Metabolites-Enabled Survival of Crucian Carps Infected by Edwardsiella tarda in High Water Temperature

Abstract

Temperature is one of the major factors that affect the outbreak of infectious disease. Lines of evidences have shown that virulence factors can be controlled by thermo-sensors in bacterial pathogens. However, how temperature influences host's responses to the pathogen is still largely unexplored, and the study of this might pave the way to develop strategies to manage pathogenic bacterial infection. In the present study, we show that finfish Carassius carassius, the crucian carp that is tolerant to a wide range of temperatures, is less susceptible to bacterial infection when grown in 20°C than in 30°C. The different responses of C. carassius to bacterial infection could be partially explained by the distinct metabolisms under the specific temperatures: C. carassius shows elevated tricarboxylic acid cycle (TCA cycle) but decreased taurine and hypotaurine metabolism as well as lower biosynthesis of unsaturated fatty acids at 30°C. The decreased abundance of palmitate, threonine, and taurine represents the most characteristic metabolic feature. Consistently, exogenous palmitate, threonine, or taurine enhances the survival of C. carassius to bacterial infection at 30°C in a dose-dependent manner. This effect could be attributed to the inhibition on the TCA cycle by the three metabolites. This notion is further supported by the fact that low concentration of malonate, a succinate dehydrogenase inhibitor, increases the survival of C. carassius at 30°C as well. On the other hand, addition of the three metabolites rescued the decreased expression of pro-inflammatory cytokines including TNF-α1, TNF-α2, IL-1β1, IL-1β2, and lysozyme at 30°C. Taken together, our results revealed an unexpected relationship between temperature and metabolism that orchestrates the immune regulation against infection by bacterial pathogens. Thus, this study shed light on the modulation of finfish physiology to fight against bacterial infection through metabolism.

Keywords: Carassius carassius; bacterial infection; innate immunity; metabolome; water temperature.

Figure 1

The survival of crucian carp to E. tarda infection cultured at 20 and 30°C. C. carassius (n = 30 per group) were acclimated at 20 or 30°C for 7 days before bacterial challenge. For bacterial infection, C. carassius were injected with 10 μl 1 × 10⁵ CFU/fish E. tarda or 10 μl saline solution as negative control. Accumulative death was monitored for a total of 15 days.

Figure 2

C. carassius cultured at 20 and 30°C had a different metabolism. (A) Categories of the different metabolites. Fifty six metabolites with different abundance were searched against in KEGG for their categories, and the pie chart was generated in Excel 2010 (Microsoft, USA). (B) The number of metabolites of different abundance in each category as shown in (A). (C) Heat map of unsupervised hierarchical clustering of different metabolites (row). Yellow and blue indicate increase and decrease of the metabolites scaled to mean and standard deviation of row metabolite level, respectively, (see color scale) (D) Z scores (standard deviation from average) of metabolites identified from 30 to 20°C, which are corresponding to the data shown in (C). Each point represents one technical repeat of metabolite.

Figure 3

Pathway enrichment analysis of metabolites with different abundance. (A) Pathway enrichment analysis of different metabolites. Metabolites demonstrating different abundance were analyzed by pathway enrichment analysis using online software (http://www.metaboanalyst.ca). Significant enriched pathways are selected to plot (p < 0.05). (B) Abundance of metabolites in the enriched pathways listed in (A). The metabolites in fish grown at 30°C were compared to that at 20°C. Different metabolites highlighted in yellow and blue indicate increased and decreased abundance, respectively.

Figure 4

Identification of crucial biomarkers. (A) The PCA analysis of metabolites with difference abundance in fish grown at the 30 and 20°C to investigate intergroup difference. Each dot represents the technique replicates in the plot. The metabolites with difference abundance in fish grown at 30 and 20°C were separated by the independent factors t[1]. (B) S-plot, generated by OPLS-DA, to identify different metabolites of intragroup as from t[1] in (A). Each triangle represents individual metabolite. The potential biomarkers are highlighted in red for metabolites whose p value is ≥0.05 and 0.5 for absolute value of covariance p and correlation p (corr), respectively. (C) Crucial biomarkers represent the metabolism of fish grown between 30 and 20°C. The abundance of the three crucial biomarkers, taurine, threonine, and palmitic acid, are compared between 20 and 30°C and presented as scatter plot. Each dot represents individual replicate. *p < 0.05; **p < 0.01.

Figure 5

Metabolic network of metabolites of different abundance, and activity of the TCA cycle. (A) Integrated metabolic network in relation to different metabolites. Different metabolites are searched against KEGG, and the metabolic network was reconstructed with ChemDraw Pro 18. The red and blue stand for the increased and decreased metabolites in fish grown at 30°C, respectively. (B)The enzymatic activity of PDH, α-KGDH, SDH, and MDH of spleen from fish grown at 30 and 20°C were quantified. Spleens were removed, lysed and extracted for enzyme analysis, and the data were shown as histogram. The significant differences are analyzed by non-parametric Kruskal-Wallis one-way analysis with Dunn multiple comparison post hoc test. **p < 0.01 indicates statistic significant.

Figure 6

Crucial biomarkers modulate the metabolism of crucian carps and promote their survival against bacterial infections. (A) The survival of crucian carps in the presence of crucial biomarkers upon E. tarda infection. C. carassius was treated with saline control or different dose crucial biomarkers at 30°C for 3 days, followed by bacterial challenge through intraperitoneal injection (1 × 10⁵ CFU). The accumulative fish death was monitored for a total of 15 days' post-infection (n = 30 per group). (B) Activity of PDH, alpha-KGDH, SDH, and MDH of spleen in the presence of crucial biomarkers (200 μg taurine, 500 μg threonine, or 14 μg palmitic acid plus 10% BSA). Values are means ± SEM(n = 6 per group), and statistic difference is analyzed with non-parametric Kruskal-Wallis one-way analysis with Dunn multiple comparison post hoc test. **p < 0.01. (C) Percent survival of crucial carps in the presence of malonate. C. carassius was treated with malonate at different doses for 12 h followed by E. tarda challenge through intraperitoneal injection (1 × 10⁵ CFU). The accumulative fish death was monitored for a total of 15 days post-infection (n = 30 per group).

Figure 7

Crucial biomarkers modulate the innate immune responses of fish at 30°C. (A,B) qRT-PCR for cytokine genes of C. carassius treated with control (saline or 10% BSA) or crucial biomarkers (200 μg taurine, 500 μg threonine, or 14 μg palmitic acid plus 10% BSA) for 3 days following E. tarda challenge through intraperitoneal injection (1 × 10³ CFU). The spleens were collected 12 h post-injection for RNA extraction and qRT-PCR. Values are means ± SEM from six biological replicates. *p < 0.05; **p < 0.01.