1 Nature Communications 2013 Vol: 4():1638-. DOI: 10.1038/ncomms2612

Expansion of host cellular niche can drive adaptation of a zoonotic malaria parasite to humans

The macaque malaria parasite Plasmodium knowlesi has recently emerged as an important zoonosis in Southeast Asia. Infections are typically mild but can cause severe disease, achieving parasite densities similar to fatal Plasmodium falciparum infections. Here we show that a primate-adapted P. knowlesi parasite proliferates poorly in human blood due to a strong preference for young red blood cells (RBCs). We establish a continuous in vitro culture system by using human blood enriched for young cells. Mathematical modelling predicts that parasite adaptation for invasion of older RBCs is a likely mechanism leading to high parasite densities in clinical infections. Consistent with this model, we find that P. knowlesi can adapt to invade a wider age range of RBCs, resulting in proliferation in normal human blood. Such cellular niche expansion may increase pathogenesis in humans and will be a key feature to monitor as P. knowlesi emerges in human populations.

Mentions
Figures
Figure 1: P. knowlesi H strain invades young human red blood cells more efficiently.(a) The density of red blood cells increases with age as their volume decreases, attributable to loss of plasma membrane and cytoplasm without concomitant expulsion of intracellular contents such as haemoglobin. They can thus be separated into four fractions by layering them on top of an optimized gradient of Percoll solutions. This is validated by measuring (b) mean cell volume (MCV) and (c) cell haemoglobin concentration mean (CHCM). MCV decreases in the older subpopulations of red blood cells while CHCM increases. Ninety-five per cent confidence interval (CI) for three replicates are shown. (d) Fold change in parasitemia following a single round of invasion, the PEMR, for P. falciparum 3D7 and P. knowlesi H strain into human red blood cell populations of varying age fractions, measured in parallel. (e) Skew in age preference measured as the normalized invasion efficiency of P. falciparum 3D7 and P. knowlesi H strain in each age category relative to the pooled control for each species. Data are from six biological replicates. Statistical significance was determined using a two-way analysis of variance with a Bonferroni correction for multiple comparisons. Ninety-five per cent CI are shown; *P<0.05; **P<0.01; ***P<0.001; P, pooled blood; VY, very young red blood cells; Y, young red blood cells; M, medium age red blood cells; O, old red blood cells. Figure 2: Continuous in vitro culture established by increasing the number of young red blood cells.(a) PEMR of P. knowlesi H strain in cultures with different proportions of very young red blood cells after one round of reinvasion, as calculated in  Statistical significance was determined using Dunnett’s multiple comparison test. Ninety-five per cent confidence interval for five replicates are shown; *P<0.05. (b) Parasitemia of P. knowlesi H strain in cultures with different proportions of very young red blood cells, compared with rhesus blood (small circles), and human blood (large circles). H was continuously cultured in the different proportions by sub-culturing them every 2 days (dashed lines) and parasitemia monitored daily, before and after the split. Figure 3: Potential mechanisms leading to increased parasite proliferation.Schematics show the density of red blood cells (vertical axis) as they age (horizontal axis), with infected cells containing parasites (in blue). (a) At baseline, P. knowlesi has a limited intrinsic invasion ability β and an invasion niche restricted to young red blood cells. Increased parasite proliferation can be achieved by (b) increasing the parasite’s intrinsic invasion ability within this niche, (c) skewing the distribution of red blood cells in the host to increase the proportion of young cells or (d) expanding the parasite’s invasion niche to include older cells. Figure 4: Comparison of different adaptive pathways on predicted in vivo peak parasite densities.(a) Relative changes in peak parasite density (initial R0=3.3 and the parasite can invade red blood cells during the first 20% of their lifespan). Increasing R0 from this baseline is achieved either by increasing the intrinsic invasion ability β of susceptible cells (dashed line) or by increasing the age range of red blood cells that are susceptible to invasion (solid line) up to 100%. (b) Parasite density as the infection progresses for parasites that have attained an R0=6 by either increasing the density of susceptible cells (corresponding to point on solid curve in a), or by increasing their intrinsic invasion ability β (corresponding to point on dashed curve in a). The peak parasite density occurs when Re=1 and is higher for the parasite with an increased density of susceptible cells (solid curve) than for the parasite with increased intrinsic invasion ability β (dashed curve). (c) Changes in susceptible cell density for infection dynamics in b. As infection progresses, the density of susceptible cells declines. The net decrease in susceptible cell density due to invasion is much greater for the parasite with a higher initial density of susceptible RBCs (solid curve) than for the parasite with increased intrinsic growth (dashed curve). Figure 5: Relative peak parasite densities for hosts with skewed red blood cell age distributions.Each curve shows the peak parasite density for a parasite restricted to very young cells, but invading hosts with different red blood cell age distributions (as distinguished by the fold increase in reticulocytes compared to a healthy host). All peak parasite densities are relative to the peak parasite density attained by a parasite that can invade cells of all ages in a healthy host and baseline intrinsic invasion