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Biophysics of malarial parasite exit from infected erythrocytes.

Chandramohanadas R, Park Y, Lui L, Li A, Quinn D, Liew K, Diez-Silva M, Sung Y, Dao M, Lim CT, Preiser PR, Suresh S - PLoS ONE (2011)

Bottom Line: 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.These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.

View Article: PubMed Central - PubMed

Affiliation: Singapore-MIT Alliance for Research and Technology Centre, Singapore, Singapore.

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.

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Related in: MedlinePlus

Protease inhibitors arrest merozoite egress.(A) Schizont-stage iRBCs (∼44 hpi) were treated with DMSO, E64d, EGTA-AM or Chymostatin and were followed by giemsa staining and microscopy. Morphology of iRBCs was analyzed at 46 hpi (Upper Panel) and 55 hpi (Lower Panel). While all schizont-stage iRBCs from DMSO-treated samples ruptured and established ring stage infections by 55th hpi, merozoites from inhibitor-treated iRBCs failed to egress. (B) Viability of inhibitor-treated iRBCs was analyzed by JC-1 staining and flow cytometry. The values are expressed as percentage of healthy schizonts gated based on JC-1 and DAPI staining before and after inhibitor treatments; error bars represent standard deviation.
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pone-0020869-g001: Protease inhibitors arrest merozoite egress.(A) Schizont-stage iRBCs (∼44 hpi) were treated with DMSO, E64d, EGTA-AM or Chymostatin and were followed by giemsa staining and microscopy. Morphology of iRBCs was analyzed at 46 hpi (Upper Panel) and 55 hpi (Lower Panel). While all schizont-stage iRBCs from DMSO-treated samples ruptured and established ring stage infections by 55th hpi, merozoites from inhibitor-treated iRBCs failed to egress. (B) Viability of inhibitor-treated iRBCs was analyzed by JC-1 staining and flow cytometry. The values are expressed as percentage of healthy schizonts gated based on JC-1 and DAPI staining before and after inhibitor treatments; error bars represent standard deviation.

Mentions: Schizont-stage P. falciparum infected RBCs, approximately 44 h post-invasion (hpi) were treated with protease inhibitors E64d, EGTA-AM and chymostatin that are known to block parasite egress. E64 or a more cell permeable version E64d primarily inactivates thiol proteases [14], [15], [16] and primarily host-calpain 1, EGTA-AM irreversibly chelates calcium ions required for calpain activation [14] while chymostatin can inactivate both cysteine and serine family proteases involved in rupture. In our experiments, all inhibitors irreversibly locked merozoites within the iRBCs at schizont stage while the DMSO-treated controls established new ring stage infections by 50–52 hpi (Fig. 1A). No food vacuole swelling effect was observed in the schizont-stage parasites treated with inhibitors. The rupture phenotypes resulted by E64d and EGTA-AM treatments appeared highly fragile structures from giemsa smears, and a fraction of them had undergone bursting when smeared. Merozoites from rupture-phenotypes appeared less invasive upon removal of inhibitors -compared to DMSO-treated iRBCs, possibly due to partial inhibition of invasion related proteases by the broad-spectrum inhibitors used in this study. However, the parasites that were locked inside the host cells were metabolically active as observed from vitality staining experiments (Fig. 1B). Minimal parasite death was observed in cultures treated with inhibitors, when iRBCs stained with JC-1 were used to monitor mitochondrial potential [20], [21], [22] as a measure of viability (Fig. S1A) before and actual rupture time points, while treatment with compounds such as chloroquine or CCCP caused death of a major fraction of parasites within 2 hours (Fig. S1B).


Biophysics of malarial parasite exit from infected erythrocytes.

Chandramohanadas R, Park Y, Lui L, Li A, Quinn D, Liew K, Diez-Silva M, Sung Y, Dao M, Lim CT, Preiser PR, Suresh S - PLoS ONE (2011)

Protease inhibitors arrest merozoite egress.(A) Schizont-stage iRBCs (∼44 hpi) were treated with DMSO, E64d, EGTA-AM or Chymostatin and were followed by giemsa staining and microscopy. Morphology of iRBCs was analyzed at 46 hpi (Upper Panel) and 55 hpi (Lower Panel). While all schizont-stage iRBCs from DMSO-treated samples ruptured and established ring stage infections by 55th hpi, merozoites from inhibitor-treated iRBCs failed to egress. (B) Viability of inhibitor-treated iRBCs was analyzed by JC-1 staining and flow cytometry. The values are expressed as percentage of healthy schizonts gated based on JC-1 and DAPI staining before and after inhibitor treatments; error bars represent standard deviation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3117855&req=5

pone-0020869-g001: Protease inhibitors arrest merozoite egress.(A) Schizont-stage iRBCs (∼44 hpi) were treated with DMSO, E64d, EGTA-AM or Chymostatin and were followed by giemsa staining and microscopy. Morphology of iRBCs was analyzed at 46 hpi (Upper Panel) and 55 hpi (Lower Panel). While all schizont-stage iRBCs from DMSO-treated samples ruptured and established ring stage infections by 55th hpi, merozoites from inhibitor-treated iRBCs failed to egress. (B) Viability of inhibitor-treated iRBCs was analyzed by JC-1 staining and flow cytometry. The values are expressed as percentage of healthy schizonts gated based on JC-1 and DAPI staining before and after inhibitor treatments; error bars represent standard deviation.
Mentions: Schizont-stage P. falciparum infected RBCs, approximately 44 h post-invasion (hpi) were treated with protease inhibitors E64d, EGTA-AM and chymostatin that are known to block parasite egress. E64 or a more cell permeable version E64d primarily inactivates thiol proteases [14], [15], [16] and primarily host-calpain 1, EGTA-AM irreversibly chelates calcium ions required for calpain activation [14] while chymostatin can inactivate both cysteine and serine family proteases involved in rupture. In our experiments, all inhibitors irreversibly locked merozoites within the iRBCs at schizont stage while the DMSO-treated controls established new ring stage infections by 50–52 hpi (Fig. 1A). No food vacuole swelling effect was observed in the schizont-stage parasites treated with inhibitors. The rupture phenotypes resulted by E64d and EGTA-AM treatments appeared highly fragile structures from giemsa smears, and a fraction of them had undergone bursting when smeared. Merozoites from rupture-phenotypes appeared less invasive upon removal of inhibitors -compared to DMSO-treated iRBCs, possibly due to partial inhibition of invasion related proteases by the broad-spectrum inhibitors used in this study. However, the parasites that were locked inside the host cells were metabolically active as observed from vitality staining experiments (Fig. 1B). Minimal parasite death was observed in cultures treated with inhibitors, when iRBCs stained with JC-1 were used to monitor mitochondrial potential [20], [21], [22] as a measure of viability (Fig. S1A) before and actual rupture time points, while treatment with compounds such as chloroquine or CCCP caused death of a major fraction of parasites within 2 hours (Fig. S1B).

Bottom Line: 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.These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.

View Article: PubMed Central - PubMed

Affiliation: Singapore-MIT Alliance for Research and Technology Centre, Singapore, Singapore.

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.

Show MeSH
Related in: MedlinePlus