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Prepublished online as a Blood First Edition Paper on January 16, 2003; DOI 10.1182/blood-2002-08-2654.
RED CELLS
From the Physiological Laboratory, University
of Cambridge, Cambridge, United Kingdom; and the Department of
Biological Chemistry, Institute of Life Sciences, The Hebrew University
of Jerusalem, Jerusalem, Israel.
During their asexual reproduction cycle (about 48 hours) in human
red cells, Plasmodium falciparum parasites consume most of
the host cell hemoglobin, far more than they require for protein biosynthesis. They also induce a large increase in the permeability of
the host cell plasma membrane to allow for an increased traffic of
nutrients and waste products. Why do the parasites digest hemoglobin in
such excess? And how can infected red cells retain their integrity for
the duration of the asexual cycle when comparably permeabilized uninfected cells hemolyse earlier? To address these questions we
encoded the multiplicity of factors known to influence host cell volume
in a mathematical model of the homeostasis of a parasitized red cell.
The predicted volume changes were subjected to thorough experimental
tests by monitoring the stage-related changes in the osmotic fragility
of infected red cell populations. The results supported the model
predictions of biphasic volume changes comprising transient shrinkage
of infected cells with young trophozoites followed by continuous volume
increase to about 10% lower than the critical hemolytic volume of
approximately 150 fL by the end of the asexual cycle. Analysis
of these results and of additional model predictions demonstrated that
the osmotic stability of infected red cells can be preserved only by a
large reduction in impermeant solute concentration within the host cell
compartment. Thus, excess hemoglobin consumption represents an
essential evolutionary strategy to prevent the premature hemolysis of
the highly permeabilized infected red cell.
(Blood. 2003;101:4189-4194) During their intraerythrocytic phase,
Plasmodium falciparum parasites grow and divide within the
red blood cells to occupy about 16- to 20-fold the volume of the
invading merozoite. The parasite ingests and digests about 70% of the
host cell hemoglobin (Hb)1 but uses only up to 16% of the
released amino acids for protein biosynthesis.2 The excess
is discharged out of the infected red blood cells (IRBCs) to the
surrounding plasma3 mainly through new permeation pathways
(NPPs) of broad solute selectivity induced by the parasite in the host
cell membrane.4-6 The reason why parasites expend so much
energy ingesting and digesting excess hemoglobin7-10 and
detoxifying the cell from toxic ferriprotoporphyrin IX11-13 remains puzzling.
Another unresolved puzzle concerns the mechanism by which parasitized
red cells are able to retain their osmotic stability for the
approximately 48-hour reproductive cycle of the parasite despite the
rapid NPP-mediated dissipation of the Na+ and
K+ gradients.14 A recent study by Staines et
al15 demonstrated that if NPPs were induced in
uninfected cells as they are in infected cells, the
uninfected cells would hemolyse by approximately 44 hours.
Since the additional volume of the parasite was excluded from their
computations, their estimates make the lysis resistance of IRBCs with
large internal parasites even harder to comprehend.
To investigate these issues we developed a mathematical model of the
homeostasis of a parasitized red cell, formulated critical predictions
on the stage-related volume changes of IRBCs, and carried out
experimental tests to determine the validity of the model. The
experimental results confirmed the predicted stage-related volume
changes, and validated the model-based suggestion that NPP-mediated
permeability and excess Hb consumption are fine-tuned to ensure the
osmotic stability and integrity of the parasitized cell for the
duration of its asexual cycle.
