Complete Plasmodium falciparum liver-stage development in liver-chimeric mice (original) (raw)
P. falciparum in vivo LS development in the FRG huHep mouse. The FRG huHep mice (all female) used in this study were obtained from the Yecuris Corp., and the huHep repopulation index of the mouse liver was estimated based on the levels of human serum albumin. Repopulation of all mice used in the study ranged between 60% and 90% (data not shown). Sporozoites used for the study were generated in Anopheles stephensi mosquitoes that had been fed on mature gametocyte cultures of P. falciparum NF54 parasites or, in one instance, the GFP-expressing NF54HT-GFP parasite. Mice were injected intravenously with sporozoites and sacrificed 3, 5, 6, and 7 days after infection, at which time liver tissue was collected for transcriptional analysis, histological evaluation and indirect immunofluorescence assays (IFAs).
IFAs on infected liver sections demonstrated that P. falciparum LS developed in the FRG huHep mice. At day 3 after infection, spherical LS were detected using a circumsporozoite protein (CSP) antibody, which localizes to the parasite surface and, as expected, was expressed in a circumferential pattern (Figure 1A). Using an antibody specific for human FAH, it was evident that P. falciparum LS always grew within the huHeps (Figure 1A). DAPI staining showed between 4 and 8 nuclear centers on day 3 of LS, indicating that parasite DNA replication was progressing. At day 5 after infection, LS had further grown and maintained their near spherical shape. The parasites were specifically labeled with an antibody to exported protein-2 (EXP-2) (26) in a pattern indicative of PV localization (Figure 1B). EXP-2 was previously localized to the PV membrane (PVM) of blood stages (27). More recently, EXP-2 was shown to be part of the P. falciparum translocon of exported proteins (PTEX) (28), a translocon that is believed to be responsible for the trafficking of exported parasite proteins into the erythrocyte cytoplasm. A further protein component of the translocon is PTEX150, and interestingly, we also detected expression of this protein at 5 days of LS development (Figure 1B). Its localization was similar to that seen for EXP-2 (Figure 1B). Thus, components of the translocon are present in LS and are localized to the PV. Day 5 LS still expressed CSP (Figure 1B), the transcript of which was also detected at this time point in the SCID Alb-uPA huHep mouse LS infections (16). Day 5 LS also expressed PF10_0164 (ETRAMP10.3) (Figure 1B) in the PVM, the syntenic ortholog of the rodent malaria parasite gene coding for UIS4 (14). At day 7 after infection, we observed large multinucleated LS schizonts that had displaced the surrounding tissue. The LS were readily detected with EXP-2 and merozoite surface protein 1 (MSP1) antibodies (Figure 1, C and D). Codetection with the FAH antibody clearly showed the LS contained within a vastly expanded huHep with only remnant cytoplasm and a single nucleus pushed to the periphery of the infected cell (Figure 1C). The pattern of EXP-2 expression ceased to be circumferential, resulting in a complex internal pattern of staining as seen in Figure 1C, likely due to EXP-2 expression being associated with the PV lumen at this developmental stage. The PVM markers EXP-1 and EXP-2 did not overlap in late LS parasites, indicating that EXP-1 remained in the PVM during late LS development, but EXP-2 did not (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI62684DS1). Furthermore, at day 7, PTEX150 (28) expression and EXP-2 expression showed a partially overlapping staining pattern (Supplemental Figure 1B). Late LS development is marked by the expression of MSP1 and subsequent formation of merozoites, with MSP1 expression localized to the merozoite surface. Indeed, at day 7 after infection, there was robust expression of MSP1 (Figure 1D). The pattern of MSP1 staining was indicative of cytomere formation, where multiple invaginations of the parasite plasma membrane eventually lead to the pinching off of membrane around each nascent exoerythrocytic merozoite. In contrast with the MSP1 staining, the PVM marker EXP-1 could clearly be seen to delineate the confines of the LS parasite where cytomere formation was still taking place (Figure 1D). All day 7 LS examined showed robust expression of MSP1, and in highly humanized parts of the liver substantial numbers of LS were observed (Figure 1E). IFA analysis also demonstrated the lack of MSP1 expression in 3 and 5 day LS and the lack of CSP expression in 7 day LS (data not shown).
