Biophysics of Malarial Parasite Exit from Infected Erythrocytes

Abstract

Upon infection and development within human erythrocytes, P. falciparum induces alterations to the infected RBC morphology and bio-mechanical properties to eventually rupture the host cells through parasitic and host derived proteases of cysteine and serine families. We used previously reported broad-spectrum inhibitors (E64d, EGTA-AM and chymostatin) to inhibit these proteases and impede rupture to analyze mechanical signatures associated with parasite escape. Treatment of late-stage iRBCs with E64d and EGTA-AM prevented rupture, resulted in no major RBC cytoskeletal reconfiguration but altered schizont morphology followed by dramatic re-distribution of three-dimensional refractive index (3D-RI) within the iRBC. These phenotypes demonstrated several-fold increased iRBC membrane flickering. In contrast, chymostatin treatment showed no 3D-RI changes and caused elevated fluctuations solely within the parasitophorous vacuole. We show that E64d and EGTA-AM supported PV breakdown and the resulting elevated fluctuations followed non-Gaussian pattern that resulted from direct merozoite impingement against the iRBC membrane. Optical trapping experiments highlighted reduced deformability of the iRBC membranes upon rupture-arrest, more specifically in the treatments that facilitated PV breakdown. Taken together, our experiments provide novel mechanistic interpretations on the role of parasitophorous vacuole in maintaining the spherical schizont morphology, the impact of PV breakdown on iRBC membrane fluctuations leading to eventual parasite escape and the evolution of membrane stiffness properties of host cells in which merozoites were irreversibly trapped, recourse to protease inhibitors. These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.

Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse in Plasmodium falciparum egress

“…2C). This loss of cell shape and alteration in mechanical properties, both of which have been observed in optical and AFM studies (46), implies that the erythrocyte cytoskeleton undergoes a very sudden, rapid loss of its mechanical integrity just before egress. Importantly, the observation is not in accord with a previous suggestion that the host cell cytoskeleton is progressively dismantled over several hours during schizont maturation (13,36).…”

Biomedical applications of holographic microspectroscopy [Invited]

“…Via the tomographic reconstruction method, the 3D RI distribution of individual biological samples can be obtained [40,69,131]. Recently, 3D RI measurements demonstrated the quantitative measurement of local RI formation of individual RBCs [68,132]. A multispectral approach in the 3D tomographic methods will open new applications by addressing 3D localization of molecular-specific optical properties.…”

Measurement Techniques for Red Blood Cell Deformability: Recent Advances

“…Recently, integrated with a mathematical model, DPM provide the mechanical properties of individual RBCs from membrane fluctuations: shear modulus, bending modulus, area expansion modulus, and cytoplasmic viscosity . Several alterations in the deformability of RBCs have been studied using DPM, including the effects of ATP Ben-Isaac, Park et al,2011), the nonlinear behavior of RBC deformability in response to different osmotic pressure , and malaria egress mechanism (Chandramohanadas, Park et al,2011). Employing spectroscopic quantitative phase imaging, cytoplasmic Hb concentration that is tightly related to the cytoplasmic viscosity, can also be simultaneously quantified (Park, Yamauchi et al,2009;Jang, Jang et al,2012).…”