Complete Plasmodium falciparum liver-stage development in liver-chimeric mice

Abstract

Plasmodium falciparum, which causes the most lethal form of human malaria, replicates in the host liver during the initial stage of infection. However, in vivo malaria liver-stage (LS) studies in humans are virtually impossible, and in vitro models of LS development do not reconstitute relevant parasite growth conditions. To overcome these obstacles, we have adopted a robust mouse model for the study of P. falciparum LS in vivo: the immunocompromised and fumarylacetoacetate hydrolase-deficient mouse (Fah-/-, Rag2-/-, Il2rg-/-, termed the FRG mouse) engrafted with human hepatocytes (FRG huHep). FRG huHep mice supported vigorous, quantifiable P. falciparum LS development that culminated in complete maturation of LS at approximately 7 days after infection, providing a relevant model for LS development in humans. The infections allowed observations of previously unknown expression of proteins in LS, including P. falciparum translocon of exported proteins 150 (PTEX150) and exported protein-2 (EXP-2), components of a known parasite protein export machinery. LS schizonts exhibited exoerythrocytic merozoite formation and merosome release. Furthermore, FRG mice backcrossed to the NOD background and repopulated with huHeps and human red blood cells supported reproducible transition from LS infection to blood-stage infection. Thus, these mice constitute reliable models to study human LS directly in vivo and demonstrate utility for studies of LS-to-blood-stage transition of a human malaria parasite.

Figure 1. 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).

Figure 2. 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.

Figure 3. 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.

Figure 4. 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.

Figure 5. 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.