Toxoplasma and Plasmodium subversion of host cell microRNAs for parasite intracellular development – APImiR

The goal of this proposal is to investigate how Plasmodium and Toxoplasma interfere with the miRNA expression of the host cells inside which they develop. We will first assess the human small RNAome alterations following T. gondii and P. berghei infection. We will then select pertinent microRNAs and validate their expression profiles following infection by Northern Blot and qRT-PCR. We will investigate how they contribute in host cell to promote or prevent parasite infection. Finally, we plan to identify parasite effector molecules that hijack the RNA silencing machinery in the host cell.

Team 1 profiled the levels of miRNAs in primary human foreskin fibroblasts 6 hours post-infection by T. gondii. It appears that 10% of the host miRNAs have a modified expression pattern after infection. These results mirror those recently published showing differential alterations in the mature miRNA expression profile in biliary epithelial cells following C. parvum infection. We propose in this project to capitalize on these findings and further the analysis of Toxoplasma-induced miRNA modifications in the host cell, and test the likely hypothesis that intrahepatocytic Plasmodium also impact the host cell miRNAs. Our project is to map the host cell miRNA profile at a single time point, prioritize potentially interesting miR candidates, and attempt to validate these changes functionally (as pro-parasite, anti-parasite, or neutral). We believe that this proposal has the potential to provide crucial new insights into how these sophisticated eukaryotic parasites of major human relevance hijack their host cells, to point to new host cell functions that are important for parasite survival and growth, and to reveal new drug targets against these parasites.

An interesting and intriguing result from our profiling studies is that some MIRNAs are transcribed but are not processed into the mature miRNA unless the fibroblast cells are infected with T. gondii. These data raise the possibility that T. gondii infection is affecting directly miRNA biogenesis, stability, or activity. miRNA are generated through the concerted action of multi-subunit complexes that promote the sequential cleavage, export, and loading of miRNA into silencing complexes. An increasing number of reports suggest that each of these steps serves as a potential point of regulation, and therefore provides additional complexity to miRNA-dependent gene regulation. Because little is yet known about the biogenesis, stability and degradation of miRNAs, this is a promising area for the discovery of novel regulatory mechanisms. The identification of more interacting partners of individual precursors will further broaden the spectrum of control mechanisms. Ultimately, the characterization of pri- or pre-miRNA/protein “interactomes” will be an invaluable tool to gain an in-depth understanding of the complex circuitries governing miRNA activity.

Hakimi MA, Cannella D. Apicomplexan parasites and subversion of the host cell microRNA pathway. Trends Parasitol. 2011 Nov;27(11):481-6.

This proposal focuses on the basis of host cell manipulation by two of the most successful human pathogens, Plasmodium and Toxoplasma, the agents of malaria and toxoplasmosis, respectively. Malaria remains one of the top human killers. Almost half of the world population is at risk of contracting the disease, which kills up to 3 million people each year, an infant every twenty seconds in Africa. Toxoplasma is the most widespread apicomplexan parasite, present virtually everywhere on earth. Although usually causing only mild symptoms in the adult, it can cause severe and life-threatening diseases in developing fetuses and in immune-compromised individuals, especially AIDS and transplant patients. Although the alterations of the host cell harboring Toxoplasma or Plasmodium (liver stage) parasites have been extensively documented at the cellular level, still little is known about how the parasite manipulates the host cell at the molecular level. So far, most of the molecular work on the host-parasite interface has focused on the role of parasite factors that are secreted by the invading parasite, as well as by the resident intracellular parasite, into the host cell.

MicroRNAs are a class of small non-coding RNAs that are found in plants, animals, and some viruses. They modulate the gene function at the post-transcriptional level and act as a fine tuner of various processes, such as development, proliferation, cell signaling, metabolism and apoptosis. When homeostatic conditions are ruptured such as when cells encounter micro-organisms, these regulatory pathways might also contribute to the mounting of host cell responses/defenses (i.e., inflammatory response) against the foreign bodies. Among examples, cell infection by mammalian viruses might be counteracted by cellular miRNAs that target either the virus or a critical host factor. Conversely, miRNAs can also act in favor of the micro-organism, either when it is pathogen-encoded (eg, mammalian virus-encoded miRNAs) or when the micro-organism subverts host miRNAs to its own benefit. Effectors from the bacteria Pseudomonas syringae have been recently shown to suppress transcriptional activation of some miRNAs generated upon sensing of PAMPs (pathogen-associated molecular patterns) by Arabidopsis.

Given the propensity of Toxoplasma and Plasmodium to co-opt cellular pathways and activities for their benefit, it is perhaps not surprising that these parasites could also reshape their cellular environment by reprogramming the host’s RNA-interference machinery. Specific host miRNAs could either counteract the intracellular growth of parasites or facilitate it, the two possibilities being not mutually exclusive and depending on the physiological context. Team 1 profiled the levels of miRNAs in primary human foreskin fibroblasts 6 hours post-infection by T. gondii. It appears that 10% of the host miRNAs have a modified expression pattern after infection. These results mirror those recently published showing differential alterations in the mature miRNA expression profile in biliary epithelial cells following C. parvum infection. We propose in this project to capitalize on these findings and further the analysis of Toxoplasma-induced miRNA modifications in the host cell, and test the likely hypothesis that intrahepatocytic Plasmodium also impact the host cell miRNAs. Our project is to map the host cell miRNA profile at a single time point, prioritize potentially interesting miR candidates, and attempt to validate these changes functionally (as pro-parasite, anti-parasite, or neutral). We believe that this proposal has the potential to provide crucial new insights into how these sophisticated eukaryotic parasites of major human relevance hijack their host cells, to point to new host cell functions that are important for parasite survival and growth, and to reveal new drug targets against these parasites.