Preparation of cells and determination of the osmotic fragility
distribution of infected red cell populations
Mathematical model of the homeostasis of P
falciparum-infected red cells (HCM)
The equations used to model stage-related Hb consumption (H)
and NPP-mediated permeabilities (NPPK) as a function of
time were of the form
Y = Ymax/{1 + exp ([t
Hb is incorporated within the parasite by endocytosis of the host
cytoplasm.1,25 Parasite volume growth is thus tightly linked to Hb digestion as follows. To digest the amount of Hb prescribed by the Y(H) function for any infinitesimal time interval (dt) the parasite would have to endocytose a volume of red
cell cytoplasm (dV') that contains the required
amount of Hb at the concentration prevailing in the host during
dt. So, for each dt, the model reduces the host
cell volume by dV' and increases parasite volume by
dV'. Thus, parasite volume is computed at each stage as the
cumulative sum of all the dV's up to that stage. Within each dt, the total change in host cell volume
(dV) will be given by the algebraic sum of dV'
and of all osmotically driven fluid transfers (dV"). Thus,
dV = dV' + dV". With this strategy,
parasite volume growth is determined by the Hb consumption function,
whereas the volume changes within the host compartment are additionally determined by all passive and active fluxes across the host cell plasma
membrane. Note that dV" and dV' are
interdependent quantities: fluid transfers determine the
dV', which contains the prescribed amount of Hb to be
digested within dt, and, in turn, dV" is
influenced by host residual volume and Hb content. At each time
(t), parasite volume (VP) and host cell volume
(VH) are computed from
VP = VPo +
An important assumption in this modeling strategy is that the homeostatic behavior of the host cell is influenced mainly through NPP induction and dV feeds. Solute exchanges between parasite and host, as well as many of the other red cell membrane alterations induced by the parasite may be important for specific roles in parasite and host, but, within current knowledge, their relevance to IRBC homeostasis is not apparent.
The stage-dependent patterns of Hb digestion and of NPP-mediated permeabilities were derived from recently published data2,15 and encoded in the model as shown in Figure 1 and detailed in "Materials and methods." The curves approach maximal values of NPPK-max, representing the maximal NPP-mediated K+ flux, and of Hmax, representing the maximal fraction of host Hb consumed. Parasite volume growth is determined by the Hb consumption curve, as explained in "Materials and methods." The time-dependent patterns in Figure 1 describe 3 stages in host permeabilization and Hb consumption: a long latency during the ring stage, a rapid exponential rise starting at the young trophozoite stage, and a leveling-off phase through to schizont and segmentor stages. This time-dependent pattern parallels other growth and metabolic activities of the parasite.31-34 Figure 2 shows the changes in the main homeostatic variables of IRBCs predicted by the HCM. The model reproduced the known patterns of change in the Na+, K+, and Hb content of host red cells2,14 (Figure 2A-B). The critical novel predictions concerned the stage-related volume changes of parasite and host cells shown in Figure 2C. Unlike with uninfected red cells,15 the model predicted that NPP-permeabilization and parasite volume growth would not induce the hemolysis of IRBCs by the end of the 48-hour asexual cycle. According to the model, IRBC volume would change little before NPP development. As NPP-mediated permeability increases, and coincidental with the highest rate of Na+ and K+ gradient dissipation (Figure 2A), excess KCl loss over NaCl gain would cause transient shrinkage, as expected from a PK/PNa permeability ratio of about 2.315 and from the high initial outward K+ gradient across the RBC plasma membrane. After the initial K+ and Na+ gradient dissipation, the inward colloid osmotic gradients would drive NaCl and water in swelling the cells toward a volume lower than the critical hemolytic volume by 48 hours, with a small but significant safety margin for additional swelling (Figure 2C). The model predicts distinct contributions of parasite and host cell to the overall volume change of IRBCs. Whereas parasite volume increases monotonically, the predicted host cell volume displays a clear wavy pattern after about 16 hours after invasion (Figure 2C), with 4 distinct phases: (1) initial shrinkage due to KCl loss exceeding NaCl gain through developing NPPs, (2) swelling due to sustained net NaCl and water gain after K+ gradient dissipation, (3) volume reduction due to transfers of cytoplasm from host to parasite exceeding swelling tendency from net NaCl gain; during this period, dilution of Hb in the host cell (Figure 2B) imposes larger dV' parasite feeds to incorporate the amounts of Hb prescribed by the Y(H) function ("Materials and methods"), and (4) a final stage in which net NaCl gain and colloid osmotic swelling prevail again over declining parasite growth. By 48 hours, host cell volume is reduced to about 80% of its original value, and parasite volume exceeds that of the host cell (Figure 2C). Before any further analysis of the factors, which according to the model ensure the integrity of the infected cell to the end of the parasite reproduction cycle, it was essential to seek experimental confirmation of the volume changes predicted in Figure 2C to establish the reliability of the model for the interpretation of the homeostatic behavior of