Dataset Information

The Three Lipocalins of Egg-White: Only Ex-FABP Inhibits Siderophore-Dependent Iron Sequestration by Salmonella Enteritidis.

ABSTRACT: Salmonella Enteritidis is the most prevalent food-borne pathogen associated with egg-related outbreaks in the European Union. During egg colonization, S. Enteritidis must resist the powerful anti-bacterial activities of egg white (EW) and overcome ovotransferrin-imposed iron-restriction (the most important anti-bacterial mechanism of EW). Many pathogens respond to iron restriction by secreting iron-chelating chemicals called siderophores but EW contains a siderophore-sequestering "lipocalin" protein (Ex-FABP) that is predicted to limit the usefulness of siderophores in EW. S. Enteritidis produces two siderophores: enterobactin, which is strongly bound by Ex-FABP; and the di-glucosylated enterobactin-derivative, salmochelin (a so-called "stealth" siderophore), which is not recognized by Ex-FABP. Thus, production of salmochelin may allow S. Enteritidis to escape Ex-FABP-mediated growth inhibition under iron restriction although it is unclear whether its EW concentration is sufficient to inhibit pathogens. Further, two other lipocalins (Cal-? and ?-1-ovoglycoprotein) are found in EW but their siderophore sequestration potential remains unexplored. In addition, the effect of EW lipocalins on the major EW pathogen, S. Enteritidis, has yet to be reported. We overexpressed and purified the three lipocalins of EW and investigated their ability to interact with the siderophores of S. Enteritidis, as well as their EW concentrations. The results show that Ex-FABP is present in EW at concentrations (5.1 ?M) sufficient to inhibit growth of a salmochelin-deficient S. Enteritidis mutant under iron restriction but has little impact on the salmochelin-producing wildtype. Neither Cal-? nor ?-1-ovoglycoprotein bind salmochelin or enterobactin, nor do they inhibit iron-restricted growth of S. Enteritidis. However, both are present in EW at significant concentrations (5.6 and 233 ?M, respectively) indicating that ?-1-ovoglycoprotein is the 4th most abundant protein in EW, with Cal-? and Ex-FABP at 11th and 12th most abundant. Further, we confirm the preference (16-fold) of Ex-FABP for the ferrated form (K d of 5.3 nM) of enterobactin over the iron-free form (K d of 86.2 nM), and its lack of affinity for salmochelin. In conclusion, our findings show that salmochelin production by S. Enteritidis enables this key egg-associated pathogen to overcome the enterobactin-sequestration activity of Ex-FABP when this lipocalin is provided at levels found in EW.

SUBMITTER: Julien LA

PROVIDER: S-EPMC7242566 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

The Three Lipocalins of Egg-White: Only Ex-FABP Inhibits Siderophore-Dependent Iron Sequestration by Salmonella Enteritidis.

Julien Louis Alex LA Fau Clémence C Baron Florence F Bonnassie Sylvie S Guérin-Dubiard Catherine C Nau Françoise F Gautier Michel M Karatzas Kimon Andreas KA Jan Sophie S Andrews Simon Colin SC

Frontiers in microbiology 20200515

Salmonella Enteritidis is the most prevalent food-borne pathogen associated with egg-related outbreaks in the European Union. During egg colonization, S. Enteritidis must resist the powerful anti-bacterial activities of egg white (EW) and overcome ovotransferrin-imposed iron-restriction (the most important anti-bacterial mechanism of EW). Many pathogens respond to iron restriction by secreting iron-chelating chemicals called siderophores but EW contains a siderophore-sequestering " ...[more]

