The Zinc Nutritional Immunity of Epinephelus coioides Contributes to the Importance of znuC During Pseudomonas plecoglossicida Infection

Abstract

Previously, the dual RNA-seq was carried out in a Pseudomonas plecoglossicida- Epinephelus coioides infection model to investigate the dynamics of pathogen-host interplay in vivo. ZnuC, a member of ZnuCBA Zn importer, was found transcriptionally up-regulated during infection. Thus, this study aimed to assess its role during the trade-off for Zn between host and P. plecoglossicida. ICP-MS analysis and fluorescent staining showed that Zn was withheld from serum and accumulated in the spleen, with increased Zn uptake in the Golgi apparatus of macrophages after infection. Additionally, growth assay, macrophage infection and animal infection after gene knockout / silencing revealed that znuC was necessary for growth in Zn-limiting conditions, colonization, intracellular viability, immune escape and virulence of P. plecoglossicida. Further analysis with dual RNA-seq revealed associations of host's Zn nutritional immunity genes with bacterial Zn assimilation genes. IL6 and ZIP4 played key roles in this network, and markedly affected znuB expression, intracellular viability and immune escape, as revealed by gene silencing. Moreover, EMSA and GFP reporter gene analysis showed that Fur sensed changes in Fe concentration to regulate znuCBA in P. plecoglossicida. Jointly, these findings suggest a trade-off for Zn between host and P. plecoglossicida, while ZnuC is important for P. plecoglossicida Zn acquisition.

Keywords: Epinephelus coioides; Pseudomonas plecoglossicida; dual RNA-seq; nutritional immunity; znuC.

Figure 1

Effect of Pseudomonas plecoglossicida infection on Zinc (Zn) concentration in serum, spleen, and macrophages. (A) Serum Zn concentrations and (B) spleen Zn concentrations were measured via inductively coupled plasma mass spectrometry (ICP-MS) in Epinephelus coioides infected with P. plecoglossicida at 48 hpi and compared to the healthy E. coioides. Values are mean ± SD (n = 3, *P < 0.05, **P < 0.01). (C) Confocal microscopy was used to visualize labile Zn with the fluorescent probe, Zinquin ethyl ester. Staining for Zn (blue) and Golgi (red) in macrophages. Purple shows a co-localization of Golgi and Zn. Scale bars represent 10 μm, 3 independent experiments.

Figure 2

ZnuC is essential for Pseudomonas plecoglossicida growth under Zinc (Zn) limiting conditions. (A) Expression of znuC during infection was determined by dual RNA-seq and quantitative real time PCR (qRT-PCR). (B) To characterize znuC’s function, five znuC-RNAi strains were constructed, and the silencing efficiencies were validated by qRT-PCR. (C) To validate whether the znuC in P. plecoglossicida is important for growth during Zn limitation, growth of wild type, znuC-50%RNAi, znuC-95%RNAi, ΔznuC and znuC ⁺ strains were compared in Zn-limiting condition with 2 μM TPEN. gyrB was used as a reference gene in the qRT-PCR analysis. Values are mean ± SD (n = 3, *P < 0.05, **P < 0.01).

Figure 3

ZnuC is essential for Pseudomonas plecoglossicida virulence. (A) To assess the importance of znuC to P. plecoglossicida pathogenesis, Epinephelus coioides were infected with P. plecoglossicida wild type and znuC-95%RNAi. Amounts of E. coioides that survived after infection with the indicated strains were compared (n=3). (B) The E. coioides infected with P. plecoglossicida were dissected and observed at 96 hpi. Symptoms of E. coioides spleen after infection with P. plecoglossicida were compared. (C) The bacterial burdens of P. plecoglossicida wild type and znuC-95%RNAi in E. coioides spleen were measured by quantitative real time PCR (qRT-PCR) at 1, 6, 12, 24, 48, 72, and 96 hpi. Temporal dynamic distribution of P. plecoglossicida in E. coioides spleen were compared. (D) To determine whether the reduced bacterial burdens of the znuC-95%RNAi in E. coioides spleen was due to the stable low expression of znuC, we compared the expression of znuC in P. plecoglossicida wild type and znuC-95%RNAi during the infection at 0, 24, 48, 72 and 96 hpi. (E, F) Given our observation of increased Zinc (Zn) uptake in the Golgi apparatus of E. coioides macrophages after infection, we sought to validate whether the znuC in P. plecoglossicida is important for intracellular survival. The intracellular viability and immune escape of wild type, znuC-50%RNAi, znuC-95%RNAi, ΔznuC and znuC ⁺ strains were compared in E. coioides macrophages. Values are mean ± SD (n = 3, **P < 0.01).