IRBCs. The volume changes predicted by the model were tested by comparing the osmotic fragility distribution of IRBC populations with that of coincubated, mostly uninfected red cells (cohorts; "Materials and methods"). If the volume of IRBCs changes as predicted by the model (Figure 2C), then the osmotic fragility of IRBCs would be expected to vary little from controls during the first 16 to 20 hours of the parasite's asexual cycle, to decrease slightly around 24 hours after invasion (late rings to young trophozoites) when IRBC volume is somewhat reduced, and to increase steadily from about 26 to 28 hours onwards (through mature trophozoite, schizont, and segmentor stages). These osmotic fragility changes were followed by comparing hemolysis curves from cohorts (controls) with those of concentrated IRBCs obtained from unsynchronized and synchronized cultures. IRBCs with young trophozoites would require more water gain than control cells to reach their critical hemolytic volume, whereas little extra swelling would be required to bring IRBCs with schizonts or segmentors to their critical hemolytic volume. This could easily be detected as left or right shifts in the hemolysis curves of IRBCs relative to those of cohorts, respectively. Hemolysis curves of IRBCs from unsynchronized cultures may be expected to show both left and right shifts with the crossover points determined by the relative proportions of young and mature parasite forms, whereas curves of IRBCs from synchronized cultures would show mostly or exclusively left or right shifts depending on how well synchronized and at what stage they were. Figure 3 reports the result of an
experiment with IRBCs and cohorts from a nonsynchronized culture,
representative of 6 with similar results. The figure shows the
hemolysis curves of cells from top and bottom gelatin fractions
containing 96% IRBCs and over 96% cohorts, respectively. The inset
(Figure 3) reports the differential cell count in unlysed cells
recovered from microplate wells with the relative tonicities
(RTs) indicated in the figure ("Materials and
methods").
It can be seen (Figure 3) that the hemolysis curve of the IRBCs is right-shifted at the higher tonicities and left-shifted at the lower tonicities, relative to the hemolysis curve of the cohorts. This pattern was reproduced in the 6 experiments of this series. The more mature the parasite population, the lower the RT at which the crossover occurred, as expected from the decline in the relative proportion of young trophozoites. Microscopic inspection of IRBCs that did not lyse in hypotonic media showed a consistent and clear tendency for the progressive decline in the proportion of mature parasite forms and enrichment in young trophozoites with decreasing tonicity (Figure 3, inset). The results of Figure 3 are typical of IRBCs concentrated from
nonsynchronized cultures. According to the model, IRBC samples containing only young trophozoites should show no right shift and those
containing only mature trophozoites should show no left crossover. This
was tested in 3 additional experiments with IRBCs harvested from
synchronized cultures, 1 containing only young trophozoites and 2 containing mature trophozoites, schizonts, and segmentors but no young
parasite forms (Figure 4). It can be seen
that IRBCs with young parasites showed no right shift, only a barely
detectable, not statistically significant left shift (Figure 4A). It
should be noted that in samples from highly synchronized cultures it
may be more difficult to generate IRBC samples with parasites at a
developmental stage precisely within the narrow 2- to 4-hour time
period for which slight dehydration is predicted (Figure 2C) than in
nonsynchronized cultures. Thus, absence of right shift is the only
significant result in Figure 4A. On the other hand, only right shifts
with no crossovers were obtained in samples of IRBCs containing only
mature parasite forms (Figure 4B). Taken together, these results reveal
a stage-related, gradually increasing osmotic fragility pattern,
reflecting the continued volume expansion of IRBCs with mature
parasites, consistent with the model predictions.
In the comparison between predicted and observed results, it is important to bear in mind the approximate nature of the model predictions. The model analyzes an oversimplified condition of identical red cells, defined by mean-valued parameters, invaded by single, identically developing parasites, whereas real IRBC populations, on which the predictions were tested, are highly heterogeneous in certain aspects relevant to volume homeostasis such as multiple invasion, intrinsic variations in membrane area, Hb content, and Hb concentration of invaded RBCs, and in the timing and extent of NPP and Hb consumption development among parasites. Within these semiquantitative constraints, the results in Figures 3 and 4 do support the overall reliability of the volume predictions advanced in Figure 2C, and we can now ask how the model accounts for the volume stability of the infected cells. To understand which are the main factors that prevent premature IRBC
lysis, exploratory simulations were performed varying parameter values
away from their experimentally based default values. The 2 most
critical parameters for the understanding of host cell homeostasis
proved to be NPPK-max and Hmax. Figure
5 shows the results of simulations in
which these 2 parameters were varied alternatively. The effects on host
cell volume of NPPK-max values higher than those measured
by Staines et al15 are illustrated in Figure 5, top panel.
