Suppression of host p53 is critical for Plasmodium liver-stage infection

Abstract

Plasmodium parasites infect the liver and replicate inside hepatocytes before they invade erythrocytes and trigger clinical malaria. Analysis of host signaling pathways affected by liver-stage infection could provide critical insights into host-pathogen interactions and reveal targets for intervention. Using protein lysate microarrays, we found that Plasmodium yoelii rodent malaria parasites perturb hepatocyte regulatory pathways involved in cell survival, proliferation, and autophagy. Notably, the prodeath protein p53 was substantially decreased in infected hepatocytes, suggesting that it could be targeted by the parasite to foster survival. Indeed, mice that express increased levels of p53 showed reduced liver-stage parasite burden, whereas p53 knockout mice suffered increased liver-stage burden. Furthermore, boosting p53 levels with the use of the small molecule Nutlin-3 dramatically reduced liver-stage burden in vitro and in vivo. We conclude that perturbation of the hepatocyte p53 pathway critically impacts parasite survival. Thus, host pathways might constitute potential targets for host-based antimalarial prophylaxis.

Figure 1. The use of protein microarrays to study liver stage malaria infection

(A) Schematic representing steps required to obtain lysate microarray data from HepG2-CD81 cells either infected or uninfected with P. yoelii-GFP liver stages. Liver stages were allowed to develop for 24 hours. (B) Representative array images from three antibodies, total p53, LC3A and p-Akt1/2/3 (pS473). (C) Graph representing the ratio of infected (GFP-positive) cells to uninfected (GFP-negative) host cells for signals obtained for 46 separate antibodies, plotted against the log of the p-value obtained. Each point represents a single antibody. Significant differences that pass multiple hypothesis testing (Holm-Bonferroni method) are shown in blue, non-significant differences are shown in red. Also see Figure S1.

Figure 2. Key host signaling pathways in Plasmodium infection

(A) Schematic showing the connectivity between host proteins significantly perturbed in liver stage-infected cells as measured by protein lysate microarrays. Proteins for which total level or post-translational modifications are increased in parasitized cells are shown in green, decreases levels in red. Select antibodies tested demonstrate a linear relationship between immunoblotting (WB) and lysate array (LA) are colored orange. Proteins for which transcripts where tested and changed are colored yellow. (B) Demonstration that total p53 antibody (#9282) produces a linear relationship for data obtained using western blotting and lysate array. Data was fit using a linear regression. (C) Quantitative PCR showing that transcript levels of Bad are decreased in infected hepatocytes (P=0.001). (D) Transcript levels of Bcl-2 are elevated in infected hepatocytes (P=0.05). Also see Figure S1.

Figure 3. Transgenic mice and pharmacological perturbations demonstrate a critical role for host p53 in liver stage infection

(A) Mice with an additional copy of p53 (‘super-p53 mice’) (n= 13), without p53 (‘p53 KO mice’) (n=7) or wild-type C57BL/6 mice (n=20) were infected with 100,000 P. yoelii sporozoites. Liver stage burden was monitored 42-44 h after infection using quantitative RT-PCR. Parasite burden was significantly reduced in super-p53 mice (P= 0.004) and significant elevated in p53 KO mice (P= 0.04). (B) p53 levels increase in response to 48 h Nutlin-3 treatment, as demonstrated by western blot using an anti-p53 antibody. An anti-αTubulin antibody was used as a loading control. Nutlin-3 treatment (20µM) 24h before and during infection (pre and post), dramatically reduces liver-stage burden of P. yoelii (C, middle) and P.berghei (D, middle). When treatment is applied begining at time of infection and continuing until 24h after infection (post) liver-stage liver stage burden is also reduced, albeit less substantially for both P. yoelii (C, right) P. berghei parasites (D, right). All liver stages were quantified 24 h post-infection in HepG2 CD81 cells. Error bars represent standard deviation between biological replicates. (E) To monitor the effects of Nutlin-3 on P. yoelii (Py) sporozoite early infection of HepG2 CD81 cells, cells were trypsinized 90 min postinfection, fixed and then stained with and antibody to CSP, and subjected to flow cytometric analysis. Wells that were not infected with sporozoites were used as a control. Error bars represent standard deviation of biological replicates. (F, G) Nutlin-3 treatment dramatically reduces liver-stage burden in mice. 50mg/kg Nutlin-3 was administered once daily for two days to BALB/cJ mice. At the time of the last administration of Nutlin-3, mice were infected with 50,000 P. yoelii sporozoites. Livers were removed at 24 h (E) or 44 h (F) post-infection and parasite 18S ribosomal RNA was assessed by qRT-PCR. Signal was normalized to mouse GAPDH. For in vivo experiments, the mean is represented by a horizontal line and the level of Py18s/GAPDH is shown for each individual mouse. Also see Figure S2.