5 LISTERIA-MONOCYTOGENES GENES PREFERENTIALLY EXPRESSED IN INFECTED MAMMALIAN-CELLS - PLCA, PURH, PURD, PYRE AND AN ARGININE ABC TRANSPORTER GENE, ARPJ

Listeria monocytogenes is a bacterial pathogen that multiplies within the cytosol of eukaryotic cells. To identify Listeria genes with prefe rentially intracellular expression (pie genes), a library of Tn917-lac insertion mutants was screened for transcriptional fusions to lacZ wi th higher expression inside a macrophagelike cell line than in a rich broth medium. Five pic genes with up to 100-fold induction inside cell s were identified. Three of them (purH, purD and pyrE) were involved i n nucleotide biosynthesis. One was part of an operon encoding an ABC ( ATP-binding cassette) transporter for arginine. The corresponding muta nts were not affected in intracellular growth, cell-to-cell spread or virulence, except for the transporter mutant, whose LD(50) after intra venous infection of mice was twofold higher than the wild-type. The fi fth gene was plcA, a previously identified virulence gene that encodes a phosphatidylinositol-phospholipase C, and is cotranscribed with prf A, a gene encoding a pleiotropic transcriptional activator of known vi rulence genes. Although plcA expression is known to depend on PrfA, a prfA promoter-lacZ fusion was highly expressed both inside and outside cells. Furthermore, in the presence of cellobiose, a disaccharide rec ently shown to repress plcA and hly expression, plcA and hly mRNA leve ls were dramatically reduced without any decrease in the monocistronic prfA mRNA levels. These results demonstrate that virulence gene activ ation does not depend only on prfA transcript accumulation.