ability (β0). Each curve represents a different intrinsic invasion ability for the restricted parasite: β0 (solid), 0.5β0 (dashed), 2β0 (dot-dashed). The baseline intrinsic invasion ability β0 was chosen so that in a healthy host, a parasite restricted to very young cells has a R0=5. Inset shows examples of different host red blood cell age distributions. A skewed host distribution is modelled by assuming that an elevated rate of RBC destruction across all cell ages is compensated for by an increased production of reticulocytes. It is assumed that the increased production of reticulocytes is sufficient to maintain a normal density of RBCs (5 million cells per μl). The fold increase in reticulocytes for the skewed distributions (inset: black curves) is calculated relative to the RBC distribution of a normal host (inset: blue curve). Figure 6: P. knowlesi H strain adapts to proliferate in normal human blood.(a) Schematic timeline for production of a P. knowlesi line (Hhu) adapted to in vitro culture in normal human red blood cells. (b) PEMR of primate-adapted P. knowlesi H strain compared with the human-adapted strain P. knowlesi Hhu into human red blood cell populations of varying age, and (c) relative invasion efficiency of P. knowlesi H and Hhu into red blood cell populations of varying age, calculated as in . Ninety-five per cent confidence interval for five biological replicates are shown. Statistical significance was determined using a two-way analysis of variance analysis with a Bonferroni correction for multiple comparisons. *P<0.05; **P<0.01; ***P<0.001. (d) Predicted in vivo infection dynamics assuming an approximately uniform distribution of RBCs for (i) P. vivax (Pv) restricted to reticulocytes (R) (red), (ii) P. falciparum (Pf) able to invade red blood cells of all ages (white), (iii) P. knowlesi (Pk) restricted to very young (VY) red blood cells (very light grey), (iv) Pk restricted to very young and young (VYY) red blood cells (light grey), (v) Pk able to invade very young to medium (VYM) red blood cells (medium grey) and (vi) Pk able to invade red blood cells of all ages (VYO) (dark grey). For ease of interpretation, we assume that (i) the initial parasite invasion rates for Pv, Pf and the Pk strain restricted to very young cells are the same and (ii) the intrinsic invasion abilities of all Pk strains are the same. Figure 7: Sensitivity analysis for peak parasite densities for an initially healthy host.Each shaded region shows the simulated peak parasite densities for different assumptions about parasite invasion. Moving to the right each shaded region shows how peak parasite density changes as the parasite adapts to be less age restricted. Moving upwards each shaded region shows how peak parasite density changes as the overall percentage of cells that are susceptible (within the susceptible age range) changes from 1 to 100%. Parameter values are chosen so that the middle shaded region corresponds to R0=5, when 40% of the RBC lifespan is susceptible. Note that this implies that the intrinsic invasion ability β is constant as the parasite age restriction changes but will depend on the maximum overall fraction of susceptible cells. For example, if only 1% of cells within the susceptible age range are susceptible (lower red circle) then β will be larger than if 100% of cells within the susceptible age range are susceptible (upper red circle)—in both cases β has been chosen so that R0=5. The intrinsic invasion ability β for the upper (lower) shaded region are twice (half) the value for the middle shaded region. All other parameter values are the same between regions.
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References
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    • . . . P. knowlesi can invade both reticulocytes and normocytes in its natural host, the long-tailed macaque15, as well as in the rhesus macaque animal model16 . . .
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    • . . . P. knowlesi can invade both reticulocytes and normocytes in its natural host, the long-tailed macaque15, as well as in the rhesus macaque animal model16 . . .
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    • . . . This type of adaptation for proliferation in human hosts may significantly increase pathogenesis17, 18, 19. . . .
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    • . . . This type of adaptation for proliferation in human hosts may significantly increase pathogenesis17, 18, 19. . . .
    • . . . Previous theoretical work has shown that only modest between-host R0 values are required for the spread and maintenance of emerging pathogens in human populations if pathogen mutability is high19 . . .
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    • . . . Parasites were maintained in vitro in rhesus (P. knowlesi) or human 0+ RBCs (P. falciparum) at 2% haematocrit using standard culture conditions20, 23 . . .
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    • . . . Owing to its low prevalence in humans, most P. knowlesi parasites are unlikely to encounter hosts with adaptive immune responses, and we focus on initial parasite replication in the absence of an immune response like previous frameworks24, 27. . . .
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    • . . . We find that the highly skewed distribution of RBCs required to attain high parasite densities, analogous to the in vitro culture system described above, would be associated with severe anaemia (Fig. 5), and this has not been observed in P. knowlesi infections5, 7, 30 . . .
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    • . . . P. knowlesi will be particularly useful as a model system for cell biological studies, due to the relatively large size and stability of the invasive merozoite form31, 32, and will provide a powerful system for genetic analyses33 . . .
