Metabolic Signatures of Cryptosporidium parvum-Infected HCT-8 Cells and Impact of Selected Metabolic Inhibitors on C. parvum Infection under Physioxia and Hyperoxia

Abstract

Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-cause of diarrhoea-induced mortality in children. In contrast to other apicomplexans, C.parvum has minimalistic metabolic capacities which are almost exclusively based on glycolysis. Consequently, C. parvum is highly dependent on its host cell metabolism. In vivo (within the intestine) infected epithelial host cells are typically exposed to low oxygen pressure (1–11% O2, termed physioxia). Here, we comparatively analyzed the metabolic signatures of C. parvum-infected HCT-8 cells cultured under both, hyperoxia (21% O2), representing the standard oxygen condition used in most experimental settings, and physioxia (5% O2), to be closer to the in vivo situation. The most pronounced effect of C. parvum infection on host cell metabolism was, on one side, an increase in glucose and glutamine uptake, and on the other side, an increase in lactate release. When cultured in a glutamine-deficient medium, C. parvum infection led to a massive increase in glucose consumption and lactate production. Together, these results point to the important role of both glycolysis and glutaminolysis during C. parvum intracellular replication. Referring to obtained metabolic signatures, we targeted glycolysis as well as glutaminolysis in C. parvum-infected host cells by using the inhibitors lonidamine [inhibitor of hexokinase, mitochondrial carrier protein (MCP) and monocarboxylate transporters (MCT) 1, 2, 4], galloflavin (lactate dehydrogenase inhibitor), syrosingopine (MCT1- and MCT4 inhibitor) and compound 968 (glutaminase inhibitor) under hyperoxic and physioxic conditions. In line with metabolic signatures, all inhibitors significantly reduced parasite replication under both oxygen conditions, thereby proving both energy-related metabolic pathways, glycolysis and glutaminolysis, but also lactate export mechanisms via MCTs as pivotal for C. parvum under in vivo physioxic conditions of mammals.

Eimeria bovis Macromeront Formation Induces Glycolytic Responses and Mitochondrial Changes in Primary Host Endothelial Cells

“…In contrast to other apicomplexan parasites, C. parvum entirely lacks enzymes necessary for TCA cycle, cytochrome-based respiratory chain, and de novo biosynthesis of fatty acids, nucleotides, and amino acids. All these findings strongly evidence C. parvum dependence on host cell metabolism to achieve its replication (Abrahamsen et al, 2004;Zhu, 2004;Veĺez et al, 2021), thereby giving novel insights into a wide array of adaptative strategies used by apicomplexans related to their modulation capacities of host cell metabolism.…”

First Metabolic Insights into Ex Vivo Cryptosporidium parvum-Infected Bovine Small Intestinal Explants Studied under Physioxic Conditions

“…Both BSI explants and BSIEC were cultured under two different oxygen atmospheresnamely at 5% (physioxia) and 21% O 2 (hyperoxia)-to mimic both the physioxic conditions of the small intestine in vivo [13,29], and hyperoxic conditions as typically applied in the lab conditions used in most studies on C. parvum-infected host cells in vitro [13,30].…”

“…Based on genome sequencing [8], knowledge of C. parvum-related survival strategies has increased tremendously in recent years, and has led to the identification of general metabolic patterns-especially with respect to glycolysis as a key source of energy supply [9][10][11][12][13]. Nevertheless, host species-related differences in the C. parvum-driven metabolic impact on host cells have also been demonstrated [10,14,15].…”

“…The complex host-and microbiome-dependent differences in C. parvum-related virulence evidence the necessity of expanding scientific efforts beyond in vitro cell culture systems under hyperoxic conditions (21% O 2 ) and immunosuppressed murine models, which reflect neither in vivo intestinal physioxia nor host innate immune reactions of humans and ruminants against C. parvum [13,21]. Similarly, it also seems relevant to include the most important zoonotic reservoir species, i.e., domestic bovine species [13,22]. As pointed out elsewhere, cryptosporidiosis is not only a neglected anthropozoonotic disease, but also of high concern in terms of the One Health concept [23,24].…”

“…To date, information on metabolic alterations/changes in C. parvum-infected cattle is scarce, probably due to both high animal maintenance costs and limited availability of speciesspecific metabolic tools [25]. To improve current knowledge on C. parvum-driven metabolic changes in bovine small intestinal epithelial cells, studies considering physiological oxygen conditions and primary host cell types are urgently needed [13]. In this sense, individual bovine small intestinal (BSI) explants, which include the individual microbiome of the host, may provide a useful and uncostly model for ex vivo analysis of C. parvum-host epithelial cell-microbial consortia interactions.…”