Immunoprotective Effects of Two Histone H2A Variants in the Grass Carp Against Flavobacterium columnare Infection

Abstract

In teleost fish, the nucleotide polymorphisms of histone H2A significantly affect the resistance or susceptibility of zebrafish to Edwardsiella piscicida infection. Whether histone H2A variants can enhance the resistance of grass carp to Flavobacterium columnare infection remains unclear. Here, the effects of 7 previously obtained variants (gcH2A-1~gcH2A-7) and 5 novel histone H2A variants (gcH2A-11, gcH2A-13~gcH2A-16) in response to F. columnare infection were investigated. It was found that these histone H2A variants could be divided into type I and II. Among them, 5 histone H2A variants had no any effects on the F. columnare infection, however 7 histone H2A variants had antibacterial activity against F. columnare infection. The gcH2A-4 and gcH2A-11, whose antibacterial activity was the strongest in type I and II histone H2A variants respectively, were picked out for yeast expression. Transcriptome data for the samples from the intestines of grass carp immunized with the engineered Saccharomyces cerevisiae expressing PYD1, gcH2A-4 or gcH2A-11 revealed that 5 and 12 immune-related signaling pathways were significantly enriched by gcH2A-4 or gcH2A-11, respectively. For the engineered S. cerevisiae expressing gcH2A-4, NOD-like receptor and Toll-like receptor signaling pathways were enriched for up-regulated DEGs. Besides NOD-like receptor and Toll-like receptor signaling pathways, the engineered S. cerevisiae expressing gcH2A-11 also activated Cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway and C-type lectin receptor signaling pathway. Furthermore, grass carp were immunized with the engineered S. cerevisiae expressing PYD1, gcH2A-4 or gcH2A-11 for 1 month and challenged with F. columnare. These grass carp immunized with gcH2A-4 or gcH2A-11 showed lower mortality and fewer numbers of F. columnare than did the control group. All these results suggest that gcH2A-4 and gcH2A-11 play important roles in evoking the innate immune responses and enhancing disease resistance of grass carp against F. columnare infection.

Keywords: Flavobacterium columnare; Saccharomyces cerevisiae; grass carp; histone H2A variants; immunoprotective effect.

Figure 1

The cloning and sequence analysis of grass carp histone H2A variants. (A) The cloning of gcH2A-11 from the gills of grass carp individual 1 infected with F. columnare. (B) The cloning of gcH2A-13 and gcH2A-14~16 from the gills of grass carp individual 2 and 3 infected with F. columnare, respectively. (C) Sequence alignments of gcH2A-4, gcH2A-11 and gcH2A-13~16. The conventional histone H2A region is underlined. (D) Phylogenetic analysis of grass carp histone H2A variants, and histone H2A from other species including shrimp H2A (GenBank accession number: XM_037921631), human H2A.1 (GenBank accession number: M60752), mouse H2A.1 (GenBank accession number: M33988), rat H2A.2 (GenBank accession number: JX661509), human H2A.2 (GenBank accession number: L19779), tongue sole H2A (GenBank accession number: KU904500) and Arabidopsis H2A (GenBank accession number: AK228272).

Figure 2

The effects of grass carp histone H2A variants in bacterial infection. (A–E) No significant effects of gcH2A-1, gcH2A-5, gcH2A-13, gcH2A-14 and gcH2A-15 on the proliferation of F. columnare in vitro. (F–J) The antibacterial effects of gcH2A-2, gcH2A-3, gcH2A-4, gcH2A-11 and gcH2A-16 both at 3 hpi and 6 hpi. (K, L) The antibacterial effects of gcH2A-6 and gcH2A-7 at 6 hpi. Data represented means ± SEM (n=3), and were tested for statistical significance. **p < 0.01; ns, not significant. The asterisk above the bracket indicates statistical significance between the two groups connected by the bracket.

Figure 3

The recombinant expressions of gcH2A-4 and gcH2A-11 in the S. cerevisiae strain. (A) Validation of the recombinant gcH2A-4−PYD1 and gcH2A-11−PYD1 plasmids using BamH I/EcoR I double restriction enzyme digestion. (B) Western Blotting of the cell lysates transformed with the PYD1 empty plasmid. (C) Western Blotting of the cell lysates transformed with PYD1, gcH2A-4−PYD1 or gcH2A-11−PYD1.

Figure 4

Differentially expressed genes and significantly enriched KEGG pathways in grass carp regulated by the engineered S. cerevisiae expressing gcH2A-4 and gcH2A-11. (A) The schematic diagram for the immunized grass carp used for illumina deep sequencing. (B) The number of DEGs regulated by the engineered S. cerevisiae expressing gcH2A-4 and gcH2A-11. (C) The significantly enriched KEGG pathways for up-regulated DEGs in intestines regulated by the engineered S. cerevisiae expressing gcH2A-4 and gcH2A-11. (D) The significantly enriched KEGG pathways for down-regulated DEGs in intestines regulated by the engineered S. cerevisiae expressing gcH2A-4 and gcH2A-11.

Figure 5

The effects of the engineered S. cerevisiae expressing gcH2A-4 on the expression patterns of DEGs involved in PRRs-mediated signaling pathways. (A) The gene cluster for DEGs involved in the NOD-like receptor signaling pathway for the samples from the intestines of grass carp after 7 days of the first immunization. (B) The gene cluster for DEGs involved in the Toll-like receptor signaling pathway for the samples from the intestines of grass carp after 7 days of the first immunization. A color key denotes the gradient scale of gene expression from low (blue) to high (red) degrees. The receptors of NLR and TLR are underlined.

Figure 6

The effects of the engineered S. cerevisiae expressing gcH2A-11 on the expression patterns of DEGs involved in PRRs-mediated signaling pathways. (A) The gene cluster for DEGs involved in the C-type lectin receptor signaling pathway for the samples from the intestines of grass carp after 7 days of the first immunization. (B) The gene cluster for DEGs involved in the Cytosolic DNA-sensing pathway for the samples from the intestines of grass carp after 7 days of the first immunization. (C) The gene cluster for DEGs involved in the RIG-I-like receptor signaling pathway for the samples from the intestines of grass carp after 7 days of the first immunization. (D) The gene cluster for DEGs involved in the Toll-like receptor signaling pathway for the samples from the intestines of grass carp after 7 days of the first immunization. A color key denotes the gradient scale of gene expression from low (blue) to high (red) degrees. The receptors of CLR and TLR are underlined.

Figure 7

Validation of transcriptome data by qRT-PCR. (A) The gene cluster for DEGs involved in the NOD-like receptor signaling pathway for the samples from the intestines of grass carp after 7 days of the first immunization using the engineered S. cerevisiae expressing gcH2A-11. (B) Validation of transcriptome data by qRT-PCR for 11 DEGs involved in the Toll like receptor pathway. Data represented means ± SEM (n=3), and were tested for statistical significance. *p < 0.05; **p < 0.01; ns, not significant.

Figure 8

The effects of the engineered S. cerevisiae expressing gcH2A-4 and gcH2A-11 on the disease resistance of grass carp against F. columnare infection. (A) The schematic diagram for the immunized grass carp used for survival analysis. (B) The effects of the engineered S. cerevisiae expressing gcH2A-4 and gcH2A-11 on the bacteria proliferation of F. columnare Data represented means ± SEM (n=3), and were tested for statistical significance. *p < 0.05; **p < 0.01. (C) The effects of the engineered S. cerevisiae expressing gcH2A-4 and gcH2A-11 on the survival of grass carp in response to F. columnare infection.