Toxoplasma gondii inhibits the expression of autophagy-related genes through AKT-dependent inactivation of the transcription factor FOXO3a

Diez, Andrés Felipe (2023). Toxoplasma gondii inhibits the expression of autophagy-related genes through AKT-dependent inactivation of the transcription factor FOXO3a Thèse. Québec, Université du Québec, Institut national de la recherche scientifique, Doctorat en immunologie et virologie, 204 p.

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Résumé

Toxoplasma gondii (T. gondii) is an intracellular protozoan parasite that can infect virtually any nucleated cell and a wide variety of warm-blooded vertebrate hosts, including humans, causing toxoplasmosis. While many cases are asymptomatic, symptoms may include fever, lymphadenopathy, and myalgia. In the United States, toxoplasmosis is a notable contributor to foodborne disease-associated mortality. Immunocompromised individuals are susceptible to severe ocular, pulmonary, and cerebral complications, and behavioral changes can occur. The disease results in an annual average of 71 deaths, with toxoplasmic encephalitis significantly impacting AIDS patients.

It is estimated that 30-50% of the world population is seropositive for T. gondii, making it one of the most prevalent infections among humans. T. gondii can be transmitted through ingestion of oocysts shed in felines feces or undercooked meat from infected animals. T. gondii undergoes various life stages, such as tachyzoites and bradyzoites, that are capable of persisting in the host's tissues; additionally, secreted virulence factors play a key role in different stages of infection and are linked to T. gondii virulence. Toxoplasmosis is an important public health concern, as there is no available treatment against all forms of the parasite or vaccine for humans. Treatment with drugs can have side effects and is unable to eliminate T. gondii cysts from the host.

T. gondii manipulates the host cell to create a safe environment for replication. To do this, the parasite scavenges essential nutrients, alters host metabolism, inhibits apoptosis, and manipulates host autophagy, immune response, and cell cycle progression. T. gondii has developed complex mechanisms to create a non-fusogenic parasitophorous vacuole (PV) that helps acquire nutrients while preventing contact with host cytoplasmic components and lysosomal content, that could trigger parasite destruction. The parasite also uses virulence factors to target various aspects of the host cell's biology, such as gene expression, transcriptional machinery, and host protein post-translational modifications.

As a key defense mechanism against infections, autophagy, a cellular process that aids in maintaining cellular homeostasis, is involved in the capture and elimination of intracellular parasites. However, intracellular parasites, such as T. gondii, Plasmodium spp, Trypanosoma spp and Leishmania spp, have developed several evasion mechanisms to manipulate the host cell autophagy. The PI3K/AKT pathway, is activated in response to physiological circumstances such as growth factor signaling, insulin binding, cell survival signals, nutrient availability, and even stress conditions. It plays a crucial role in regulating cell survival, growth, and metabolism in various cell types. PI3K/AKT pathway also plays a role in inhibiting autophagy while promoting cell growth, differentiation, and survival. Inhibition of this pathway can lead to a significant increase in autophagy. Interestingly, T. gondii exploits the host's PI3K/AKT signaling to evade the killing effects of autophagy. This evasion strategy is particularly activated during the early stages of the invasion, where the parasite activates AKT. By doing so, T. gondii prevents the accumulation of autophagosome and lysosome components around its PV, which ultimately reduces parasite replication. However, the specific targets of AKT responsible for T. gondii's ability to hinder the host's autophagy machinery are not yet fully understood.

Amongst the different AKT targets, FOXOs (Forkhead Box O subfamily) have been shown to be a major node regulating autophagy. FOXO transcription factors (TFs) are essential regulators of cellular homeostasis and autophagy that are controlled by the PI3K/AKT signaling pathway. When AKT phosphorylates FOXO TFs, it causes them to move out of the nucleus inhibiting their transcriptional activity and, in some cases, promoting their degradation in the cytoplasm or enabling cytoplasmic functions such as binding with other proteins or their entry into other organelles like the mitochondria. Interestingly, autophagy dysregulation is linked to various human diseases, with the FOXO-autophagy axis playing a crucial role in several of these diseases. Therefore, different pathogens manipulate FOXO proteins. As a result, various intracellular parasites use the manipulation of the host PI3K/AKT/FOXO pathway to survive within the host cell.

Using a combination of pharmacological and genetic approaches, herein we investigated whether T. gondii hijacks the PI3K/AKT pathway to suppress FOXO3a-regulated autophagy-related transcriptional programs, hindering the activation of the host autophagic response against the parasite. To corroborate this, we first investigated the molecular mechanisms responsible for AKT-dependent phosphorylation of FOXO3a. Then, we determined whether AKT-dependent repression of FOXO3a during T. gondii infection affects autophagy-related transcriptional programs. Finally, we established whether FOXO3a-regulated autophagy-related functions are altered during T. gondii infection.

We found that T. gondii triggers a gradual and sustained AKT-dependent phosphorylation of FOXO3a at residues S253 and T32 in both (human foreskin fibroblasts, HFF) and murine 3T3 fibroblasts. Additionally, live infection is necessary to manipulate and activate the PI3K/AKT signaling pathway, leading to the phosphorylation of FOXO3a. Interestingly, this phosphorylation process is independent of the plasma membrane receptor EGFR and the kinase PKCα. Furthermore, T. gondii causes nuclear exclusion of FOXO3a in infected HFF, which is directly correlated with its phosphorylation. However, the parasite cannot drive cytoplasmic localization of FOXO3a when AKT is blocked pharmacologically or when an AKT-insensitive mutant form of FOXO3a is overexpressed. During T. gondii infection, the transcription of a subset of autophagy-related target genes of FOXO3a is reduced in an AKT-dependent manner because FOXO3a is inactivated. However, the parasite's ability to repress autophagy-related genes remains unaffected by AKT inhibition in cells where Foxo3 transcripts are silenced (knockdown). As a result, T. gondii fails to inhibit the recruitment of acidic organelles and LC3, an autophagy marker, to the PV when FOXO3a is chemically or genetically retained in the nucleus. In all, we provide evidence that T. gondii suppresses FOXO3a-regulated transcriptional programs to prevent autophagy-mediated killing.

In conclusion, autophagy is a cellular process that can be hijacked by several pathogens for replication and survival, but the host has developed countermeasures to prevent its survival. The complex interplay between autophagy and microbial adaptations determines the outcome of host-pathogen encounters. The FOXO family of TFs is crucial for regulating autophagy, and T. gondii manipulates host PI3K/AKT signaling to promote its survival and replication while subverting the host's autophagy process, partly by inhibiting the transcriptional activity of FOXO3a. However, inhibition of the PI3K/AKT pathway or expression of an AKT-resistant form of FOXO3a can promote host cell autophagy and target T. gondii, making autophagy an attractive therapeutic strategy against the parasite. Our findings indicate that T. gondii hijacks the PI3K/AKT pathway to suppress autophagy-related transcriptional programs under the control of FOXO3a, thereby hindering the activation of the host autophagic response against the parasite. Further characterization of altered transcriptional networks under the control of FOXO3a, and potentially other FOXO family members, during T. gondii infection will yield invaluable health-related knowledge to develop effective and safe host-directed strategies for better treatment or prevention of toxoplasmosis.

Type de document:	Thèse Thèse
Directeur de mémoire/thèse:	Jaramillo, Maritza
Mots-clés libres:	-
Centre:	Centre INRS-Institut Armand Frappier
Date de dépôt:	09 juill. 2024 15:35
Dernière modification:	09 juill. 2024 15:35
URI:	https://espace.inrs.ca/id/eprint/15801

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