Role of yoaE Gene Regulated by CpxR in the Survival of Salmonella enterica Serovar Enteritidis in Antibacterial Egg White

Abstract

The survival ability of Salmonella enterica serovar Enteritidis in antibacterial egg white is an important factor leading to Salmonella outbreaks through eggs and egg products. In this study, the role of the gene yoaE, encoding an inner membrane protein, in the survival of Salmonella Enteritidis in egg white, and its transcriptional regulation by CpxR were investigated. Quantitative reverse transcription-PCR (RT-qPCR) results showed that the yoaE gene expression was upregulated 35-fold after exposure to egg white for 4 h compared to that in M9FeS medium, and the deletion of yoaE (ΔyoaE) dramatically decreased the survival rate of bacteria in egg white to less than 1% of the wild type (WT) and the complementary strain at both 37 and 20°C, indicating that yoaE was essential for bacteria to survive in egg white. Furthermore, the ΔyoaE strain was sensitive to a 3-kDa ultrafiltration matrix of egg white because of its high pH and antimicrobial peptide components. Putative conserved binding sites for the envelope stress response regulator CpxR were found in the yoaE promoter region. In vivo, the RT-qPCR assay results showed that the upregulation of yoaE in a ΔcpxR strain in egg white was 1/5 that of the WT. In vitro, results from DNase I footprinting and electrophoretic mobility shift assays further demonstrated that CpxR could directly bind to the yoaE promoter region, and a specific CpxR binding sequence was identified. In conclusion, it was shown for the first time that CpxR positively regulated the transcription of yoaE, which was indispensable for survival of Salmonella Enteritidis in egg white.IMPORTANCESalmonella enterica serovar Enteritidis is the predominant Salmonella serotype that causes human salmonellosis mainly through contaminated chicken eggs or egg products and has been a global public health threat. The spread and frequent outbreaks of this serotype through eggs correlate significantly with its exceptional survival in eggs, despite the antibacterial properties of egg white. Research on the survival mechanisms of S. Enteritidis in egg white will help develop effective strategies to control the contamination of eggs by this Salmonella serotype and help further elucidate the complex antibacterial mechanisms of egg white. This study revealed the importance of yoaE, a gene with unknown function, on the survival of S. Enteritidis in egg white, as well as its transcriptional regulation by CpxR. Our work provides the basis to reveal the mechanisms of survival of S. Enteritidis in egg white and the specific function of the yoaE gene.

Keywords: Salmonella; alkaline pH; antimicrobial peptide; cpxR; egg white; ultrafiltration matrix; yoaE.

FIG 1

Survival of yoaE derivatives in egg white. (A) Survival rate of yoaE derivatives incubated with egg white for 24 h at 37°C and for 5 days at 20 and 4°C, respectively. (B) Growth curve of yoaE derivatives in egg white at 37°C for 12 h. The incubation temperatures were the same as the egg storage temperature. Mean values were taken from three independent experiments, and the standard errors of the means (error bars) are shown. **, P < 0.01.

FIG 2

Survival of yoaE derivatives in egg white and its ultrafiltration matrix. (A) Survival of the ΔyoaE strain in egg white and its ultrafiltration matrix with different molecular weight cutoffs. (B) Survival of ΔyoaE strain in a 3-kDa ultrafiltration matrix digested by different proteases. (C) Survival of a ΔyoaE strain in 3-kDa ultrafiltration matrix of different pHs. (D) Viability of yoaE derivatives in physiological saline of different pHs. (E) Growth curve of yoaE derivatives in neutral-pH LB medium. (F) Growth curve of yoaE derivatives in alkaline-pH LB medium (9.5).

FIG 3

Relative expression changes of genes yoaE and cpxR at mRNA level in egg white compared to that of M9FeS medium in both wild-type and ΔcpxR strains. The expression of yoaE and cpxR gene in the single strain in M9FeS was set to 1. Mean values are taken from three independent experiments, and the standard errors of the means (error bars) are shown. **, P < 0.01.

FIG 4

Verification of the directly binding of CpxR-P to the yoaE promoter regions by EMSA. Lanes 1 to 4, FAM-labeled yoaE Promoter (P_yoaE-FAM) incubated with different amounts of phosphorylated CpxR (CpxR-P, 0, 0.5, 1.0, and 2 μg); lanes 5 and 6, FAM-labeled cpxP promoter (P_cpxP-FAM) incubated without (lane 5) or with (lane 6) CpxR-P, with P_cpxP-FAM used as a positive control; lane 7, 50× unlabeled yoaE promoter probe (P_yoaE) competed with P_yoaE-FAM. For every lane, 0.5 μg of poly(dI-dC) was added to decrease nonspecific binding.

FIG 5

Identification of phosphorylated CpxR binding sequence in yoaE promoter and alignment analysis in different species. (A) DNase I footprinting assays of phosphorylated CpxR binding sequence on yoaE promoter region. A 250-bp FAM-labeled yoaE promoter DNA fragment was incubated without or with 2 μg pf phosphorylated CpxR (CpxR-P) and then subjected to DNase I digestion and fragment length analysis. The fluorescence signal of the labeled DNA fragments is plotted against the sequence of the fragment. The sequence of protected region bound by CpxR-P is shown. (B) Alignment of CpxR binding site on yoaE promoter region in the investigated species.

FIG 6

YoaE protein structure predicted by SMART (http://smart.embl-heidelberg.de/).