturbation of CISH alone is not a major contributor for severe malaria in an acute model of infection.<br /

Differential Dynamical Effects of Macromolecular Crowding on an Intrinsically Disordered Protein and a Globular Protein: Implications for In-Cell NMR Spectroscopy

In-cell NMR provides a valuable means to assess how macromolecules, with concentrations up to 300 g/L in the cytoplasm, affect the structure and dynamics of proteins at atomic resolution. Here an intrinsically disordered protein, alpha-synuclein (alphaSN), and a globular protein, chymotrypsin inhibitor 2 (CI2) were examined by using in-cell NMR. High-resolution in-cell spectra of alphaSN can be obtained, but CI2 leaks from the cell and the remaining intracellular CI2 is not detectable. Even after stabilizing the cells from leakage by using alginate encapsulation, no CI2 signal is detected. From in vitro studies we conclude that this difference in detectability is the result of the differential dynamical response of disordered and ordered proteins to the changes of motion caused by the increased viscosity in cells

The cysteine protease dipeptidyl aminopeptidase 3 does not contribute to egress of Plasmodium falciparum from host red blood cells

The ability of Plasmodium parasites to egress from their host red blood cell is critical for the amplification of these parasites in the blood. Previous forward chemical genetic approaches have implicated the subtilisin-like protease (SUB1) and the cysteine protease dipeptidyl aminopeptidase 3 (DPAP3) as key players in egress, with the final step of SUB1 maturation thought to be due to the activity of DPAP3. In this study, we have utilized a reverse genetics approach to engineer transgenic Plasmodium falciparum parasites in which dpap3 expression can be conditionally regulated using the glmS ribozyme based RNA-degrading system. We show that DPAP3, which is expressed in schizont stages and merozoites and localizes to organelles distinct from the micronemes, rhoptries and dense granules, is not required for the trafficking of apical proteins or processing of SUB1 substrates, nor for parasite maturation and egress from red blood cells. Thus, our findings argue against a role for DPAP3 in parasite egress and indicate that the phenotypes observed with DPAP3 inhibitors are due to off-target effects

Knockdown of DPAP3 does not affect maturation of SUB1 substrates.

<p>Representative western blots (n = 3–5) of schizont (Sch) and merozoite (M) lysates and culture supernatant (CS) collected after egress (SERA5) or schizont lysates (RAP1, RAMA) made from infected RBCs grown in the presence (+) or absence (-) of 2.5 mM GlcN. Densitometry of bands relative to the EXP2 loading control and between treatment groups revealed no significant difference in the processing of SERA5, RAP1 and RAMA as a result of DPAP3 knockdown.</p

The localization of DPAP3 is distinct from proteins that localize to the apical organelles.

<p><b>A.</b> IFA on RBCs infected with PfDPAP3-HAglmS parasites and fixed with acetone/methanol. DPAP3 is labeled with the anti-HA antibody. The scale bars represent 5 μm. <b>B.</b> Immuno-electron microscopy of PfDPAP3-HAglmS parasites with anti-HA antibody shows labelling for DPAP3 (as indicated by the arrowheads) in a mature schizont at the periphery of the rhoptry bulb (RB), rhoptry neck (RN) as well as at the PV/parasitophorous vacuole membrane (PVM).</p

DPAP3 expression in <i>P</i>. <i>falciparum</i> can be conditionally regulated.

<p><b>A.</b> Western blot analysis showing dose-dependent PfDPAP3-HA protein expression in two independent clones after treatment with glucosamine (GlcN) for one (upper panel) or two (lower panel) cell cycles (Cyc1 and Cyc2, respectively). EXP2 serves as the loading control. <b>B.</b> Densitometry of PfDPAP3 expression levels in PfDPAP3-HAglmS Clone 1 parasites grown in the presence or absence of glucosamine for one cycle revealed DPAP3 expression levels were knocked down by 91%. Error bars represent ± SEM from three independent experiments. <b>C.</b> IFA showing DPAP3-HA expression is significantly reduced within 24 hrs after addition of 2.5 mM GlcN when the parasites are at schizont stage. Scale bars represent 5 μm.</p