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. IMPORTANCE Salmonella 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.

Egg-White Proteins Have a Minor Impact on the Bactericidal Action of Egg White Toward Salmonella Enteritidis at 45°C

“…Moreover, it is possible that EW contains unknown antimicrobial polypeptides of low mass (<10 kDa) which could also play a part in the bactericidal activity of EW at 45°C. Studies on the survival of an S. Enteritidis yoaE mutant found that proteinase K treatment eradicated the antibacterial activity of a 3-kDa EW filtrate ( Huang et al, 2020 ). This result suggests that antimicrobial polypeptides of <3 kDa (and therefore < 10 kDa) play an active role in the antibacterial defense of EW, highlighting the need for further investigation.…”

The Role of Ovotransferrin in Egg-White Antimicrobial Activity: A Review

“…Upon egg white exposure, the psp genes where shown to be subject to rapid (≤7 min) up-regulation followed by an expression decrease from 25–45 min [ 83 ]. In apparent contradiction to this finding, Huang et al (2019) failed to observe any induction of the psp genes by egg white [ 96 ]; this discrepancy likely arises from the longer incubation times (6 and 24 h) employed.…”

“…Enteritidis to egg white and by the egg white sensitivity of mutants with defects in cell envelope systems. Approaches used to demonstrate such effects have included in vivo expression technology (IVET) [ 30 ], microarray- and RNAseq-based transcriptomic analysis [ 83 ], quantitative proteomics [ 84 ], directed mutagenesis [ 31 , 33 , 96 ], random mutagenesis [ 13 ], and microarray-based transposon-library screening [ 32 ].…”

“…Many of the S. Enteritidis genes/proteins highlighted by these studies have roles in periplasm homeostasis and degradation of abnormal proteins [ 33 , 83 ], membrane permeability and replacing the general-diffusion porins with porins of smaller pore-size [ 13 , 83 ], maintenance of cell envelope integrity [ 33 , 83 , 84 , 96 ], LPS biosynthesis [ 13 , 31 , 32 ], cell-wall integrity and biosynthesis [ 30 ], remodelling of the peptidoglycan [ 83 , 96 ], and removal of antimicrobial compounds through multidrug-efflux [ 33 , 83 ]. The majority of these genes are under the control of the CpxAR regulator that responds to a wide variety of envelope perturbations, including those induced by exposure to antimicrobial molecules or peptides ([ 97 ] for a review).…”

“…Important new insight into the components of egg white responsible for induction of the CpxAR controlled genes in S . Enteritidis was recently obtained by utilisation of egg white ultrafiltrates (3 and 9 kDa cut-off membranes) [ 96 ] whereby similar CpxAR responses were achieved with egg white and the egg white filtrates. Thus, since the filtrates lacked ovotransferrin yet still induced the CpxAR regulon, it is clear that neither ovotransferrin (nor any other protein above 3 kDa) are strictly required for the observed CpxAR induction.…”