Genetic Determinants of Resistance of Salmonella Enteritidis to Chicken Egg Albumen

In this proposal, we will screen a transposon library to systematically identify genes necessary for S. enteritidis to survive in egg albumen and characterize the mechanisms of resistance mediated by these genes. The specific objectives are as follows: 1. To screen a transposon library to identify genes necessary for S. enteritidis to survive in egg albumen. We will screen a transposon mutant library to select mutants that are more susceptible to egg albumen than the wild type parental strain of S. enteritidis. The mutants will be categorized by their susceptibility to different antimicrobial activities of egg albumen including: iron restriction, cell lysis by lysozyme and DNA damage. 2. To identify genes interrupted in the egg susceptible mutants and complement the mutants. We will identify the genes inactivated by the transposon insertions in the mutants selected from the library screening. Mutants will be complemented to confirm that the susceptible phenotype observed is due to gene inactivation by transposon.
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We will screen a transposon mutant library to select mutants that are more susceptible to egg albumen than the wild type parental strain of S. enteritidis. The mutants will be categorized by their susceptibility to different antimicrobial activities of egg albumen including: iron restriction, cell lysis by lysozyme and DNA damage. We will then identify the genes inactivated by the transposon insertions in the mutants selected from the library screening. Mutants will be complemented to confirm that the susceptible phenotype observed is due to gene inactivation by transposon.
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Salmonella enteritidis is a major foodborne pathogen. Its incidence has increased from 5% of all Salmonella infections to approximately 20% in the last 30 years. Salmonella enteritidis is the only bacterium that routinely causes human infection through intact chicken eggs. However, little is understood on how this bacterium survives and persists in the eggs. We seek to understand how S. enteritidis resists the killing by egg albumen by identifying the bacterial genes necessary for their survival in eggs. Insights obtained from the research will facilitate the development of strategies to control and prevent human diseases caused by S. enteritidis.
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The long-term goal of this proposal is to understand how S. enteritidis survives in eggs, a characteristic important for the transmission of this bacterium. In this project, we have screened a transposon library to systematically identify genes necessary for S. enteritidis to survive in egg albumen and characterize the mechanisms of resistance mediated by these genes. We screened approximately 3,000 mutants and identified 33 egg-susceptible (ES) mutants that are more susceptible to egg albumen than the wild type parental strain SE2472. Genes inactivated by the Tn insertion were identified either by a rescue cloning procedure or a 3-step PCR procedure (http://www.epicentre.com/pdfforum/9 2rapidident.pdf).
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The mutants were then categorized by the genes interrupted by Tn insertions into several different groups (Table 1). Of 33 ES mutants we identified, 16 (48.5%) have Tn insertions in genes expected to be involved in the structure and function of bacterial cell wall. Eight ES mutants (24.2%) have Tn insertions in genes involved in the metabolism, especially amino acid metabolism. Two ES mutants (ES16 and ES47) have insertions in genes unique to S. enteritidis; ES16 has a Tn insertion in a restriction endonuclease, and ES47 has a Tn insertion in the Prot6e that appears to be in an operon for fimbrial biosynthesis. Analysis of genes interrupted by Tn insertion in the ES mutants demonstrate that two systems, cell wall and amino acid metabolisms, play important roles in the survival of S. enteritidis in egg albumen. Twenty-four of the 33 ES mutants (72.7%) have Tn insertion in genes involved in either cell wall structure/function or amino acid metabolism. In addition, the two systems are related to one another. For example, ES10 and ES15 have insertions in cell wall proteins that function in the transport of glutamine and proline respectively; ES25 and ES52, which have Tn insertions in genes involved in lysine and cadaverine metabolism, are likely to have cadaverine deficiency that may destabilize the LPS of Salmonella and render it more susceptible to the antimicrobial components of egg albumen. Therefore, cell wall structural and functional integrity and amino acid metabolism are essential for S. enteritidis to survive the stress of exposure to egg albumen. Characterization of the mechanisms of how these ES mutants become susceptible to egg albumen is ongoing.
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S. enteritidis is a major food-borne pathogen that causes a significant amount of human infection and economical loss both to the agricultural industry and society in general. S. enteritidis is uniquely associated with intact shell eggs. Since the majority of human infections of S. enteritidis are caused by consumption of chicken eggs, the ability to survive the hostile environment of the egg albumen must be crucial for S. enteritidis. However, little is understood about the interaction of the egg albumen and S. enteritidis. We have initiated a series of studies on the molecular mechanisms of the bactericidal activities of egg albumen and the survival of S. enteritidis in egg albumen. Our studies on the molecular mechanisms of egg resistance will generate insight into the development of novel strategies to control this important food-borne pathogen. By identifying the bacterial factors important for its survival in eggs, it is possible to generate safer vaccines that do not contaminate eggs. These studies will provide critical information for the control of human infections caused by S. enteritidis and facilitate the development of strategies to improve food safety and minimize the economic loss caused by this bacterium.