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J.R. Cursino-Santos et al.
invasion of the Babesia merozoite. Thus, the lack of osmotic pressure in the infected sickle RBC may not impact Babesia parasite egress. Additionally, FACS analy- sis of the cultures at later time points do not support the typical loss of egress phenotype, which presents as a build-up of 4N and >4N populations in a single cell, as seen in our previous work with egress inhibitors.31 Although there were cells that hosted multiple parasites (>4N), these appeared to be a result of the normal prolif- erative cycle to build the population structure, but they were not the majority of infected cells. However, the novel holding pattern reported here (Online Supplementary Figure S1), where the sub-populations are maintained after 24 h in the same ratios, indicate that egress may be com- promised in these cells. Thus, it is possible that HbSS cells were not as efficient in supporting egress as HbAA cells, but other factors also contribute to the low infection rates seen in these cultures. Production of viable, infective merozoites within HbSS cell appeared to be another limi- tation of these cultures. Although free merozoites were seen in most HbSS culture supernatants, they did not appear to be able to successfully initiate new rounds of infection. Rescue experiments with both fresh HbSS and HbAA cells indicate that the defect may not lie in the unavailability of optimal host cells but rather with the merozoite, despite them presenting normal morphology (Online Supplementary Table S2). Detailed morphological examination of these zoites by electron microscopy may provide clues to their lack of infectivity.
Despite years of research effort, the mechanism of pro- tection of the sickle trait in malaria remains unclear. A number of cellular, biochemical and immune-mediated mechanisms have been proposed, and it is likely that mul- tiple complex mechanisms are responsible for the observed protection. Invasion and growth of P. falciparum in vitro within HbAS RBC is reduced in low oxygen tension growth conditions (<5% O2).43,49 Some have proposed that the increased sickling of infected HbAS RBC, due to poly- merization of sickle Hb, may be a mechanism for impaired growth under low O2 conditions.50,51 However, in our in vitro assays, which utilize micro-aerophilus condi- tions with 5% O2, no inhibition of Babesia growth was observed in the HbAS cells, in which all five samples yielded comparable support of parasite growth as that seen in control HbAA RBC. Unlike Plasmodium, which digests Hb to meet its nutritional needs, Babesia does not digest any of the Hb, which may account for the lack of
inhibition of intra-erythrocytic proliferation seen in the HbAS cells (no significant difference between HbAA and HbAS cells; P>0.05) (Table 3). As the assays reported here are purely in vitro assays, the effect of in vivo parameters like increased cytoadhesion,52 splenic retention,53 and altered immune system response, as seen in enhanced phagocytosis and induction of inflammatory cytokines following endothelial activation, are not factored in, which may lead to an overall protection against the para- site in vivo, as seen in malaria. Future studies documenting the incidence of babesiosis in the sickle cell trait popula- tion compared to that in the HbAA individuals will con- firm these findings.
In this paper, we present evidence for altered parasite population progression caused potentially by defective merozoite maturation and/or defective egress from the sickle cell anemia RBC. The latter two phenomena when present alone or together may explain the inability of HbSS cells to support high infection rates in vitro. It is not clear whether the parasite would behave similarly in vivo. We have recently shown in a mouse SCD model similar inhibition of parasite population progression, and the results presented in this study confirm the inability of the Babesia parasite to thrive in the sickle cell anemia setting.40 In a significant contrast to malaria, sickle trait cells seemed to sustain parasite infection rates comparable to those of wild-type RBC, and, once again, these in vitro studies need to be supplemented by comparable in vivo analyses under conditions of differing oxygen tension, which has been shown to play a role in suppressing malaria parasitemia. Patients with SCD are on chronic transfusion therapy and may develop severe transfusion-associated Babesia infec- tion.29 Studies dissecting the cellular and molecular mech- anisms of parasite growth within sickle cell patients that may impact the pathogenesis of babesiosis in sickle cell and other hemoglobinopathies are needed to define potential novel therapies against this disease.
Acknowledgments
We thank Vijay Nandi, PhD (Laboratory Data Analytic Services, NYBC) for help with the statistical analysis.
Funding
This research was funded by NIH grant to CL-HL140625 and Hugoton Foundation grant to JRCS-HUG575.
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