The Broad-Spectrum Endolysin LySP2 Improves Chick Survival after Salmonella Pullorum Infection

Abstract

Salmonella pullorum causes typical "Bacillary White Diarrhea" and loss of appetite in chicks, which leads to the death of chicks in severe cases; thus, it is still a critical issue in China. Antibiotics are conventional medicines used for Salmonella infections; however, due to the extensive long-term use and even abuse of antibiotics, drug resistance becomes increasingly severe, making treating pullorum disease more difficult. Most of the endolysins are hydrolytic enzymes produced by bacteriophages to cleave the host's cell wall during the final stage of the lytic cycle. A virulent bacteriophage, YSP2, of Salmonella was isolated in a previous study. A Pichia pastoris expression strain that can express the Salmonella bacteriophage endolysin was constructed efficiently, and the Gram-negative bacteriophage endolysin, LySP2, was obtained in this study. Compared with the parental phage YSP2, which can only lyse Salmonella, LySP2 can lyse Salmonella and Escherichia. The survival rate of Salmonella-infected chicks treated with LySP2 can reach up to 70% and reduce Salmonella abundance in the liver and intestine. The treatment group showed that LySP2 significantly improved the health of infected chicks and alleviated organ damage caused by Salmonella infection. In this study, the Salmonella bacteriophage endolysin was expressed efficiently by Pichia pastoris, and the endolysin LySP2 showed good potential for the treatment of pullorum disease caused by Salmonella pullorum.

Keywords: Pichia pastoris; Salmonella pullorum; endolysin; expression.

Figure 1

Neighbor-joining phylogenetic tree based on the amino acid fragment of LySP2 and related sequences. Bootstrap values > 50% (based on 1000 replicates) are shown at branch points. GenBank accession numbers are given in parentheses following phage names.

Figure 2

Construction and expression of LySP2. (A) Verification of pPICZ–LySP2 by enzyme digestion. Lanes: 1, DL 15,000 DNA marker; 2, H₂O; 3, pPICZ–LySP2 digestion with XhoI and XbaI; 4, pPICZ–LySP2 PCR amplification product; 5, DL 2000 DNA marker. (B) Identification of X33–pPICZ–LySP2 recombinant yeast by PCR. Lanes: 1, DL 2000 DNA marker; 2–3, negative control; 4–6, pPICZ–LySP2 PCR amplification product. (C) Fermentation products analyzed by SDS-PAGE. Lanes: 1, protein ladder; 2, fermentation products of X33–pPICZ after 4 days; 3, X33–pPICZ yeast cells after 4 days of fermentation; 4, 6, and 8, fermentation products of X33–pPICZ–LySP2 after 4, 3, and 2 days; 5, 7, and 9, X33–pPICZ–LySP2 yeast cells after 4, 3, and 2 days of fermentation.

Figure 3

General biological characteristics of LySP2. (A) Activity of LySP2; add pPICZ to hole 1 and LySP2 to hole 2. (B) Effect of temperature on LySP2 activity. (C) Effect of pH on LySP2 activity. (D) Antibacterial activity of LySP2 against test bacteria. (E) Minimal inhibitory concentration of LySP2. Values are the mean ± SD of three determinations.

Figure 4

LySP2 improves the survival and health of infected chicks. (A) Chick survival rate from Salmonella pullorum infection in different groups. (B) Health status of Salmonella pullorum-infected chicks in different groups. Values are the mean ± SD of three determinations.

Figure 5

Bacterial counts. Liver (A) and intestinal (B) Salmonella count. Values are the mean ± SD of three determinations.

Figure 6

Histopathologic H&E assessment of liver and tissue samples (100×). (A) Control group, (B) pPICZ group, (C) Phage group, (D) LySP2 group, (E) Normal group.

Figure 7

Intestinal H&E staining (100×). (A) Control group, (B) pPICZ group, (C) Phage group, (D) LySP2 group, (E) Normal group.