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    • . . . P. knowlesi will be particularly useful as a model system for cell biological studies, due to the relatively large size and stability of the invasive merozoite form31, 32, and will provide a powerful system for genetic analyses33 . . .
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    • . . . P. knowlesi will be particularly useful as a model system for cell biological studies, due to the relatively large size and stability of the invasive merozoite form31, 32, and will provide a powerful system for genetic analyses33 . . .
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    • . . . Coevolution between humans and malaria parasites has led to a range of human genetic polymorphisms encoding RBC heterogeneity on a population level34 . . .
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    • . . . These mutations may restrict parasite proliferation in vivo, as exemplified by the reduced parasite densities and disease severity afforded by the sickle cell trait35, 36, and the selection against the DARC surface receptor, which is required for P. vivax invasion, in large areas of sub-Saharan Africa37, 38 . . .
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    • . . . These mutations may restrict parasite proliferation in vivo, as exemplified by the reduced parasite densities and disease severity afforded by the sickle cell trait35, 36, and the selection against the DARC surface receptor, which is required for P. vivax invasion, in large areas of sub-Saharan Africa37, 38 . . .
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    • . . . These mutations may restrict parasite proliferation in vivo, as exemplified by the reduced parasite densities and disease severity afforded by the sickle cell trait35, 36, and the selection against the DARC surface receptor, which is required for P. vivax invasion, in large areas of sub-Saharan Africa37, 38 . . .
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    • . . . These mutations may restrict parasite proliferation in vivo, as exemplified by the reduced parasite densities and disease severity afforded by the sickle cell trait35, 36, and the selection against the DARC surface receptor, which is required for P. vivax invasion, in large areas of sub-Saharan Africa37, 38 . . .
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    • . . . Differences between host species in RBC surface receptors required for invasion are also likely to be a major barrier for zoonotic transmission of malaria parasites between species39 . . .
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    • . . . It is likely that the shift in age preference exhibited by our adapted line is linked to changes in the utilization of P. knowlesi invasion ligands, as has been observed in rodent malaria parasites40 . . .
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    • . . . The analyses presented here focus on single species infections, but coinfection with other human malaria parasites is relatively common for P. knowlesi infections41, 42 and could also impact the severity of infection43, as could other underlying conditions causing anaemia . . .
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    • . . . The analyses presented here focus on single species infections, but coinfection with other human malaria parasites is relatively common for P. knowlesi infections41, 42 and could also impact the severity of infection43, as could other underlying conditions causing anaemia . . .
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    • . . . The analyses presented here focus on single species infections, but coinfection with other human malaria parasites is relatively common for P. knowlesi infections41, 42 and could also impact the severity of infection43, as could other underlying conditions causing anaemia . . .
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    • . . . Although human infections are still limited in their geographical range by vector distributions44, 45, the continuing encroachment of human populations on macaque habitat provides increasing exposure to non-human primate malaria parasites . . .
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    • . . . Although human infections are still limited in their geographical range by vector distributions44, 45, the continuing encroachment of human populations on macaque habitat provides increasing exposure to non-human primate malaria parasites . . .
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    • . . . Strikingly, although humans are likely to be frequently exposed to numerous related cynomolgous malaria parasites, such as P. cynomolgi and P. inui, P. knowlesi is unique in its emergence into human populations2, 46 . . .
  47. Ciucă, M.; Romîne, A. R. P. , (1955) .
    • . . . Of concern, parasites that are more virulent were readily obtained following continuous in vivo passage of P. knowlesi in humans for the treatment of neurosyphilis47. . . .
  48. Golenda, C. F.; Li, J.; Rosenberg, R. Continuous in vitro propagation of the malaria parasite Plasmodium vivax Proc. Natl Acad. Sci. USA 94, 6786-6791 (1997) .
    • . . . Whole blood from haemochromatosis patients undergoing phlebotomy therapy was enriched for very young human RBCs as described previously48, 49 with minor modifications . . .
  49. Russell, B. A reliable ex vivo invasion assay of human reticulocytes by Plasmodium vivax Blood 118, e74-e81 (2011) .
    • . . . Whole blood from haemochromatosis patients undergoing phlebotomy therapy was enriched for very young human RBCs as described previously48, 49 with minor modifications . . .
  50. Gravenor, M. B.; McLean, A. R.; Kwiatkowski, D. The regulation of malaria parasitaemia: parameter estimates for a population model Parasitology 110, 115-122 (1995) .
    • . . . Our mathematical model is similar to previous models that focus on the impact of resource limitation on within-host infection dynamics24, 27, 50 . . .
    • . . . A common feature of mathematical models focusing on the impact of resource limitation on within-host malaria infection dynamics is that they overestimate the initial peak parasiteamias24, 25, 50 because they ignore immunity and assume homogenous mixing of merozoites and RBCs . . .
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