Listeria monocytogenes pathogenesis: Elucidation of novel virulence factors through exploration of an alternative infection model

Listeria monocytogenes is a facultative intracellular bacterial pathogen that is the leading cause of food-borne deaths in the United States. This pathogen thrives in diverse environmental habitats and is capable of replicating in a broad range of host species. To better understand the complex interactions involved in host-pathogen relationships, it is advantageous to genetically manipulate both the pathogen and the host. The focus of this dissertation is the establishment of the fruit fly Drosophila melanogaster as a model for the exploration of L. monocytogenes pathogenesis and host responses to infection.; L. monocytogenes established lethal infections in fruit flies with extensive bacterial replication occurring before host death. Bacteria localized to the cytosol of insect cells and directed actin polymerization. Bacterial gene products necessary for intracellular replication and cell-to-cell spread within mammalian cells were similarly found to be required within insect cells, and although it has been reported that L. monocytogenes virulence gene expression requires temperatures above 30°C, bacteria within insect cells were found to express virulence determinants at 25°C. Drosophila mutants compromised for innate immunity demonstrated increased susceptibility to L. monocytogenes infection. These data indicate that L. monocytogenes infection of fruit flies shares numerous features of mammalian infection, and thus Drosophila may serve as a powerful model for examining new areas of bacterial infection.; D. melanogaster was used to screen L. monocytogenes transposon insertion libraries to identify bacterial mutants altered for virulence. Over 3000 bacterial transposon insertion mutants were screened for virulence in flies of which 27 proved to be either attenuated for virulence or hypervirulent. Transposon insertions were identified in genes predicted to encode products that function in amino acid metabolism, oxidative stress, plasmid replication, ATP-dependent transport, and cell surface adhesion. Further characterization of the mutants led to the discovery of a putative glutathione reductase whose absence increased bacterial susceptibility to hydrogen peroxide and a gene product of unknown function that contributes to L. monocytogenes virulence in mice via oral infection. These data underscore the utility of using D. melanogaster as a model host for the identification of gene products that contribute to bacterial life within vertebrate and invertebrate hosts.