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

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.

Huang X ，Hu M ，Zhou X ，Liu Y ，Shi C ，Shi X ... -  《mSphere》

Transcriptional Sequencing Uncovers Survival Mechanisms of Salmonella enterica Serovar Enteritidis in Antibacterial Egg White.

The survival mechanism of Salmonella enterica serovar Enteritidis in antibacterial egg white is not fully understood. In our lab, an egg white-resistant strain, S. Enteritidis SJTUF 10978, was identified. Cell envelope damage and osmotic stress response (separation of cell wall and inner membrane as well as cytoplasmic shrinkage) of this strain surviving in egg white were identified through microscopic observation. RNA-Seq analysis of the transcriptome of Salmonella survival in egg white showed that a considerable number of genes involved in DNA damage repair, alkaline pH adaptation, osmotic stress adaptation, envelope damage repair, Salmonella pathogenicity island 2 (SPI-2), iron absorption, and biotin synthesis were significantly upregulated (fold change ≥ 2) in egg white, indicating that these pathways or genes might be critical for bacterial survival. RNA-Seq results were confirmed by qRT-PCR, and the survival analysis of six gene deletion mutants confirmed their importance in the survival of bacteria in egg white. The importance of alkaline pH adaptation and envelope damage repair for Salmonella to survive in egg white were further confirmed by analysis of nhaA, cpxR, waaH, and eco deletion mutants. According to the RNA-Seq results, we propose that alkaline pH adaptation might be the cause of bacterial osmotic stress phenotype and that the synergistic effect between alkaline pH and other inhibitory factors can enhance the bacteriostatic effect of egg white. Moreover, cpxR and sigE were recognized as the central regulators that coordinate bacterial metabolism to adapt to envelope damage and alkaline pH.IMPORTANCESalmonella enterica serovar Enteritidis is a major foodborne pathogen that causes salmonellosis mainly through contaminated chicken eggs or egg products and has been a worldwide public health threat since 1980. Frequent outbreaks of this serotype through eggs correlate significantly with its exceptional survival ability in the antibacterial egg white. Research on the survival mechanism of S. Enteritidis in egg white will help to further understand the complex and highly effective antibacterial mechanisms of egg white and lay the foundation for the development of safe and effective vaccines to prevent egg contamination by this Salmonella serotype. Key pathways and genes that were previously overlooked under bactericidal conditions were characterized as being induced in egg white, and synergistic effects between different antimicrobial factors appear to exist according to the gene expression changes. Our work provides new insights into the survival mechanism of S. Enteritidis in egg white.

Huang X ，Zhou X ，Jia B ，Li N ，Jia J ，He M ，He Y ，Qin X ，Cui Y ，Shi C ，Liu Y ，Shi X ... -  《mSphere》

Quantitative proteomics reveals the crucial role of YbgC for Salmonella enterica serovar Enteritidis survival in egg white.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a food-borne bacterial pathogen that can cause human salmonellosis predominately by contamination of eggs and egg products. However, its survival mechanisms in egg white are not fully understood, especially from a proteomic point of view. In this study, the proteomic profiles of S. Enteritidis in Luria-Bertani (LB) broth containing 50% and 80% egg white, and in whole egg white were compared with the profile in LB broth using iTRAQ technology to identify key proteins that were involved in S. Enteritidis survival in egg white. It was found that there were 303, 284 and 273 differentially expressed proteins in S. Enteritidis after 6 h exposure to whole, 80% and 50% egg white, respectively. Most of up-regulated proteins were primarily associated with iron acquisition, cofactor and amino acid biosynthesis, transporter, regulation and stress responses, whereas down-regulated proteins were mainly involved in energy metabolism, virulence as well as motility and chemotaxis. Three stress response-related proteins (YbgC, TolQ, TolA) of the tol-pal system responsible for maintaining cell membrane stability of Gram-negative bacteria were up-regulated in S. Enteritidis in response to whole egg white. Interestingly, deletion of ybgC resulted in a decreased resistance of S. Enteritidis to egg white. Compared with the wild type and complementary strains, a 3-log population reduction was observed in △ybgC mutant strain after incubation in whole egg white for 24 h. Cellular morphology of △ybgC mutant strain was altered from rods to spheres along with cell lysis in whole egg white. Furthermore, deletion of ybgC decreased the expression of tol-pal system-related genes (tolR, tolA). Collectively, these proteomic and mutagenic analysis reveal that YbgC is essential for S. Enteritidis survival in egg white.

Qin X ，He S ，Zhou X ，Cheng X ，Huang X ，Wang Y ，Wang S ，Cui Y ，Shi C ，Shi X ... -  《-》

[sRNA (sraB) regulate the resistant ability of Salmonella enterica serovar enteritidis to egg albumen].

Jiang H ，Cao M ，Cao X ，Gu H ，Zeng K ... -  《-》

The Salmonella Enteritidis TolC outer membrane channel is essential for egg white survival.

Salmonella Enteritidis has developed the potential to contaminate eggs by surviving in the antimicrobial environment of the hen's egg white. This has led to a worldwide pandemic of foodborne salmonellosis infections in humans due to the consumption of contaminated eggs and egg-derived products. The molecular mechanisms of Salmonella Enteritidis egg white survival are not fully clear. Using in vivo expression technology and promoter-reporter fusions we showed that the promoter of the tolC gene, encoding the TolC outer membrane channel that is used by multidrug efflux pumps to export harmful molecules and to secrete bacterial products, is activated by egg white at the chicken body temperature. Using a Salmonella Enteritidis tolC deletion mutant we showed that TolC has an important role in egg white survival. Chromatographic separation techniques and subsequent testing of antimicrobial activities of separated egg white fractions led to the identification of ovotransferrin as the egg white antimicrobial factor which is capable of inhibiting growth of a tolC deletion strain but not the wild type strain. We provide evidence that TolC protects Salmonella Enteritidis against ovotransferrin-mediated growth inhibition in egg white.

Raspoet R ，Eeckhaut V ，Vermeulen K ，De Smet L ，Wen Y ，Nishino K ，Haesebrouck F ，Ducatelle R ，Devreese B ，Van Immerseel F ... -  《-》