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B. divergens infection in HbSS
can be visualized by both FACS analysis and Giemsa stained smears, was not seen in these sickle cell cultures (Figures 3 and 4). In fact, the presence of free merozoites was noted in most culture supernatants (data not shown), although it is difficult to quantitatively estimate if these numbers are normal as compared to wild-type RBC. However, unlike the fluctuating proportions of the para- site sub-populations seen in both HbAA and HbAS cul- tures, relatively stable proportions of parasite sub-popula- tions in the sickle RBC cultures, after the 24 h time point was a characteristic noted in all 12 HbSS cultures (Online Supplementary Figure S1). This novel holding pattern could potentially signify an inability of the parasites to egress, which in turn could cause the relatively static parasitemia observed in these cultures. Thus, inefficient parasite egress from the host HbSS cells infected in the first round of invasion may not permit new cycles of invasion to take place.
Discussion
Variant RBC are produced from some of the most com- mon human genetic polymorphisms, and their high inci- dence has been ascribed to the evolutionary selection by life-threatening falciparum malaria.38,39 The sickle hemo- globin genotype (HbAS/HbSS) is the best-characterized human genetic polymorphism associated with malaria. In this paper, we have explored the effect of the sickle hemo- globin mutation on a related intra-erythrocytic apicom- plexan parasite, B. divergens. There are several stages in the parasite’s erythrocytic cycle at which RBC mutations can affect parasite infection. The first would be erythrocyte invasion by Babesia merozoites; the next stage susceptible to inhibition is the intra-erythrocytic parasite develop- ment. This category would include impairments in the parasite’s ability to meet its nutritional requirements or changes in the host cell milieu that would be cidal to the parasite. Finally, impairment of red cell rupture and release of infective merozoites at parasite maturity could inhibit increase in infection. Use of in vitro growth assays as our primary outcome, along with a robust sample size of SCD RBC, provided a rare opportunity to systematically exam- ine the cellular determinants of parasite growth in the sickle setting. We present here evidence for atypical pop- ulation progression, a potential loss of merozoite infectiv- ity, and defective egress of the parasite in these hemoglo- binopathic cells (Tables 1-4 and Online Supplementary Table S2). Interestingly, using the Townes mouse model of SCD, and B. microti, we have shown that a similar inhibition results in dramatically low infection rates in HbSS mice as compared to HbAS and HBAA mice.40
Invasion of the human RBC is the central pathogenic step in the life-cycle of Babesia. When Babesia spp. sporo- zoites are first injected into the human host with a tick bite, they target the host RBC immediately, unlike Plasmodium spp. which are required to undergo an exo-ery- throcytic phase in hepatic cells. It is the parasite's ability to first recognize and then invade host RBC that is central to symptomatic human babesiosis, and the parasites invade RBC using multiple complex interactions between parasite proteins and the host cell surface, which have not been fully elucidated.2,41,42 Like Plasmodium, B. divergens has been shown to use GPA and GPB as invasion receptors.33 In vitro studies with P. falciparum have suggested a
decreased invasion and growth of the parasite in sickle cells;43-45 however, some of the older studies have not been able to differentiate between the various phases of the parasite lifecycle. Hence, a deficiency in egress or invasion would both be visualized as an overall decrease in para- sitemia. The use of our platform combining in vitro inva- sion and growth assays with synchronized cultures mon- itored by both FACS analysis and Giemsa smears provid- ed an excellent system to systematically dissect the phase of the erythrocytic cycle impacted by the HbSS environ- ment. The increased DNA content of HbSS cells that con- tributed to the subtle higher invasion percentages was found to be due to the presence of the Howell-Jolly bodies which artefactually increased the DNA load of the cell. When the contribution of the bodies was subtracted, a similar rate of invasion was obtained in all Hb genotypes, indicating a potential difference between malaria and Babesia mechanisms of invasion in sickle cells.
The development of the parasite in HbSS cells in the first 24 h was normal and exhibited all conventional forms reported in vitro cultures by us earlier.34 Thus, rings, paired figure and Maltese Cross forms were all documented in culture. However, a larger than expected proportion of cells hosted detached rings (Figure 4). The multiple unat- tached parasites feature assumed by parasites in HbSS cul- tures suggests that the parasite is able to complete cytoki- nesis shortly after the nuclear duplication, allowing the conclusion of cell division which may not allow the accu- mulation of attached morphological stages such as Maltese-Cross or paired-figures, as seen in HbAA cultures. After 24 h, the progression of cultures is stalled in HbSS cells as seen by FACS analysis (Figure 3A and Online Supplementary Figure S1B-E) where the sub-populations remain in static proportions unlike the dynamic move- ments seen among sub-populations in HbAA (Figure 3C and Online Supplementary Figure S1A) and AS cells (Figure 3B). As this profile is representative of the parasite popu- lation and not individual parasites, it is apparent that, overall, there is no growth in the population despite small increases seen in parasitemia, reflecting a minority of par- asites successfully initiating new cycles of infection. Microscopic analysis of the parasites in HbSS cells reveals normal morphology in terms of size, shape and staining patterns. Ultra-structural analysis may shed more light on potential defects in these parasites, if present.
Egress is a phase of the cycle that, if impacted, can lead to disastrous outcomes for the parasite population pro- gression. In vitro studies with P. falciparum suggest a link between the hydration status of the host RBC and parasite invasion and egress.46,47 The high water-permeability of the RBC ensures their continued osmotic equilibrium in plasma so that they can shrink or swell by the loss or gain of a fluid isosmotic with surrounding plasma. This home- ostatic balance is disrupted in HbSS cells, resulting in altered ion fluxes, ion content regulation, and hydration states in the circulation.48 Malaria parasites have to breach both the parasitophorous vacuolar membrane (PVM) and erythrocyte membrane in order to egress. The altered exit of malaria merozoites from the dehydrated RBC was linked to the reduction of osmotic pressure within the par- asitophorous vacuole that was needed to lyse the com- partment prior to lysis of the RBC membrane. However, intra-erythrocytic Babesia parasites are free in the cyto- plasm of the RBC without being enclosed in a vacuole as the PVM is a transient structure found fleetingly after
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