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A Novel Approach for Screening Salmonella Enteritidis for In Vitro Invasion of Laying Hen Follicles


Construction of a collection of Salmonella Enteritidis mutants with the TnAraOut transposon. Perform an in vitro assay with the mutants to determine their ability to adhere and invade ovarian follicles. Identification of affected genes by amplification, sequencing, and comparison to known genomic sequences. Confirmation of mutant phenotypes will be performed by selective isolation of Tn mutants with the putative phenotype from output pool and performing an in vitro competition infection assay.

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NON-TECHNICAL SUMMARY: Infection by the bacterium Salmonella Enteritidis (SE) is a serious threat to both animal and human health. It can be transmitted from an infected hen to her eggs, where humans can be infected by consuming undercooked eggs. Since SE colonization of ovarian follicles are a primary factor in the cycle of infection, understanding the underlying mechanisms may lead to preventative strategies. The objective of this research is to identify the genes necessary for the adherence and invasion of follicular tissue by SE. This will be accomplished by creating a collection of SE mutants. These mutants will be incubated with follicles to determine if they are able to bind and invade the tissue. The genes of the non-infectious mutants will be amplified and compared to known DNA sequences to identify the affected genes.


APPROACH: Transposon footprinting is a rapid PCR-based approach for screening an organism's genome by differentiation distrinct transposon (Tn) insertions in a complex pool of Tn mutants by amplifying the flanking regions of each mutant. The ability of a collection of Tn Salmonella Enteritidis mutants to adhere and invade ovary follicles will be determined in an in vitro assay. The footprinting technique will be used to compare the banding patterns of the DNA from input and output pools of bacterial cells. Bands of genomic DNA which are present in the input pool and absent in the output pool represent mutants that were unable to bind or invade the follicular tissue. The bands from the non-invasive mutants will be excised from the gel of the input pool, amplified by PCR, sequenced, and compared to the genomic sequences of Salmonella Enteritidis at Genbank to identify the affected genes. Confirmation of the mutant phenotypes will be performed by selectively isolating the Tn mutants with the identified mutations and performing a competitive ovarian follicle infection experiment.


PROGRESS: 2007/01 TO 2007/12<BR>
OUTPUTS: Initial adhesion and invasion assays performed with the first mutant library of Salmonella enteritidis produced inconsistent results. It was determined that although the strain of Salmonella enteritidis used is an aggressive colonizer of the gastrointestinal tract, it was a relatively poor invader of egg follicles. Twelve strains of Salmonella enteritidis were screened using a modified version of the invasion assay to find invasive strains. Two of the most invasive strains were picked for the creation of mutation libraries, each of which will consist of approximately 1056 mutants. The creation of the mutation library for one of the strains is completed; the second library is nearly completed. The screening of the first mutant library for strains deficient in the ability to bind and/or invade the follicles is on-going. As new strains are identified the genomic DNA has been collected and amplified using PCR. The flanking regions surrounding the transposons in these strains are being sequenced. The sequences are being compared with published gene sequence in order to make a presumptive identification of the disrupted genes. Confirmation of the importance of these candidate genes for follicle binding and invasion will be performed both by comparing with the results of the screening of the second mutant library and the DNA microarrays. The laboratory has been developing assays to measure the internal volumes and surface areas of the various size classes of the egg follicles. We believe there are differences in the binding and invasion of Salmonella enteritidis to follicles during different stages of development. In order to quantify this effect, it is necessary to incubate the bacterial suspensions with equivalent amounts of the different egg follicle stages. As attachment of the bacteria to the follicles is an important step in both the process of adherence and invasion, measuring the surface area of the follicles allows us to calculate the number of bacteria bound per mm2 of the egg follicles. Likewise, estimates of the internal volumes of the follicles, allows for the calculation of the number of bacteria internalized per ml of the egg follicles. This information is also useful for quantifying the colonization capabilities of mutants in which the ability to colonize follicular tissue is only partially inhibited. Use of these binding and invasion indices will be used in trials used to confirm the binding and invasion capabilities of suspect mutants. Finally, this laboratory has been working to confirm the findings of the adhesion and invasion assays using a DNA microarray technology. The gene array for Salmonella enteritidis is being prepared. Using modified adhesion and invasion assays, we hope to determine which genes are up- and down- regulated during the processes involved in the colonization of egg follicles. These studies will commense when the creation of the microarrays has been completed. <BR>
PARTICIPANTS: Turki Dawoud, a graduate student in Dr. Young M. Kwon laboratory. Aiding me in the creation of the mutation libraries and in the running of the binding and invasion assays. He will be continuing on with this project to gain training in molecular methods as part of his professional development. <BR>
TARGET AUDIENCES: The data generated will be of interest to microbiologist interested in food borne and reproductive disease. The libraries will be of interest to microbiologists and molecular biologist working with the genus Salmonella <BR>
PROJECT MODIFICATIONS: Development of methods to measure the internal volume and surface area of the egg follicles used in the colonization assays. As some of the mutants exhibit subtle changes in their ability to bind and invade the follicular tissue, we need more quantifiable means to measure these mechanisms.

IMPACT: 2007/01 TO 2007/12<BR>

The general purpose of this research is to identify the genes responsible for the binding to and invasion of Salmonella enteritidis in egg follicles. We have produced 2 mutant libraries of S. enteritidis. These libraries will be valuable tools in the characterizing the mechanisms involved in the pathogenesis by this bacteria. As we screen the mutant libraries with the binding and invasion assays, we have discovered that the disruption of some genes by the transposon reduces but does not totally inhibit the bacteria's infective capabilities. Identification of these support genes aid in the understanding the mechanisms involved in adherence and invasion of follicular tissue by S. enteritidis. Likewise, we have found that some strains of S. enteritidis are more aggressive colonizers of follicular materials than others. Comparison of the sequences virulence genes from the aggressive strains to the milder strains, will aid in determining what domains of the genes are important in the pathogenesis of egg follicle invasion. We have been developing methods to measure the internal volume and surface area of the follicles used in the binding and invasion assays. Adopting these methods will better quantify the number of bacteria colonizing the follicular tissues and aid in determining the effects of egg follicle developement on the colonization by this bacteria. It is hoped that the DNA microarray being developed in our laboratory will aid in the confirmation of data obtained in our colonization assays.

Herrera, Paul
University of Arkansas
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