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The mammalian intracellular response to Microsporidia infection

Periodic Reporting for period 1 - MicroMAM (The mammalian intracellular response to Microsporidia infection)

Reporting period: 2019-07-01 to 2021-06-30

Maintaining protein homeostasis (proteostasis) after exposure to chemical, physical or microbial stressors is critical for organismal survival. It relies on complex cellular regulatory networks that include two major interconnected proteolytic degradation pathways: the ubiquitin-proteasome system (UPS) and the autophagy lysosome (autophagy) system. Genetic predispositions, aging, infectious agents and abiotic environmental factors, can all lead to dysregulation of these two pathways which underpins the pathobiology of numerous infectious (e.g. HIV), neurodegenerative (e.g. Alzheimer's disease), chronic inflammatory (e.g. inflammatory bowel diseases [IBD]), metabolic syndrome associated diseases (e.g. type II diabetes) and cancers. Microsporidia, are a group of highly diverse obligate intracellular pathogens, infecting most animal lineages including economically and ecologically important species. Several species can infect humans and represent a well-established threat to immunocompromised patients (e.g. HIV-AIDS, organ transplant recipients). Moreover, recent studies have also shown that asymptomatic Microsporidia infections are more common in healthy immune-competent individuals than previously thought. Microsporidia infections are also associated with chronic conditions including Crohn’s disease (a form of inflammatory bowel disease – IBD) and cancer, suggesting a broader relevance to human health that requires further investigation. In the C. elegans model, both autophagy and the UPS pathways play a role in controlling Microsporidia infections through the selective autophagy pathway called Xenophagy. In the mammalian model, my analyses of the unpublished results data from Robert P. Hirt’s lab have shown the upregulation of several autophagy and UPS related genes in mammalian cells infected by the Microsporidia suggesting that proteostasis could be perturbed Microsporidia infection in mammalian organisms too.
Given the lack of effective treatments, the common occurrence of Microsporidia infection and the potential impact on host proteostasis, obtaining a better understanding of the molecular interactions between Microsporidia and their mammalian hosts is of increasing importance. Therefore, the objectives of the project were to investigate mammalian cells proteostasis pathways interplay with microsporidia infection and compare it with knowledge from the C. elegans model.
Our results show that, as in the C. elegans model, proteostasis in involved in in the response to Microsporidia infection. We detected a mild increase in autophagy flux at various time point post infection as well as a targeting of the parasite with ubiquitin, suggesting that it could be part of the host cell innate immune response. However, in mammalian cells Microsporidia seems to have developed a way to escape that mechanism and even use it to divert nutrients from the host metabolism to promote their own proliferation and eventual differentiation into the infectious spore stage.
During these 2 years we split the project in two work packages:
In the first work package, we used biochemistry approaches to investigate the impact of Microsporidia infection on the activation of both the UPS and the autophagy pathways using biochemistry approaches. Our results shows that while the level of UPS activation remains mostly stable the autophagy flux is increased upon infection, suggesting that the host cell is either using it to fight the infection or to compensate the nutrient stealing by the parasite. Subsequently, we used super-resolution microscopy coupled with immunochemistry approaches to investigate if the parasites were directly targeted by these pathways. We were able to show that the host cell targets the Microsporidia via UPS (xenophagy targets) in the early stages of infection, but that the parasite can escape it avoiding internalisation by the xenophagy/autophagy vacuoles as attested by the lack of late xenophagy marker (LC3) and the effective proliferation of the parasites.
In the second work package, we investigated the effect of the modulation of the host cell autophagy on the parasite development. We used drugs (torin-1, rapamycin, bafilomycin) and RNA silencing (of atg-5) to modulate host cell autophagy flux either negatively or positively and monitored the effect on these treatments on the parasite proliferation and spore production. We were able to demonstrate that both Microsporidia proliferation and spore production are favoured by autophagy flux drug induction. We also showed that the parasite growth is slowed by autophagy flux reduction using RNA silencing of a key gene of autophagy (atg-5). Finally, we used Short Chain Fatty Acid (SCFA), which are secondary metabolites produce by the gut microbiota upon fermentation of glycans, to mimic a more natural/physiological autophagy repression of the gut epithelial cells. SCFAs are known to play important roles in promoting gut homeostasis through multiple mechanisms including down regulating of autophagy, which promotes barrier function of the epithelium, and promotion of Treg cells differentiation key immunocytes downregulating the inflammatory tone of the gut. Notably the SCFA treatment significantly decreases the growth of the parasite in the tested gut epithelial cell line, consistent with the results derived from our genetic down regulation of autophagy. These data highlight a very important framework to study the potential clinical relevance of Microsporidia among IBD patients and how these might contribute to trigger the disease or worsen the prognosis.
Taken together our results suggest that in contrast to the C. elegans model, Microsporidia infecting mammalian cells evolved a strategy to evade the xenophagy pathway of the host cells and are in contrast able to benefit from the resulting autophagy flux induction. We hypothesize that Microsporidia are exploiting the autophagy flux to divert nutrient, energy and building blocks from the host cell to support their own biomass production.
This is the first study investigating the interplay between proteostasis and microsporidia infection in the mammalian model and it will be of major interest to the Microsporidia community by its comparison with the C. elegans model. However, even if Microsporidia were disregarded for very long time, it is now known that these parasites are largely spread in all the animal kingdom including human where they have been shown to be more common than initially though. Showing that these parasites can influence the proteostasis pathways of host gut epithelium, as well as benefit from dysregulated autophagy, will therefore interest a wider community of scientist focused on the gut epithelium immunity and interaction with microbiota. Finally, our results could also explain why microsporidia are more prevalent in Crohn diseases patient, as this disease is well known to be linked with autophagy dysregulation, and further study to investigate a potential link between them should be done.