Abstract:Hematin crystallization, the primary heme detoxification mechanism of malaria parasites infecting human erythrocytes, most likely requires the participation of lipid structures.
“…High-resolution electron microscopy and X-ray tomography, however, have failed to detect lipid phases larger than 25 nm, interpreted in favor of growth from the aqueous environment (10)(11)(12), where crystallization may be assisted by a protein complex that catalyzes hematin dimerization (45). In a previous paper, we reconciled these seemingly opposite viewpoints by suggesting that β-hematin crystals grow from a thin shroud of lipid that coats their surface (46), a mechanism that is qualitatively consistent with experimental observations (47). Driven by the evidence favoring neutral lipids as a preferred environment for hematin crystallization in vivo (46), we use a solvent comprising octanol saturated with citric buffer (CBSO) at pH 4.8 as a growth medium.…”
In malaria pathophysiology, divergent hypotheses on the inhibition of hematin crystallization posit that drugs act either by the sequestration of soluble hematin or their interaction with crystal surfaces. We use physiologically relevant, time-resolved in situ surface observations and show that quinoline antimalarials inhibit β-hematin crystal surfaces by three distinct modes of action: step pinning, kink blocking, and step bunch induction. Detailed experimental evidence of kink blocking validates classical theory and demonstrates that this mechanism is not the most effective inhibition pathway. Quinolines also form various complexes with soluble hematin, but complexation is insufficient to suppress heme detoxification and is a poor indicator of drug specificity. Collectively, our findings reveal the significance of drug-crystal interactions and open avenues for rationally designing antimalarial compounds.
“…High-resolution electron microscopy and X-ray tomography, however, have failed to detect lipid phases larger than 25 nm, interpreted in favor of growth from the aqueous environment (10)(11)(12), where crystallization may be assisted by a protein complex that catalyzes hematin dimerization (45). In a previous paper, we reconciled these seemingly opposite viewpoints by suggesting that β-hematin crystals grow from a thin shroud of lipid that coats their surface (46), a mechanism that is qualitatively consistent with experimental observations (47). Driven by the evidence favoring neutral lipids as a preferred environment for hematin crystallization in vivo (46), we use a solvent comprising octanol saturated with citric buffer (CBSO) at pH 4.8 as a growth medium.…”
In malaria pathophysiology, divergent hypotheses on the inhibition of hematin crystallization posit that drugs act either by the sequestration of soluble hematin or their interaction with crystal surfaces. We use physiologically relevant, time-resolved in situ surface observations and show that quinoline antimalarials inhibit β-hematin crystal surfaces by three distinct modes of action: step pinning, kink blocking, and step bunch induction. Detailed experimental evidence of kink blocking validates classical theory and demonstrates that this mechanism is not the most effective inhibition pathway. Quinolines also form various complexes with soluble hematin, but complexation is insufficient to suppress heme detoxification and is a poor indicator of drug specificity. Collectively, our findings reveal the significance of drug-crystal interactions and open avenues for rationally designing antimalarial compounds.
“…We observed a smooth to rough transition [Figs. 1(c)-1(f)] during the growth of hematin crystals from a biomimetic mixed organic-aqueous solvent [23][24][25]; for experiment details, see Supplemental Material [26]. Hematin crystallization is the main pathway employed by malaria parasites to sequester toxic hematin, released during hemoglobin digestion [31]; its inhibition is considered the most successful target for antimalarial drugs [32].…”
The structure of the interface of a growing crystal with its nutrient phase largely determines the growth dynamics. We demonstrate that hematin crystals, crucial for the survival of malaria parasites, transition from faceted to rough growth interfaces at increasing thermodynamic supersaturation Δμ. Contrary to theoretical predictions and previous observations, this transition occurs at moderate values of Δμ. Moreover, surface roughness varies nonmonotonically with Δμ, and the rate constant for rough growth is slower than that resulting from nucleation and spreading of layers. We attribute these unexpected behaviors to the dynamics of step growth dominated by surface diffusion and the loss of identity of nuclei separated by less than the step width w. We put forth a general criterion for the onset of kinetic roughening using w as a critical length scale.
“…The subsequent growth of hemozoin crystals follows the vapor-liquid-solid crystallization mechanisms described by Vekilov et al . 29 . Figure 7 Assembly line model for hemozoin (Hz) crystal formation in the digestive vacuole of a RBC infected by the malaria parasite P. falciparum , in the trophozoite stage.…”
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 development.
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