Multiple alignments of C-terminal amino acid sequences contained conserved amidase domains in zebrafish (Danio rerio, Dr), ricefish (Oryzias latipes, Ol), human (Homo sapiens, Hs) and mouse (Mus musculus, Ms) PGRPs.

(A) The multiple sequence alignment of PGRPs was presented. The conserved amino acid residues were shaded in red, and similar amino acids were marked with red characters. (B) The homology percentage of PGRPs gene sequences was presented. The protein sequences were listed as followed: Zebrafish (PGRP2: NP_001038631; PGRP5: NP_001037786; PGRP6:NP_001038687), ricefish (PGRP2: NP_004065551; PGRP5: NP_004075905; PGRP6:NP_004071889), human (PGRP1: NP_005082; PGRP2: NP_443122; PGRP3: NP_443123; PGRP4: NP_065126) and mouse (PGRP1: NP_033428; PGRP2: NP_067294; PGRP3: NP_997130; PGRP4: NP_997146).

Phylogenetic tree analysis of DrPGRPs with other known PGRPs form various species.

Phylogenetic tree was obtained from a CLUSTALW alignment and MEGA6 Neighbor-joining of 14 sequences. The number at each node indicates the bootstrap analysis from 1000 replicates.

Structural characteristics and expression patterns of zebrafish PGRPs genes.

(A) Schematic representations of the zebrafish PGRP structures. Characteristic domains and lengths of the amino acid sequences are indicated. The green boxes represent the low complexity region, and pink boxes stand for PGRP domains that contained a conserved N-acetylmuramoyl-L-alanine amidase (ami_2) region. (B) The mRNA expression levels of zebrafish PGRPs in different tissues. The relative transcript levels of DrPGRP2, 5, 6 in brain, eye, muscle, egg, liver and intestine were presented. The values in each group were presented as means ± SEM (Each value contains at least four biological replicates, and each biological replicate contains three technical replicates). For all experiments, *p < 0.05; **p <0 .01.

Zebrafish PGRP5 exhibits differential expression characteristics under different bacterial stimuli.

(A) The survival rate of zebrafish embryos at 72 hpf after Gram-negative E.coli (A) and Gram-positive M. luteus (B) challenge. (B) The hatching rate of zebrafish embryos at 24 hpf after E.coli and M. luteus challenge. (C) The temporal mRNA expression profiles of zebrafish PGRP5 in zebrafish larvae after E. coli challenge. (D) The temporal mRNA expression profiles of zebrafish PGRP5 in zebrafish larvae after M. luteus challenge. We detected the expression of PGRP5 gene under different stimuli at time points 6, 24, 48, and 72 hours, respectively. The values in each group were presented as means ± SEM (Each value contains at least four biological replicates, and each biological replicate contains three technical replicates). For all experiments, *p < 0.05; **p <0.01.

The pro-inflammatory cytokines were mainly inhibited but anti-inflammatory genes were significantly activated in zebrafish embryos under PGRP5-KD conditions.

(A) The relative mRNA levels of IL-1β at 6, 24, 48, and 72 hpf both in the mock and PGRP5-KD conditions. (B) The relative mRNA levels of IL-6 at 6, 24, 48, and 72 hpf both in the mock and PGRP5-KD conditions. (C) The relative mRNA levels of TNF-α at 6, 24, 48, and 72 hpf both in the mock and PGRP5-KD conditions. (D) The relative mRNA levels of TGF-β at 6, 24, 48, and 72 hpf both in the mock and PGRP5-KD conditions. The values in each group were presented as means ± SEM (Each value contains at least four biological replicates, and each biological replicate contains three technical replicates). For all experiments, *p < 0.05 and **p <0 .01 for intra-group comparison; #p < 0.05 and ##p <0 .01 for inter-group comparison.

PGRP5 plays an important role in neurobehavioral alteration and oxidative stress in zebrafish embryos.

(A) The movement tracks of zebrafish at 5 dpf larvae that exposed to mock and PGRP5-KD conditions. Three representative photographs are shown in each group. (B) The total distance moved of zebrafish larvae that both in control and PGRP5-KD conditions. (C) The average velocity of zebrafish larvae that both in mock and PGRP5-KD conditions. (D) Detection the contents of ROS, along with enzymatic activities of CAT, SOD and AChE in larval zebrafish that both in mock and PGRP5-KD conditions. The values are presented as means ± SEM (Each value contains at least four biological replicates, and each biological replicate contains three technical replicates). *, p < 0.05; **, p < 0.01. (E) The behavioral trajectory characteristics of zebrafish under 10 μM minocycline drug exposure. (F-G) The total distance and mean velocity of zebrafish larvae that exposed to mock and 10 μM minocycline.