Altered parasite life-cycle processes characterize Babesia divergens infection in human sickle cell anemia

Abstract

Babesia divergens is an intra-erythrocytic parasite that causes malaria-like symptoms in infected people. As the erythrocyte provides the parasite with the infra-structure to grow and multiply, any perturbation to the cell should impact parasite viability. Support for this comes from the multitude of studies that have shown that the sickle trait has in fact been selected because of the protection it provides against a related Apicomplexan parasite, Plasmodium, that causes malaria. In this paper, we examine the impact of both the sickle cell anemia and sickle trait red blood cell (RBC) environment on different aspects of the B. divergens life-cycle, and reveal that multiple aspects of parasite biological processes are altered in the mutant sickle anemia RBC. Such processes include parasite population progression, caused potentially by defective merozoite infectivity and/or defective egress from the sickle cell, resulting in severely lowered parasitemia in these cells with sickle cell anemia. In contrast, the sickle trait RBC provide a supportive environment permitting in vitro infection rates comparable to those of wild-type RBC. The elucidation of these naturally occurring RBC resistance mechanisms is needed to shed light on host-parasite interaction, lend evolutionary insights into these related blood-borne parasites, and to provide new insights into the development of therapies against this disease.

Figure 1.

The expansion of B. divergens population was inhibited in HbSS cultures. Comparison of the growth inhibition rate between parasite cultures grown in HbSS cells and HbAS cells relative to growth in wild-type HbAA as measured by parasitemia. (A) Between cultures originating from a single HbSS and single HbAS donor. Values calculated based on data from Table 1. (B) Among HbSS cells from 11 different sickle cell disease patients, showing that, despite variation in parasitemia in individual cultures, all exhibit high degrees of inhibition of culture progression. Values calculated based on data from Table 2.

Figure 2.

Invasion efficiency is similar in HbAA, HbAS and HbSS cells. FACS analysis of DNA content in representative (A,C,E) uninfected red blood cells (RBC) and (B,D,F) infected RBC, along with Giemsa stained smears. (A) HbSS cells reveal DNA positive population (0.1-0.3%) from Howell Jolly (H-J) Bodies in cells, Giemsa image shows a dense H-J body, (marked by asterisk). (C) HbAS cells and (E) HbAA cells do not have DNA positive population and Giemsa smear shows absence of bodies. (B) Parasite cultures in the same HbSS cells show higher Vybrant positive population than uninfected, being composed of both cells containing H-J bodies and parasites. The difference between panels (B) and (A) yields invasion parasitemias (see Table 4 for data). Giemsa smear shows distinct ring formed 1 hour (h) post invasion, presenting a clear difference from H-J body. (D and F) 1 h post-invasion cultures of (D) HbAS cells and (F) HbAA cells showing Vybrant positive population, and 1 h post-invasion Giemsa image showing newly invaded parasites (marked by arrows).

Figure 3.

Distribution of parasite sub-populations cultured in three types of red blood cells (RBC), based on genome content reveals all sub-populations are represented in the cultures. (A) HbSS. (B) HbAS. (C) HbAA. Samples were collected at 1 hour (h), 24 h and 48 h and the percentage of each infected RBC sub-population was determined by FACS using VybrantRDyeCycleTMGreen dye to quantify the amount of parasitic DNA within infected cells, where (N) is the number of parasite genomes. (Top left) HbSS cells show higher numbers of 2N cells at 1 h time point compared to (left middle) HbAS and (left bottom) HbAA cells confirming a higher frequency of multiple invasion events in HbSS. (Middle and right panels) Parasite population structure at 24 and 48 h showing all sub-populations represented in all three cell genotypes.

Figure 4.

Parasites grown in HbSS cells exhibit atypical morphology although normal parasite forms are also seen in the same cultures. (A-C) Normal morphology of parasite seen as paired figures or Maltese Crosses or double paired figures (D-M) Unusually high numbers of detached rings seen at high frequency in all HbSS parasite cultures.