P. falciparum LS development in FRG huHep mice. Infected liver sections were assayed by indirect immunofluorescence using antibodies specific to P. falciparum for parasite detection. (A) LSs at day 3 of infection were visualized using antibodies to parasite CSP, which localizes to the parasite surface. (B) LSs at day 5 of infection were visualized with antibodies to EXP-2 and PTEX150, components of the Plasmodium translocon of exported proteins (28), which were both robustly expressed (3 panels on the left), as well as the PVM protein PF10_0164 (14) and CSP (3 panels on the right). LSs at day 7 of infection were visualized with antibodies to EXP-2 (C), MSP1 (E), and in combination with MSP1 and EXP-1 (D). huHeps were visualized with antibody to human FAH in A, C, and E, and the liver sections were visualized by differential interference contrast microscopy (DIC) in C and D. DNA was visualized with DAPI in all panels. Note the nucleus of the infected hepatocyte in C, which has been pushed to the extremity of the infected hepatocyte (white arrow in the DNA panel). Scale bars: 10 μm (A, B, and D); 20 μm (C); 100 μm (E).
Previous studies using intravital microscopy of fluorescent rodent malaria LS (both P. berghei and P. yoelii) have shown that merozoites are frequently released from infected hepatocytes as merosomes: packets of multiple merozoites held together by a surrounding host hepatocyte-derived membrane (9, 29, 30). The rodent malaria merosomes bud off into the sinusoidal lumen and are carried in the bloodstream to the lung, where they burst, releasing erythrocyte-infectious merozoites (29). However, it remains unknown whether this key feature of late LS development is conserved in human malaria parasites. Here, we observed, for what we believe is the first time, budding of P. falciparum exoerythrocytic merozoite aggregates at day 7 after infection (Figure 2A), suggesting that merosome formation is conserved in human malaria parasites and can be modeled in the FRG huHep mouse model. Individual, fully segregated merosomes that appeared to be surrounded by host hepatocyte–derived membrane were observed (Figure 2B), and individual merozoites were visualized within the mature LS as well as within the merosomes (Figure 2C). Merosomes sometimes stayed in close proximity to the mature LS and appeared to retain a narrow connection (Figure 2C). However, often, less organized release of merozoites from late LS into the adjacent tissue was seen, and these releases contained individual merozoites (Figure 2D and Supplemental Figure 2). The full maturation of rodent malaria parasite LS in vitro and in vivo was shown to be accompanied by the breakdown of the PVM (29, 31), and we also saw this process in P. falciparum LS. This was indicated by the observation that the PVM marker EXP-1 in a fully mature LS labeled only a small PVM remnant (Figure 2E and Supplemental Figure 3).
Maturation of P. falciparum LSs and exoerythrocytic merozoite release in FRG huHep mice. Indirect immunofluorescent images of mature P. falciparum LS parasites were captured at day 7 of infection. The merozoites were localized with antibodies to MSP1. The PVM was localized with antibodies to EXP-1. DNA was visualized with DAPI, and differential interference contrast microscopy images of the liver sections were captured. (A) Appearance of a budding merosome (white arrow, MSP1 panel) is associated with a perturbation in the membrane surrounding the mature LS (white arrow, DIC panel). (B and C) Merosomes adjacent to mature LS parasites (white arrows, MSP-1 panels). Note that the DIC image in B suggests that the merosome is ensconced within a membrane (white arrow, DIC panel and in the magnification shown in the lower right of the panel). The DIC image inset in B shows that the membranes of the mature LS and the membrane of the merosome have completely separated. (D) Unorganized merozoite masses appear to be spilling into the surrounding liver tissue, indicating that merozoite release occurs not only in merosomes. Note that individuated merozoites are visible. (E) A mature LS with multiple merozoite release events (white arrows, MSP1 panel) shows that the PVM has broken down (31), resulting in the presence of a small EXP-1–positive PVM remnant (white arrow, EXP-1 panel). Scale bars: 10 μm.
