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Perry, Kyle James

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Perry

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Kyle James

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Perry, Kyle James
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    Identification of Listeria monocytogenes Determinants Required for Biofilm Formation
    (Public Library of Science, 2014) Alonso, Almaris N.; Perry, Kyle James; Regeimbal, James M.; Regan, Patrick M.; Higgins, Darren
    Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.
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    Differential fluorescence-based genetic screens to identify novel Listeria monocytogenes virulence determinants
    (2015-01-23) Perry, Kyle James; Bernhardt, Thomas; Camilli, Andrew
    Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen that causes gastroenteritis, which in the young, the elderly, and the immunocompromised can progress to severe invasive disease with high mortality. While previous studies have largely elucidated the bacterial and host mechanisms necessary for the bacterium to access its replicative niche in the host cell cytosol, the L. monocytogenes factors required for adaptation to life within this restrictive environment are poorly understood. In this dissertation, I describe a fluorescence-activated cell sorting (FACS)-based differential fluorescence genetic screening technique for the identification of L. monocytogenes genes necessary for optimal intracellular replication. Bacteria harboring deletions in identified genes were defective for intracellular replication, plaque formation, and in vivo virulence, validating the ability of the screening method to identify novel intracellular replication-defective mutants. Minor alteration of the FACS-based screening strategy allowed the detection and differentiation of bacterial mutants displaying varying severities of actin-based motility defects. A preliminary FACS-based genetic screen to identify actin-based motility mutants isolated multiple independent insertions within internal control genes, demonstrating the potential utility of FACS-based differential fluorescence genetic screening methods for the identification of L. monocytogenes genes important for multiple virulence phenotypes. Lastly, my characterization of the X-prolyl aminopeptidase PepP, a novel virulence factor identified by the FACS-based genetic screen to discover genes necessary for optimal intracellular replication, revealed this enzyme plays an unexpected role in L. monocytogenes virulence gene regulation.