Elsevier

Molecular Immunology

Volume 154, February 2023, Pages 69-79
Molecular Immunology

Cryptosporidium parvum maintains intracellular survival by activating the host cellular EGFR-PI3K/Akt signaling pathway

https://doi.org/10.1016/j.molimm.2023.01.002Get rights and content

Highlights

  • C. parvum could activate EGFR-PI3K/Akt signaling pathway to promote intracellular survival in HCT-8 cells.

  • C. parvum triggered complete autophagy and enhanced autophagic flux in HCT-8 cells.

  • C. parvum inhibited autophagy killing by activating EGFR-Akt signaling pathway.

Abstract

Autophagy is a critical cellular mechanism in helping infected cells remove intracellular pathogens and is countered by pathogens maintaining intracellular survival by regulating autophagy through the manipulation of the host cellular signal transduction pathway. Cryptosporidium parvum is a zoonotic intracellular but extracytoplasmic protozoon that causes diarrhea in infants and young children worldwide. However, it is still unclear how Cryptosporidium adapts to intracellular survival. In the present study, we demonstrated that C. parvum could activate the EGFR-PI3K/Akt signaling pathway to promote intracellular survival in HCT-8 cells. The western blot results showed that C. parvum induced EGFR and Akt phosphorylation in HCT-8 cells. The EGFR inhibitor AG1478 decreased EGFR and Akt phosphorylation, and the PI3K inhibitor LY294002 impaired Akt phosphorylation induced by C. parvum in HCT-8 cells. Inhibition of EGFR or Akt decreased the number of intracellular parasites. Second, low-dose infection of C. parvum triggered complete autophagy and enhanced autophagic flux in HCT-8 cells. The expressions of mTOR and p62 were decreased, and the expressions of LC3 and Beclin1 were increased in C. parvum-infected HCT-8 cells. Transfection with siBeclin1 or siATG7 reduced LC3 accumulation, while lysosome inhibitor E64d+pepA increased LC3 accumulation induced by C. parvum in HCT-8 cells. Intracellular parasite proliferation was decreased when treated with autophagy inducer rapamycin, whereas autophagy inhibitor 3-MA, E64d+pep A, siBeclin1 or siATG7 increased intracellular parasites. Third, C. parvum inhibited autophagy killing to promote its own intracellular survival by activating EGFR-Akt signaling pathway. The EGFR inhibitor AG1478 enhanced autophagic flux, and Akt inhibitor IV increased LC3 accumulation and inhibited C. parvum proliferation in HCT-8 cells. Akt inhibitor IV-inhibited C. parvum proliferation was attenuated by E64d+pepA. In summary, C. parvum could maintain intracellular survival by inhibiting autophagy via EGFR-PI3K/Akt pathway. These results revealed a new mechanism for the interaction of C. parvum with host cells.

Introduction

Cryptosporidium, the cause of cryptosporidiosis, belongs to the phylum apicomplexa, which is widely distributed in more than 90 countries, including China. The host range includes more than 240 species, such as mammals, poultry, reptiles, amphibians and fish (O'Donoghue, 1995). C. parvum is one of the zoonotic Cryptosporidium that infects intestinal epithelial cells in mammals, including humans, and is thought to be a significant cause of diarrheal disease in developing and industrialized nations. C. parvum transmission occurs through a fecal-oral route by accidental ingestion, and water contamination is the main cause of cryptosporidiosis outbreaks, which is a considerable public health concern(Bouzid et al., 2013). Cryptosporidiosis is usually self-limiting in immunocompetent adults. In immunocompromised individuals such as those with AIDS, C. parvum infection can be life-threatening (Dumaine et al., 2019). Cryptosporidiosis is an important water-borne protozoan disease and is listed in the standard of urban water supply quality (CJ/T206-2005) and the standard of drinking water sanitation (GB 5749-2006 instead of GB 5749-85) published in China. More than 200 drugs have been screened for cryptosporidiosis, but there is no ideal clinical treatment. Some vaccine candidate genes have been reported, and some protective effects have been observed but are not ideal. To date, there are no effective treatments for cryptosporidiosis. A better understanding of the molecular mechanism of interaction between parasites and hosts will help in deciphering the molecular basis of pathogenesis and developing vaccines and therapeutic drugs(Checkley et al., 2015; Love and McNamara, 2021). At present, the mechanism of C. parvum intracellular survival is not yet clearly understood.