LS growth and gene transcription in the FRG huHep mouse. To further investigate LS growth in FRG huHep infections, the maximum area of multiple LS cross sections (n ≥ 14) was determined (Figure 3A). In day 3 infections, maximum LS diameter ranged from 5.0 to 8.4 μm, and the maximum LS area was 42.2 ± 9.6 μm2; at day 5, maximum LS diameter ranged from 17 to 26 μm, and the maximum LS area was 322 ± 71 μm2; and finally at day 7, maximum LS diameter ranged from 50 to–80 μm, and the maximum LS area was 3020 ± 730 μm2. These measurements demonstrate the remarkable growth acceleration during the final 2 days of LS development as well as the relative uniformity of LS schizont size at distinct developmental time points; the SD as a percentage of the mean was similar throughout development. Importantly, growth-retarded P. falciparum LSs, as often seen in in vitro culture, were never observed in day 7 FRG huHep infections.
LS growth and parasite gene expression in infected FRG huHep mice. (A) LS size was measured based on indirect immunofluorescence analysis of infected liver sections using the maximal diameters of parasites at 3, 5, and 7 days after infection. At least 14 LSs were analyzed for each time point and the results represented by LS parasite area. Data represent mean ± SD. (B) RT-PCR on RNA isolated from infected FRG huHep livers demonstrates transcription of hapoAI, mGAPDH, and P. falciparum 18S rRNA (Pf 18S) at 3, 5, and 7 days after infection with sporozoites. (C) Transcripts for the parasite merozoite-stage proteins MSP1, EBA-175, and AMA-1 are detected in LS at day 7 after infection (+) and are not present in the minus reverse transcriptase control (–). A 100-bp DNA ladder was run in the far left lanes of the gels as shown in B and C, and pertinent fragment sizes are shown to the left of the gel images.
To amplify P. falciparum transcripts using RT-PCR, RNA was isolated from infected liver tissue, reverse transcribed, and subject to amplification with oligonucleotide primers specific for human apoAI (hapoAI), mouse GAPDH (mGAPDH), and P. falciparum 18S rRNA. Parasites were detectable by RT-PCR at all time points of development, and amplification of transcribed parasite 18S rRNA was greatest in day 7 infections compared with either day 5 or day 3 infections, proportional to the massive increase in parasite biomass (Figure 3B). To further analyze LS maturation, we amplified transcripts for 3 genes indicative of merozoite maturation: MSP1, erythrocyte-binding antigen-175 (EBA-175), and apical membrane antigen-1 (AMA-1) (Figure 3C) at day 7 of infection. This is encouraging in that it not only implies that LS infection rates are high enough in the FRG huHep mice to produce sufficient transcript for amplification from whole infected tissue, but also that stage-appropriate transcripts are being expressed during LS maturation.
Ex vivo visualization of fluorescent P. falciparum LSs in the FRG huHep mouse. Previous analyses of rodent malaria LS transcriptomes and proteomes have relied on FACS of fluorescent parasites from hepatocytes isolated from perfused, collagenase-treated, infected livers (10). P. falciparum LS transcriptomic and proteomic analyses would be invaluable for research into this life-cycle stage, and the FRG huHep mouse could be utilized for FACS of fluorescent P. falciparum LS. A fluorescent P. falciparum 3D7 transgenic parasite was recently created that expressed GFP under control of the EF1α promoter throughout the life cycle (32). However, the P. falciparum 3D7 parasite line is not a robust gametocyte producer. Therefore, we recreated the transgenic parasite in the P. falciparum NF54 parasite line. Blood stages, oocysts, and salivary gland sporozoites from this line (NF54HT-GFP) were fluorescent (data not shown). NF54HT-GFP sporozoites (2 million) were injected into a FRG huHep mouse, and 6 days later, the mouse was sacrificed. The liver was removed, and lobes were immediately sectioned ex vivo without fixation and analyzed by fluorescent microscopy. GFP-fluorescent LS parasites were readily visualized (Supplemental Figure 4A). IFA following fixation and sectioning of liver lobes also showed the robust expression of GFP in the day 6 LS (Supplemental Figure 4B).