PMID: 32477312

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Project description:Salmonella enterica serovar Enteritidis is noted for its ability to survive the harsh antibacterial activity of egg white which is presumed to explain its occurrence as the major food-borne pathogen associated with the consumption of eggs and egg products. Liquid egg white is a major ingredient for the food industry but, because of its thermal fragility, pasteurization is performed at the modest temperature of 57°C (for 2-6 min). Unfortunately, such treatment does not lead to sufficient reduction in S. Enteritidis contamination, which is a clear health concern when the product is consumed without cooking. However, egg white is able to limit S. Enteritidis growth due to its alkaline pH, iron deficiency and multiple antimicrobial proteins. This anti-Salmonella activity of egg white is temperature dependent and becomes bactericidal once the incubation temperature exceeds 42°C. This property is exploited in the highly promising pasteurization treatment (42-45°C for 1-5 days) which achieves complete killing of S. Enteritidis. However, the precise mechanism and the role of the egg-white proteins are not fully understood. Here, the impact of exposure of S. Enteritidis to egg white-based media, with or without egg-white proteins (>10 kDa), under bactericidal conditions (45°C) was explored by measuring survival and global expression. Surprisingly, the bactericidal activity of egg white at 45°C was only slightly affected by egg-white proteins indicating that they play a minor role in the bactericidal activity observed. Moreover, egg-white proteins had minimal impact on the global-gene-expression response to egg white such that very similar, major regulatory responses (20% genes affected) were observed both with and without egg-white proteins following incubation for 45 min at 45°C. Egg-white proteins caused a significant change in expression for just 64 genes, including the psp and lysozyme-inhibitor responses genes which is suggestive of an early membrane perturbation effect. Such damage was supported by disruption of the proton motive force by egg-white proteins. In summary, the results suggest that low-mass components of egg white are largely responsible for the bactericidal activity of egg white at 45°C.

Project description: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.IMPORTANCE Salmonella 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.

Project description:Purpose: To elucidated key metabolic pathways important for S. Enteritidis survival in egg white. Methods: RNA sample of Mid-log phase bacteria cultures that grown in M9FeS medium were taken as 0h sample, and bacteria incubated with egg white for 6h 12h and 24h were taken as 6,12,24h RNA sample respectively. Three biological replicates were carried out per sample. RNA sample was sequenced with Illumina HiSeq 2000 System. DESeq2 method was used to calculate the differentially expressed genes. qRT–PCR and gene mutation were used to validate RNA-seq result. Results: A total of 12 samples were sequenced. The average output of each sample was 1.33 Gb, and the average comparison rate with the reference genome was 97.51%. M9FeS and egg whites cultivation resulted in 2400 to 2700 expressed genes accounting for 53 to 58% of the whole genome. In this group, 1240 to 1470 genes had significant levels of log2 fold changes ≥1, (Padj <0.05) compared with control. These accounted for 27.3 to 32.2% of the whole genome indicating a large shift in the transcriptional response to egg whites. qRT–PCR result of 21 selected genes highly consistent with the RNAseq results at all three timepoints. 8 out of 12 DEGs mutants showed decreased survival ability in egg white, suggested that the DEGs from the RNAseq results correlated significantly with the egg white resistance phenotype. Conclusions: Using strand-specific RNA sequencing, we found pathways significantly changed in egg white. Genes involved in SOS-dependent and independent DNA damage repair, alkaline pH adaption, osmotic stress adaption, envelope damage repair including maintaining periplasm homeostasis and reinforcing peptidoglycan layer, nitrogen assimilation, Salmonella pathogenicity island 2, iron absorption and biotin synthesis, were significantly up regulated in bacteria surviving in egg whites. Differentially expressed genes involved in energy metabolism, translation, and cell motility, Salmonella pathogenicity island 1 were significantly down-regulated in egg whites.

Project description: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.

Dataset Information

The Three Lipocalins of Egg-White: Only Ex-FABP Inhibits Siderophore-Dependent Iron Sequestration by Salmonella Enteritidis.

Publications

The Three Lipocalins of Egg-White: Only Ex-FABP Inhibits Siderophore-Dependent Iron Sequestration by <i>Salmonella</i> Enteritidis.

Similar Datasets

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