Autophagy is a process that relies on the lysosomal pathway for the degradation of cytoplasmic proteins and organelles and is widely present in eukaryotic cells(Choi et al., 2013). It is well known that autophagy emerges as an important immune defense against intracellular pathogens. However, intracellular pathogens can escape autophagy or use autophagy to achieve proliferation (Mizushima, 2007). Several protozoa, such as Plasmodium, Toxoplasma gondii, Trypanosoma cruzi and Neospora caninum, can induce host cell autophagy (Onizuka et al., 2017, Prado et al., 2015, Py et al., 2007; Wang et al., 2021, Wang et al., 2009). The invading Plasmodium was eliminated by autophagy at the very beginning (Prado et al., 2015). Cellular infection by L. monocytogenes induced an autophagic response that inhibited parasite growth (Py et al., 2007). T. gondii manipulates host autophagy machinery to obtain nutrients required for parasite growth (Wang et al., 2009). Recently, C. parvum was found to promote autophagy in Caco-2 cells (Priyamvada et al., 2021), but little is known about whether C. parvum manipulates autophagy to prevent its degradation. It is still not clear whether the invading Cryptosporidium induces HCT-8 cell autophagy and how Cryptosporidium regulates host cell autophagy.

Autophagy is regulated by complex signaling pathways, including mTOR-dependent signaling pathways(Li et al., 2022), mTOR-independent pathways and others (Yang et al., 2005). The EGFR pathway is an important cellular signaling pathway, and its downstream signaling pathways mainly include the MAPK pathway and PI3K/Akt/mTOR pathway, which regulate autophagy, apoptosis and proliferation of cells (Wee and Wang, 2017). EGFR-PI3K/Akt signaling has been shown to be a key pathway in the negative regulation of autophagy. Human cholangiocytes infected with C. parvum showed a significant increase in phosphorylation of p-38 but not JNK or ERK1/2(Chen et al., 2005). However, little is known about the mechanism by which the EGFR-PI3K/Akt pathway contributes to host defense against C. parvum. The potential role of EGFR-PI3K/Akt signaling in the regulation of autophagy during C. parvum intracellular survival warrants further investigation.

As an intracellular parasite, C. parvum’s survival relies on the nutrient environment provided by host cells. Studies have shown that autophagy has profound effects on parasite development and proliferation. T. gondii can use host cell autophagy to provide itself with nutrients, thereby promoting its own proliferation(Krishnamurthy et al., 2017). In addition, short periods of starvation-induced autophagy contribute to P. falciparum development and invasion(Joy et al., 2018).

In the current study, we aimed to determine whether C. parvum manipulates host cell autophagy by activating the EGFR-PI3K/Akt pathway. The activation of the EGFR-PI3K/Akt pathway and autophagy in HCT-8 cells induced by C. parvum were examined, and intracellular parasite proliferation was determined.

Section snippets

Cells and parasites

HCT-8 cells (obtained from ATCC) were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10 % fetal bovine serum (Biological Industries, USA) and 1 % penicillin streptomycin (Gibco, USA). This study used C. parvum oocysts sourced from Changchun, China, and propagated through C. parvum-free neonatal Holstein calves. Oocysts were purified with sieve filtration, discontinuous sucrose density gradient centrifugation and washing and then stored at 4 °C in PBS (Schupfner et al., 2013).

C. parvum could activate the EGFR-PI3K/Akt signaling pathway in HCT-8 cells

HCT-8 cells displayed elevated levels of p-EGFR expression, after incubation with C. parvum for 2–8 h (Fig. 1 A and B). To determine whether phosphorylation of EGFR was caused by invasion of C. parvum, we assessed the effect of viable or dead parasites on the phosphorylation of EGFR in cells. We found viable but not dead sporozoites induced EGFR phosphorylation at 2 h (Fig. 1 D and E). The Akt-mediated pathway is the major downstream pathway of EGFR, which phosphorylates within the carboxy

Discussion

Autophagy has been widely reported to play an essential role in resistance to infection by most intracellular pathogens, such as bacteria, viruses, and protozoa. Our studies indicated that C. parvum activated the EGFR-PI3K/Akt signaling pathway in HCT-8 cells to prevent autophagy-related processes from promoting parasite proliferation.

Several reports have shown that EGFR-Akt can be activated during parasite-host cell interactions, such as T. gondii (Lopez Corcino et al., 2019, Quan et al., 2013

Funding

This research was funded by the National Natural Science Foundation of China (No. 31872465). The experiments conducted in this study comply with the current laws of China.

Author contributions

Heng Yang, Mengge Zhang and Jianhua Li drafted the main manuscript and performed the data analysis; Heng Yang, Mengge Zhang and Xiaocen Wang planned and performed the experiments; Jianhua Li and Xin Li were responsible for the experimental design; Pengtao Gong and Xichen Zhang were responsible for guiding and supporting the experiments and manuscript revisions.

Competing financial interests

The authors declare they have no competing financial interests.

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    These authors contributed equally.

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