Quantification of P. falciparum LS burden in the FRG huHep mouse and the level of LS infection in the FRG huHep and FRG NOD huHep mice. We tested to determine whether the level of huHep repopulation within the FRG huHep mouse liver correlated with LS burden. We reasoned that the higher the repopulation, the larger the LS burden would be in any one part of the liver. Thus, on the same day, we injected 2 FRG huHep mice with approximately 80% repopulation with equal numbers of P. falciparum sporozoites (4 million) and sacrificed the mice at 7 days after injection. The mice were littermates and had received the same donor hepatocytes on the same day, but varied in their total human albumin levels. Liver sections (between 16 and 21) were taken from multiple lobes, and RNA was isolated from each individual section. After DNase treatment and reverse transcription, equal amounts of cDNA from each section were subjected to quantitative RT-PCR (qRT-PCR) analysis of parasite burden (based on parasite 18S rRNA transcription) and humanization (based on a ratio of hapoAI transcripts to mGAPDH transcripts). This allowed us to plot LS biomass with the extent of humanization. LS burden directly correlated in a linear fashion to the degree of humanization in a given sample of liver tissue (Figure 4A). The results also showed that once repopulation falls below a certain threshold, LS burden is undetectable, as demonstrated by a positive, non-zero X-intercept. Moreover, the slope of the best-fit line was similar for the 2 mice, suggesting reproducibility.
Correlation of LS burden with liver humanization in FRG huHep mice and comparison of LS density in _P. falciparum_–infected FRG huHep mice, FRG NOD huHep mice, and _P. yoelii–_infected BALB/cJ mice. (A) Liver tissue fragments (each point on the graph represents a single sample) taken from a 7-day LS infection of 2 FRG huHep mice (female littermates who received the same human donor hepatocytes) were analyzed by qRT-PCR for P. falciparum 18S rRNA burden (Pf 18S, arbitrary units) as well as the level of humanization based on the ratio of hapoAI transcripts relative to mGAPDH transcripts (arbitrary units). The results show a statistically significant, linear relationship (coefficient of determination, R2 = 0.87–0.89) between LS burden and liver humanization in the 2 mice. (B) The level of P. falciparum LS burden in the FRG huHep mouse was compared with that of the FRG NOD huHep mouse and P. yoelii rodent malaria LS burden in BALB/cJ mice. LS burden is shown as LS/cm2 50-μm liver section/106 sporozoites injected. Average LS counts per liver section were determined by analyzing at least 6 nonserial 50-μm liver sections from 3 individual mice. Humanized mice had huHep repopulation levels above 80%. The results show that the FRG huHep and FRG NOD huHep mice support robust P. falciparum LS infections. Data for B represent mean ± SD.
We next set out to determine the P. falciparum LS infection densities in the FRG huHep mice and FRG NOD huHep mice and compared them with a rodent malaria/mouse combination — the P. yoelii parasite in the BALB/cJ mouse. Nonserial 50-μm liver slices were analyzed by IFA for LS numbers, and these numbers were related to the areas of the liver slices and the numbers of sporozoites injected. Calculations were made on 3 FRG huHep and FRG NOD huHep mouse livers (with huHep repopulation levels above 80%) assayed at 7 days after injection of between 2 and 4 million P. falciparum sporozoites. The FRG huHep mouse was approximately 50% as susceptible to a P. falciparum LS infection (21 LS/cm2 50-μm liver section/106 sporozoites injected) as the BALB/cJ mouse was to P. yoelii LS infection (46 LS/cm2 50-μm liver section/106 sporozoites injected), whereas LS infection in the FRG NOD huHep mouse, although robust, was lower (8 LS/cm2 50-μm liver section/106 sporozoites injected) (Figure 4B).
