Identification of Listeria monocytogenes in vivo-induced genes by fluorescence-activated cell sorting

Listeria monocytogenes is a gram-positive, intracellular, food-borne pathog en capable of causing severe infections in immunocompromised or pregnant in dividuals, as well as numerous animal species. Genetic analysis of Listeria pathogenesis has identified several genes which are crucial for virulence. The transcription of most of these genes has been shown to be induced upon entry of Listeria into the host cell. To identify additional genes that ar e induced in vivo and may be required for L. monocytogenes pathogenesis, a fluorescence-activated cell-sorting technique was initiated. Random fragmen ts of the L. monocytogenes chromosome were cloned into a plasmid carrying a promoterless green fluorescent protein (GFP) gene, and the plasmids were t ransformed into the L. monocytogenes actA mutant DP-L1942. Fluorescence-act ivated cell sorting (FACS) was used to isolate L. monocytogenes clones that exhibited increased GFP expression within macrophage-like J774 cells but h ad relatively low levels of GFP expression when the bacteria were extracell ular. Using this strategy, several genes were identified, including actA, t hat exhibited such an expression profile. In-frame deletions of two of thes e genes, one encoding the putative L. monocytogenes uracil DNA glycosylase (ung) and one encoding a protein with homology to the Bacillus subtilis Yhd P hemolysin-like protein, were constructed and introduced into the chromoso me of wild-type L. monocytogenes 10403s. The L. monocytogenes 10403s ung de letion mutant was not attenuated for virulence in mice, while the yhdP muta nt exhibited a three- to sevenfold reduction in virulence.