The impact of Lactococcus lactis KUST48 on the transcription profile of Aeromonas hydrophila-infected zebrafish spleen

Abstract

Aeromonas hydrophila, an aquatic pathogenic bacterium, has been found to infect many fish species and cause huge aquaculture losses. Antibiotics are the most common drugs used to treat these infections. However, antibiotic abuse can lead to the development of antibiotic resistance. Probiotics have the potential to replace antibiotics for preventing infections. Zebrafish (Danio rerio) is a model organism used to study the innate immune system and host-pathogen interactions. Currently, there is little information on how the fish immune system responds to A. hydrophila and probiotic treatment. To increase the understanding of the molecular mechanisms behind the zebrafish defense against A. hydrophila and provide evidence that antibiotics can be replaced by probiotics, a transcriptome analysis of the zebrafish spleen was conducted 48 hours after infection by A. hydrophila, as well as after treatment using Lactococcus lactis KUST48 4 hours after infection. A total of 36,499 genes were obtained. There were 3,337 genes found to have significant differential expression between treatment and control groups. According to further annotation and enrichment analysis, differentially expressed genes (DEGs) were involved in signal transduction, endocrine system cancer, and the immune system. Insulin resistance disappeared in the zebrafish after treatment. Quantitative real-time PCR was performed to confirm the significant regulation of immune defense DEGs, the results of which were consistent with the RNA-sequencing data. These results could serve as a basis for future studies on the immune response to A. hydrophila and provide suggestions for probiotic alternatives to antibiotics, which will be of great significance to aquaculture and environmental protection.IMPORTANCEIn recent years, the unreasonable use of antibiotics has led to the emergence of drug-resistant pathogenic bacteria, antibiotic residues, cross infection, toxic side effects, and so on, which has caused a serious threat to human food safety and life health. In recent years, many studies have demonstrated the potential of probiotics as a substitute for antibiotics, but there is still a lack of understanding of the molecular mechanisms underlying probiotic therapy. We conduct a research on the impact of Lactococcus lactis KUST48 on the transcription profile of Aeromonas hydrophila-infected zebrafish spleen. Mortality of zebrafish infected with A. hydrophila was significantly reduced after treatment with L. lactis KUST48. Our results can help to strengthen our understanding of the pathogenic mechanisms of zebrafish and provide a valuable reference for the molecular mechanisms of probiotic therapy.

Keywords: Aeromonas hydrophila; Lactococcus lactis; immune response; transcriptome; zebrafish.

Fig 1

The effect of A. hydrophila on zebrafish. (A) Survival of zebrafish in different group. (B) Representative morphology and spleen H&E staining of zebrafish. Tissue relaxation (red arrow) and congestion were observed in zebrafish spleen from the AHI group as well as increased number of macrophages and hemosiderin (red circle).

Fig 2

Activity of enzyme in the spleen of A. hydrophila-infected zebrafish at 48 hours. (A) SOD activity. (B) LZM activity. (C) AKP activity. (D) ACP activity. N.S. means no significance; *P < 0.05; **P < 0.01.

Fig 3

Volcano plot of differently expressed genes. (A) AHI vs CT. (B) LLT vs CT. (C) LLT vs AHI.

Fig 4

GO classifications of DEGs among different groups. DEGs were annotated into three GO categories: biological process, MF, and CC. (A) AHI vs CT. (B) LLT vs CT. (C) LLT vs AHI.

Fig 5

KEGG annotation of DEGs among different groups. Different colored bars represent different groups. (A) AHI vs CT. (B) LLT vs CT. (C) LLT vs AHI.

Fig 6

Bubble diagrams of the top 20 enriched KEGG pathways among different groups. (A) AHI vs CT. (B) LLT vs CT. (C). LLT vs AHI.

Fig 7

Heatmap of DEGs in insulin resistance pathway (A) and immune-related pathway (B).

Fig 8

Validation of RNA-seq results by qPCR. (A) LLT vs CT. (B). LLT vs AHI.