Complete P. falciparum LS development in the FRG NOD huHep mouse and transition from LS–to–blood-stage infection. Demonstrating the completion of P. falciparum LS development with formation of infectious exoerythrocytic merozoites and the subsequent transition to blood-stage infection is an important next step. Following this transition in an animal model would further aid studies into the biology of late LS and exoerythrocytic merozoite infection of hurbc. Ultimately it might even allow for conducting parasite genetic crosses in a combined huHep/hurbc model. However, severe challenges have been encountered in the creation of immunocompromised mice that can support human hematopoietic development through the xenotransplantation of human hematopoietic stem cells, and this is especially true for erythrocyte development (33). The C57BL/6 background of the FRG huHep mouse does not support engraftment with hurbc, and this is partly due to the incompatibility of mouse macrophage–expressed signal regulatory protein α (SIRPα) with human CD47, which leads to the rapid clearance of hurbc by mouse macrophages (33, 34). A mutation in the SIRPα gene in the NOD mouse prevents this incompatibility, and thus, clearance of hurbc is reduced. We thus injected FRG NOD huHep mice with P. falciparum sporozoites to ensure they were able to support complete LS development. Indeed, at 7 days after sporozoite injection, mature LSs were seen in the livers of the mice, comparable to those observed in FRG huHep mice (Supplemental Figure 5).
We next explored whether the transition of the P. falciparum LS infection to a blood-stage infection in the FRG NOD huHep mouse is possible. Six days after sporozoite injection, FRG NOD huHep mice were injected intravenously with hurbc and again on day 7 after sporozoite injection. Blood was removed from the mouse by cardiac puncture at day 7. The buffy coat was removed, and the blood was washed with standard in vitro P. falciparum culture medium, supplemented with an equal volume of hurbc, and cultured at 4% hematocrit. In 2 independent experiments with a total of 4 FRG NOD huHep mice, blood-stage parasites were consistently detected by Giemsa-stained thin blood smears from 1 to 5 days after the initiation of the in vitro culture. The parasites were subsequently maintained in continuous in vitro culture, and growth rates (Figure 5) were comparable to those of the parent NF54 strain used for mosquito infections and sporozoite production. In addition, the mouse-derived asexual parasite cultures successfully converted to produce gametocytes (Supplemental Figure 6A), which when fed to mosquitoes, resulted in the production of oocysts (Supplemental Figure 6B). Furthermore, salivary gland sporozoite numbers between the NF54 strain routinely used for our mosquito infections and the humanized mouse-transitioned NF54 parasites were similar (data not shown).
P. falciparum LS infection in FRG NOD huHep mice transitions to blood-stage infection. Growth of blood-stage P. falciparum parasites in in vitro culture that were obtained from infected FRG NOD huHep mice 7 days after sporozoite injection is shown. Infected mice were injected with hurbc on day 6 and 7 after sporozoite injection to allow asexual erythrocytic infection. Parasite-infected blood was removed from the mice and placed in in vitro rbc culture. Humanized mouse infection-derived asexual blood-stage parasites from 3 individual FRG NOD huHep mice (white bars) and parent NF54 parasites (black bars) were assayed for growth over 4 days in triplicate. Giemsa-stained thin blood smears were assayed for percentage of parasitemia and also to demonstrate the presence of healthy parasites in the culture (inset, left panel, ring stage; middle panel, trophozoite; right panel, schizont). Black arrows point to infected cells. Blood-stage parasites derived from sporozoite-induced FRG NOD huHep mouse infections show normal in vitro growth characteristics. Scale bar: 10 μm. Data represent mean ± SD.