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. 2021 Jan 15;10(1):60.
doi: 10.3390/biology10010060.

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

Affiliations

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

Juan Vélez et al. Biology (Basel). .

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.

Keywords: Cryptosporidium parvum; cryptosporidiosis; glutaminolysis; glycolysis; hyperoxia; physioxia.

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Conflict of interest statement

The authors declare that they have no competing interest.

Figures

Figure 1
Figure 1
3D-holotomographic illustration of C. parvum-infected HCT-8. (a) C. parvum-infected HCT- 8 cells were stained by biotinylated VVL and Hoechst 33258 at 24 and 48 h p. i. (n = 3): (a) 3D-holotomographic images were obtained by using 3D Cell Explorer microscope (Nanolive) at 60× magnification (λ = 520 nm, sample exposure 0.2 mW/mm2) and a depth of field of 30 µm. (b) Holotomography of C. parvum-infected HCT-8 and identification of trophozoite, meront and merozoite stages (Scale bar = 20 µm).
Figure 2
Figure 2
C. parvum development in HCT-8 cells under hyperoxic (21% O2) conditions (n = 6). SEM-based illustration of C. parvum-infected HCT-8 evidenced rapid C. parvum development and infection kinetics: (a) Overviews at different time points post infection (4–24 h p. i.). (b,c) Closer views of parasite stages-host cell interactions, i.e., column at 16 h p. i. shows meront-infected cells, row b evidenced hole-like damage on host cell surface induced by merozoites release at the same time point (white arrow). Row c (4 h p. i.) reveals early induction of villi-like structures on surface of infected cells (black arrows).
Figure 3
Figure 3
Glucose, lactate, pyruvate, aspartate, glutamine, glutamate, alanine and serine conversion rates in uninfected and C. parvum-infected HCT-8 cultivated in glutamine supplemented medium (2 mM) under hyperoxic (21% O2) and physioxic (5% O2) conditions (n = 6, each group). Metabolite conversion rates were analyzed at 24 h p. i. in the cultivation supernatants of the cells and presented as interleaved box and whiskers plots with line at median, bars indicating maximum and minimum values. Statistical significance (* = p ≤ 0.05, ** = p ≤ 0.01, **** = p ≤ 0.0001) was evaluated by t-test for independent samples.
Figure 4
Figure 4
Glucose, lactate, pyruvate, aspartate, glutamine, glutamate, alanine and serine conversion rates in uninfected and C. parvum-infected HCT-8 cultivated in glutamine-starved medium (0.02 mM) at hyperoxia (21% O2) and physioxia (5% O2) (n = 6, each group). In comparison to glutamine-supplemented cultivation conditions the parasite infection rate slightly dropped in glutamine starved medium (21% O2: 40 ± 3%; 5% O2: 28 ± 7%). Conversion rates were analysed at 48 h p. i. in the cell culture supernatants of the cells. Metabolic conversion rates are plotted as interleaved box and whiskers plots showing minimum to maximum values with line at the medians. Statistical significance (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001) was determined by t-test for independent samples.
Figure 5
Figure 5
Effect of C. parvum infection on glycolysis and glycolytic ATP production in HCT-8 cells under hyperoxic (21% O2) conditions. (a) C. parvum infection-triggered upregulation of glycolysis in HCT-8 cells as evidenced by Seahorse Glycolysis Stress Test Kit. (b) Likewise, significant infection-induced upregulation of glycolysis-derived ATP was observed. Bar graph shows mean ± SD, (n = 3). t-test was use for evaluation of significance (* = p ≤ 0.05, ** = p ≤ 0.01).
Figure 6
Figure 6
Schematic summary of the metabolic impact of C. parvum infection on HCT-8 cells cultivated in presence of 2 mM glutamine and 21% oxygen and targets of selected inhibitors. Bold arrows indicate impact on metabolic conversion rates of C. parvum-infected host cells (compare Figure 3). Based on C. parvum-induced alterations of host cell metabolism, the indicated metabolic inhibitors were selected to investigate their potential to inhibit C. parvum infection in HCT-8 cells. LND: lonidamine, MPC: mitochondrial pyruvate carrier, GLS: glutaminase, LDH: lactate dehydrogenase, MCT: monocarboxylate transporter.
Figure 7
Figure 7
Effects of galloflavin, lonidamine, syrosingopine and compound 968 treatments on glycolysis in uninfected HCT-8 cells cultivated at hyperoxic (21% O2) condition. Glycolysis stress test revealed a significant reduction of glycolytic responses by selected inhibitors. Bars represents mean ± SD, (n = 3). Data were evaluated for significance by one-way analyses of variance (ANOVA) followed by Dunnett’s test. n.s = non-significant, n.d. = non-detectable, * = p ≤ 0.05, *** = p ≤ 0.001, **** = p ≤ 0.0001.
Figure 8
Figure 8
Effects of galloflavin (400 µM), lonidamine (150 µM) (a), syrosingopine (10 µM) and compound 968 (10 µM) (a,b) treatments on C. parvum infection rates in HCT-8 cells cultivated at 21% O2 and 5% O2, and exemplary illustrations of respective inhibition assays. C. parvum was stained with VVL, green; host -cell membranes and nuclei were labelled with anti-ß-catenin (red) and Hoechst (blue), respectively. Bars show means ± SD, (n = 6). For evaluation of significance t-test was performed. ** = p ≤ 0.01.
Figure 9
Figure 9
Impact of C. parvum infection and inhibitor treatments on HCT-8 energetic profiles: (First column) The current energetic profiles are plotted by presenting OCR- and ECAR values in HCT-8 (n = 3, each condition). (Second column) Effect of galloflavin (a), lonidamine (b), syrosingopine (c) and compound 968 (d) treatments on energetic profiles of HCT-8 infected and non-infected controls: The glycolytic function was evaluated in C. parvum-infected host cells under 21% O2 by means of Seahorse Glycolysis Stress Test. Glycolytic activities in treated and infected cells were compared with untreated and infected cells, revealing a significant reduction in glycolytic capacity. (Third column) Thus, reduced glycolytic capacity corresponds with significant reduction of infection rates. Bars present means ± SD, (Energy maps, First column, n = 3; Glycolysis, second column n = 3, Infection rates, third column, n = 6). For evaluation of significance on glycolytic function (second column) one-way analyses of variance (ANOVA) followed by Dunnett’s test was performed. Significances of inhibitors effects on infection rates (third column) were estimated by means of t-test. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.
Figure 9
Figure 9
Impact of C. parvum infection and inhibitor treatments on HCT-8 energetic profiles: (First column) The current energetic profiles are plotted by presenting OCR- and ECAR values in HCT-8 (n = 3, each condition). (Second column) Effect of galloflavin (a), lonidamine (b), syrosingopine (c) and compound 968 (d) treatments on energetic profiles of HCT-8 infected and non-infected controls: The glycolytic function was evaluated in C. parvum-infected host cells under 21% O2 by means of Seahorse Glycolysis Stress Test. Glycolytic activities in treated and infected cells were compared with untreated and infected cells, revealing a significant reduction in glycolytic capacity. (Third column) Thus, reduced glycolytic capacity corresponds with significant reduction of infection rates. Bars present means ± SD, (Energy maps, First column, n = 3; Glycolysis, second column n = 3, Infection rates, third column, n = 6). For evaluation of significance on glycolytic function (second column) one-way analyses of variance (ANOVA) followed by Dunnett’s test was performed. Significances of inhibitors effects on infection rates (third column) were estimated by means of t-test. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

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