31 research outputs found
Unraveling heme detoxification in the malaria parasite by in situ correlative X-ray fluorescence microscopy and soft X-ray tomography
A key drug target for malaria has been the detoxification pathway of the iron containing molecule heme, which is the toxic byproduct of hemoglobin digestion. The cornerstone of heme detoxification is its sequestration into hemozoin crystals, but how this occurs remains uncertain. We report new results of in vivo rate of heme crystallization in the malaria parasite, based on a new technique to measure element specific concentrations at defined locations in cell ultrastructure. Specifically, a high resolution correlative combination of cryo soft X ray tomography has been developed to obtain 3D parasite ultrastructure with cryo X ray fluorescence microscopy to measure heme concentrations. Our results are consistent with a model for crystallization via the heme detoxification protein. Our measurements also demonstrate the presence of considerable amounts of non crystalline heme in the digestive vacuole, which we show is most likely contained in hemoglobin. These results suggest a tight coupling between hemoglobin digestion and heme crystallization, highlighting a new link in the crystallization pathway for drug developmen
Progression of herpesvirus infection remodels mitochondrial organization and metabolism
Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection
Malaria Pigment Crystals: The AchillesⲠHeel of the Malaria Parasite
The biogenic formation of hemozoin crystals, a crucial process in heme detoxification by the malaria parasite, is reviewed as an antimalarial drug target. We first focus on the in-vivo formation of hemozoin. A model is presented, based on native-contrast 3D imaging obtained by X-ray and electron microscopy, that hemozoin nucleates at the inner membrane leaflet of the parasitic digestive vacuole, and grows in the adjacent aqueous medium. Having observed quantities of hemoglobin and hemozoin in the digestive vacuole, we present a model that heme liberation from hemoglobin and hemozoin formation is an assembly-line process. The crystallization is preceded by reaction between heme monomers yielding hematin dimers involving fewer types of isomers than in synthetic hemozoin; this is indicative of protein-induced dimerization. Models of antimalarial drugs binding onto hemozoin surfaces are reviewed. This is followed by a description of bromoquine, a chloroquine drug analogue, capping a significant fraction of hemozoin surfaces within the digestive vacuole and accumulation of the drug, presumably a bromoquineâhematin complex, at the vacuole's membrane
Aligned hemozoin crystals in curved clusters in malarial red blood cells revealed by nanoprobe X-ray Fe fluorescence and diffraction
The human malaria parasite Plasmodium falciparum detoxifies the heme byproduct of hemoglobin digestion in infected red blood cells by sequestration into submicron-sized hemozoin crystals. The crystal is composed of heme units interlinked to form cyclic dimers via reciprocal FeâO (propionate) bonds. Templated hemozoin nucleation was envisaged to explain a classic observation by electron microscopy of a cluster of aligned hemozoin crystals within the parasite digestive vacuole. This dovetails with evidence that acylglycerol lipids are involved in hemozoin nucleation in vivo, and nucleation of β-hematin, the synthetic analogue of hemozoin, was consistently induced at an acylglycerolâwater interface via their {100} crystal faces. In order to ascertain the nature of hemozoin nucleation in vivo, we probed the mutual orientations of hemozoin crystals in situ within RBCs using synchrotron-based X-ray nanoprobe Fe fluorescence and diffraction. The X-ray patterns indicated the presence of hemozoin clusters, each comprising several crystals aligned along their needle c axes and exposing {100} side faces to an approximately cylindrical surface, suggestive of nucleation via a common lipid layer. This experimental finding, and the associated nucleation model, are difficult to reconcile with recent reports of hemozoin formation within lipid droplets in the digestive vacuole. The diffraction results are verified by a study of the nucleation process using emerging tools of three-dimensional cellular microscopy, described in the companion paper
Biochemistry of malaria parasite infected red blood cells by X-ray microscopy
Red blood cells infected by the malaria parasite Plasmodium falciparum are correlatively imaged by tomography using soft X-rays as well as by scanning hard nano-X-ray beam to obtain fluorescence maps of various elements such as S and Fe. In this way one can deduce the amount of Fe bound either in hemoglobin or in hemozoin crystals in the digestive vacuole of the malaria parasite as well as determine the hemoglobin concentrations in the cytosols of the red blood cell and of the parasite. Fluorescence map of K shows that in the parasite's schizont stage the K concentration in the red blood cell cytosol is diminished by a factor of seven relative to a pristine red blood cell but the total amount of K in the infected red blood cell is the same as in the pristine red blood cell
Digestive Vacuole Membrane in <i>Plasmodium falciparum</i>-Infected Erythrocytes: Relevance to Templated Nucleation of Hemozoin
Crystallization
of the malaria pigment hemozoin sequesters the
toxic heme byproduct of hemoglobin digestion in Plasmodium-infected red blood cells (RBCs). Recently, we applied electron and
X-ray imaging and diffraction methods to elucidate this process. We
observed crystals oriented with their {100} faces at the inner membrane
surface of the digestive vacuole (DV) of Plasmodium
falciparum in parasitized RBCs. Modeling of the soft
X-ray tomographic (SXT) images of a trophozoite-stage parasite indicated
a 4â16 nm DV membrane thickness, suggesting a possible role
for lipid multilayers. Here, we reanalyzed the trophozoite SXT images
quantitatively via X-ray absorption to map the DV membrane thickness.