It can be seen that the measured NPPK-max permeability of
1.1 h
Figure 5, bottom panel, illustrates the predicted effects of reduced Hb consumption on host cell volume. Model simulations using Hmax values lower than those measured by Krugliak et al,2 exemplified by the curve with 50% Hmax, predict early host lysis. Thus, integrity of IRBCs by 48 hours requires a sustained and substantial decline in the concentration of Hb, to reduce the colloid osmotic pressure and consequently the rate of swelling of the permabilized host cell. These results provide a novel insight into the significance of the NPPK-max value measured by Staines et al15 suggesting that NPPK-max represents a finely tuned compromise between the need to ensure an optimal traffic route for nutrient and waste product transport6 and the need to preserve the osmotic integrity of the host to the end of the asexual reproduction cycle, with a reasonable safety margin. Could osmotic swelling determine the timing of rupture and merozoite release? In all the experiments of this series no significant lysis was observed until the relative tonicity was reduced lower than 0.9 (Figures 3 and 4B), in broad agreement with the predicted safety margin of about 0.94 (1.6/1.7; Figures 2C, 5). Therefore, it is unlikely that osmotic effects could play any part in the process of rupture. At face value, the prediction that reduced Hb consumption could cause premature host cell lysis (Figure 5) may appear counterintuitive and surprising. After dissipation of the Na+/K+ gradients, the main driving force for IRBC swelling is the colloid osmotic pressure exerted by the impermeant solutes, mostly Hb, in the host cell cytoplasm. A high residual Hb would therefore speed up swelling of the host toward early lysis. The predictions in Figure 5, bearing in mind their approximate nature as discussed above, indicate that for the observed value of NPPK-max, premature hemolysis of IRBCs can be prevented only if the Hb content of the host is reduced by more than 50%. NPPK-max and Hmax are linked in the preservation of IRBC integrity: the lower NPPK-max, the lower the need to consume excess Hb. The evolutionary pressures that set the observed values of NPPK-max and Hmax suggest that the need to ensure high nutrient and waste traffic via NPPs took priority over the energy investments required for host cell ingestion and digestion and to prevent ferriprotoporphyrin IX-induced damage. In conclusion, the high measured values of NPPK-max make excess hemoglobin consumption necessary to ensure the osmotic stability of IRBCs throughout the asexual reproduction cycle of the parasite.
We are grateful to the Wellcome Trust, United Kingdom, for funds, and to Mrs Lynn Macdonald for excellent technical assistance.
Submitted August 29, 2002; accepted January 8, 2003.
Prepublished online as Blood First Edition Paper, January 16, 2003; DOI 10.1182/blood-2002- 08-2654.
Supported by grants 061269 and 059725 from the Wellcome Trust (United Kingdom).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: V. L. Lew, Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom; e-mail: vll1{at}cam.ac.uk.
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K. C. Pandey, S. X. Wang, P. S. Sijwali, A. L. Lau, J. H. McKerrow, and P. J. Rosenthal The Plasmodium falciparum cysteine protease falcipain-2 captures its substrate, hemoglobin, via a unique motif PNAS, June 28, 2005; 102(26): 9138 - 9143. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
= 415 nm) in a microplate reader (Molecular
Devices, Sunnyvale, CA).
, Na+ and K+), and
parasite volume growth. The measured amino acid efflux from IRBCs with
mature trophozoites was 13.5 nmol (108
cells)
t1/2]/s)}
(Figure 
dV' and
VH = VH o + 
) and
relative host (
) and parasite (
) volumes; all volumes are
expressed relative to RBC volume at the time of invasion, defined as 1. The horizontal top line in panel C indicates the mean critical
hemolytic RCV,
): 97% uninfected cohorts and 3%
rings.