Figure 4

Dual RNA-seq reveals differentially expressed genes (DEGs) associated with the trade-off for Zinc (Zn) between host and pathogen during Pseudomonas plecoglossicida infection. (A, B) Dual RNA-seq was carried out to assess spleen specimens from Epinephelus coioides upon infection with P. plecoglossicida for 48h. Compared to the wild-type P. plecoglossicida group, spleen specimens from animals infected with the znuC-95%RNAi strain had 23,059 DEGs, including 12,005 downregulated and 11,054 upregulated genes. Totally 5,103 genes were detected in the transcriptome of P. plecoglossicida from E. coioides spleen. In comparison to wild-type P. plecoglossicida in E. coioides spleen, the znuC-95%RNAi strain had 88 DEGs, including 74 downregulated and 14 upregulated genes. Volcano plot indicated upregulated and downregulated genes of E. coioides (A) and P. plecoglossicida (B) detected by dual RNA-seq here. (C, D) Among all the DEGs, 15 Zn nutritional immunity related genes were identified in E. coioides, while 4 DEGs related to Zn acquisition were found in P. plecoglossicida. Heat maps indicated E. coioides Zn nutritional immunity related genes and P. plecoglossicida Zn acquisition genes (adjusted FDR < 0.05; | log2FC | ≥ 1; n=3) compared to healthy E. coioides. Upregulated and downregulated genes are colored in red and green, respectively. (E, F) quantitative real time PCR (qRT-PCR) analysis of E. coioides Zn nutritional immunity related genes and P. plecoglossicida Zn acquisition genes in E. coioides injected with PBS, wild type and znuC-RNAi P. plecoglossicida, respectively. Values are mean ± SD (n = 3, *P < 0.05, **P < 0.01).

Figure 5

IL6 and ZIP4 play key roles in the trade-off for Zinc (Zn) between Epinephelus coioides and Pseudomonas plecoglossicida. (A) Predicted regulation network among E. coioides Zn nutritional immunity related genes and P. plecoglossicida Zn acquisition genes. Occurrence of infection, E. coioides genes and P. plecoglossicida genes are colored in yellow, blue and red, respectively. Positive and negative correlations are represented by lines. (B) To characterize the importance of IL6 and ZIP4, five IL6-RNAi and ZIP4-RNAi E. coioides macrophages were constructed, and the silencing efficiencies were validated by quantitative real time PCR (qRT-PCR). (C) According to the pathogen-host gene regulatory network, IL6 and ZIP4 were supposed to promote and repress znuB expression, respectively. To validate this, qRT-PCR was carried out on wild type, IL6-RNAi-2325 and ZIP4-RNAi-954 E. coioides macrophages. (D, E) In order to validate whether the IL6 and ZIP4 in E. coioides macrophages is important for intracellular killing of P. plecoglossicida, the intracellular viability and immune escape of wild type P. plecoglossicida was compared in wild type, IL6-RNAi-2325 and ZIP4-RNAi-954 E. coioides macrophages. Values are mean ± SD (n = 3, *P < 0.05, **P < 0.01).

Figure 6

ZnuCBA is negatively regulated by Fur. (A) In order to validate whether znuA, znuB and znuC were transcribed into a single mRNA molecule, reverse transcription PCR (RT-PCR) was carried out to assess RNA isolated from Pseudomonas plecoglossicida using four pairs of primers. Pair A was designed to amplify the region between WP_016394241.1 and znuC, pair B between znuC and znuB, pair C between znuB and znuA, and pair D between znuA and katE. Primer pairs A and D produced no bands, while primer pairs B and C produced bands of correct sizes. (B) Genetic organization of the znuCBA operon. (C) To validate the binding of Fur on the znuC promoter, the electrophoretic mobility shift assay (EMSA) was carried out. 6-carboxyfluorescein-labeled DNA fragment from the promoter region of znuC (upper panel) or DNA fragment with the same boundaries but with Fur box removal (lower panel) was added to the reaction mixture containing different concentrations of the Fur protein. (D) In order to assess the function of Fur, five fur-RNAi strains were constructed, and the silencing efficiencies were validated by quantitative real time PCR (qRT-PCR). (E) To validate whether the Fur in P. plecoglossicida is important for the regulation of znuCBA, qRT-PCR was carried out in wild type, fur-50%RNAi, fur-95%RNAi, Δfur and fur ⁺ strains. (F) For further validating znuCBA downregulation via Fur, a znuCBA-reporter gene fusion controlled by the putative Fur promoter (nt -900 to +150) was constructed. Expression was assessed by measuring fluorescence with fur knocked down or knocked out. (G) To validate the effect of Iron (Fe) limitation on znuCBA expression, expression of znuCBA in wild type were compared in LB and Fe-limiting condition with 2’,2-Dipyridyl. (H) For further validating znuCBA upregulation under Fe limitation, transcription levels of the znuC-GFP reporter gene fusion in P. plecoglossicida under Fe limiting conditions was detected. Values are mean ± SD (n = 3, *P < 0.05, **P < 0.01).

Figure 7

Working model of the struggle for Zinc (Zn) between Epinephelus coioides and Pseudomonas plecoglossicida.