Single-cell imaging of host-parasite metabolic interactions in the liver – iMET

- microscopy
- FRET analysis
- intravital imaging
- transgenic animals
- single-cell RNA sequencing
- micropatterning and cell culture
- flow cytometry

WP 1. Single-cell live imaging of metabolic heterogeneity and infection
For the in vivo studies, a new colony of transgenic mice expressing an AMPK sensor (AMPKAR-EV) has been successfully established in a C57BL/6 background. A pilot infection experiment has been performed using Plasmodium berghei sporozoites delivered by intradermal injection. We compared transgenic animals to wild-type mice from the same breading and C57BL/6 wild-type known to be susceptible to P. berghei infection. All mice showed parasites in the blood after 3-5 days of infection, indicative of a successful liver-stage development. These transgenic animals can now be used for the initially planned intravital imaging analysis of infection. Another pilot study has also been performed in non-transgenic mice to optimize conditions for the live imaging. For the in vitro experiments, the plasmid encoding AMPKAR-EV has been obtained from addgene, expanded, purified, and successfully transfected into primary human hepatocytes.

WP 2. Mechanisms of host-parasite metabolic interaction
Work carried out during the pandemic for another project pointed us to CD36, a lipid transporter that is regulated by AMPK. We found that Plasmodium falciparum sporozoites interact directly with CD36 on the host cell surface, and loss of CD36 in the membrane of human hepatocytes results in reduced infection. We are currently pursuing the link with AMPK. Our hypothesis is that the interaction of the parasite with CD36 in the host cell membrane triggers a signaling cascade leading to suppression of AMPK activity, which we previously found to be favorable for Plasmodium infection (Ruivo et al 2016).

WP3. Impact of host metabolic heterogeneity on dormancy and drug effectiveness
Here, we have analyzed a previously collected single-cell transcriptomic dataset of P. vivax parasites in human hepatocytes cultured in vitro. We found that hepatocytes hosting dormant hypnozoites are transcriptionally distinct from those containing replicative parasites. Specifically, gene transcripts encoding proteins implicated in iron and lipid metabolism, as well as several genes encoding drug metabolizing CYP450 enzymes, were detected at lower levels in hypnozoite-containing hepatocytes (Mancio-Silva et al 2022). These results support the hypothesis that the differential metabolic state may induce parasite dormancy and confer host resistance to treatment. In parallel, we have been working with the group of Sylvie Garcia (Institut Pasteur) on developing a new mouse model that by enabling human erythropoiesis, supports continuous P. vivax blood-stage asexual replication, sexual differentiation in the bone marrow, and transmission to mosquitoes (Luiza-Batista et al., under revision in Nat Comm). We expect this work will improve our access to P. vivax sporozoites.

WP 1. Single-cell live imaging of metabolic heterogeneity and infection
Infections with Plasmodium sporozoites and FRET analysis in vivo and in vitro are planned for the second semester of 2022. Once infection conditions are optimised with wild-type parasites, we will move to perform infections with transgenic parasites and use AMPK modulating drugs (metformin and Compound C) to further understand the mechanisms.

WP 2. Mechanisms of host-parasite metabolic interaction
Following on the results obtained for CD36, siRNA silencing and overexpression of this host factor will be performed in cells transfected with AMPKAR-EV and infection will be assessed by live imaging. For the overexpression, we will use mCherry-CD36-C-10 (55011) plasmid from addgene. A potential role of autophagy involving CD36 and AMPK will also be investigated.

WP3. Impact of host metabolic heterogeneity on dormancy and drug effectiveness
To validate the results obtained by single-cell RNA sequencing, we will now perform gene silencing (by siRNA) of selected candidates with and without drug treatments and quantify the numbers of hypnozoite forms in the P. vivax infected cultures. Drugs that will be tested include primaquine, tafenoquine and AMPK modulators (as WP1 above).

Single-cell views of the Plasmodium life cycle.
Real E, Mancio-Silva L. Trends in Parasitology. 2022 Jun 4:S1471-4922(22)00112-X. doi: 10.1016/j.pt.2022.05.009. PMID: 35672200

A single-cell liver atlas of Plasmodium vivax infection.
Mancio-Silva L, Gural N, Real E, Wadsworth MH 2nd, Butty VL, March S, Nerurkar N, Hughes TK, Roobsoong W, Fleming HE, Whittaker CA, Levine SS, Sattabongkot J, Shalek AK, Bhatia SN. Cell Host Microbe. 2022 Apr 13:S1931-3128(22)00164-0. doi: 10.1016/j.chom.2022.03.034. PMID: 35443155

Humanized mice for sustained Plasmodium vivax blood-stage infection and transmission.
Luiza-Batista C, Thiberge S, Serra-Hassoun M, Nardella F, Claes A, Nicolete VC, Commère PH, Mancio-Silva L, Ferreira MU, Scherf A,Garcia S. bioRxiv 2022.03.22.485265; doi: doi.org/10.1101/2022.03.22.485265

Hepatocytes support the first massive amplification of malaria-causing Plasmodium parasites in the mammalian host. Despite clear parasitism and subversion of host cell resources, the molecular mechanisms implicated in favorable replication inside hepatocytes remain largely unknown. Metabolic signaling pathways appear to be modestly different in Plasmodium-infected hepatic cells compared to uninfected cells. However, these differences were measured by averaging cell populations and are likely masked by the high metabolic cell-to-cell heterogeneity recently unveiled in human and mouse hepatocytes. Also, in the previous studies it was not possible to discriminate whether the metabolic differences were a response to the parasite, or the parasite had selected to home in a hepatocyte where those conditions where already in place. In this research proposal, we aim to understand whether the invading parasite i) uses sensing mechanisms to select the hepatocyte where replication will unfold and/or ii) promotes host metabolic reprograming upon invasion. To address these questions, we will employ the most-advanced single-cell live imaging approaches and bioengineered in vitro liver systems to probe the subpopulation of Plasmodium infected cells. Plasmodium liver-stage is the prime target for antimalarial therapeutic intervention, thus understanding the molecular basis and significance of hepatocyte homing signals and host-parasite metabolic interactions is fundamental in defining novel strategies.