Making use of the chemical structure and crystal density of the lipid,
we found, predominantly, a bilayer 4.2 nm thick, and the remainder
was interpreted as patches âź8 nm thick. Image analysis of electron
micrographs also yielded a 4â5 nm DV membrane thickness. The
DV lipid membrane is thus mainly a bilayer, so induced hemozoin nucleation
occurs primarily via the inner of the membraneâs two leaflets.
We argue that such a leaflet embodying mono- and di-acyl lipids with
appropriate OH or NH bearing head groups may catalyse hemozoin nucleation by
stereochemical and lattice match
to the {100} crystal face, involving a two-dimensional nucleation
aggregate of âź100 molecules
The need to freezeâDehydration during specimen preparation for electron microscopy collapses the endothelial glycocalyx regardless of fixation method
Objective The endothelial glycocalyx covers the luminal surface of the endothelium and plays key roles in vascular function. Despite its biological importance, ideal visualization techniques are lacking. The current study aimed to improve the preservation and subsequent imaging quality of the endothelial glycocalyx. Methods In mice, the endothelial glycocalyx was contrasted with a mixture of lanthanum and dysprosium LaDy . Standard chemical fixation was compared with highpressure frozen specimens processed with freeze substitution. Also, isolated brain microvessels and cultured endothelial cells were high pressure frozen and by transmission soft x rays, imaged under cryogenic conditions. Results The endothelial glycocalyx was in some tissues significantly more voluminous from chemically fixed specimens compared with high pressure frozen specimens. LaDy labeling introduced excessive absorption contrast, which impeded glycocalyx measurements in isolated brain microvessels when using transmission soft x rays. In non contrasted vessels, the glycocalyx was not resolved. LaDy contrasted, cultured brain endothelial cells allowed to assess glycocalyx volume in vitro. Conclusions Both chemical and cryogenic fixation followed by dehydration lead to substantial collapse of the glycocalyx. Cryogenic fixation without freeze substitution could be a way forward although transmission soft x ray tomography based solely on amplitude contrast seems unsuitabl
The infection leads to an increase in the mitochondrial proton leakage and Ca<sup>2+</sup> content.
(a) Seahorse real-time cell metabolic analysis of oxygen consumption rate (OCR) traces of noninfected and infected MEF cells at 4 and 8 hpi. The arrows mark the addition of oligomycin, FCCP, and rotenoneâ+âantimycin A (n = 30 000 cells, 6 replicates). Box plots showing (b) basal OCR, (c) ATP production, (c) proton leak, and (e) coupling efficiency (ATP/O). (f) The live cell analysis of the mitochondrial calcium labelled by calcium ion indicator Rhod2-AM (green) selectively accumulating within mitochondria, and mitotracker (red). The infected cells selected for imaging were identified by the expression of HSV-1 ICP4 (not shown). Scale bars, 10 Îźm. (g) The quantitative analysis of mitochondrial [Ca2+] in noninfected and infected cells at 4 and 8 hpi (n = 207, 97, and 96 for NI, 4, and 8 hpi, respectively). The box plots show the mean (dashed line) and the interquartile range. Statistical significance was determined using the Studentâs t-test. The significance values shown are denoted as **** (p<0.0001), ** (p<0.01), *(p<0.05), or ns (not significant).</p