title: Phosphatidylinositol-specific phospholipase C from Listeria monocytogenes contributes to intracellular survival and growth of Listeria innocua.
abstract: Listeria monocytogenes is a facultative intracellular organism that is capable of replicating within macrophage and macrophage-like cells. The species secretes a phosphatidylinositol-specific phospholipase C (PI-PLC) encoded by the plcA gene. A plcA gene from L. monocytogenes was cloned downstream of a gram-positive promoter in the plasmid pWS2-2. To determine what effect plcA would have on intracellular survival when introduced into Listeria innocua, a species that does not growth intracellularly or contain plcA, transformation with the recombinant pWS2-2 plasmid was performed. Phospholipase C activity in Listeria innocua/pWS2-2 was confirmed on a brain heart infusion-phosphatidylinositol agar plate, whereas wild-type L. innocua did not produce PI-PLC activity. Intracellular growth of L. innocua/pWS2-2 was subsequently measured in the macrophage-like cell line J774 by Giemsa staining and viable count determinations at specific time points following infection. The J774 cells infected with wild-type L. innocua showed a falling viable count through 8 h postinfection. Although J774 cells infected with L. innocua/pWS2-2 also initially displayed reduced viable counts, the viable count rose after 6 h postinfection and increased further at 8 h postinfection before a subsequent decline again at 16 h postinfection. Giemsa staining revealed fewer than 6 bacteria in individual macrophage cells at 2 h postinfection, and yet approximately 15% of the J774 cells had 6 to 12 bacteria localized to one area of the macrophage cell after 6 h; moreover, electron micrographs showed that the L. innocua/pWS2-2 cells were replicating inside the phagosome of the host cell. Furthermore, Thoria Sol labeling demonstrated that lysosomes had fused with these phagosomes, and acridine orange staining revealed that the compartments were acidified. These results demonstrate that L. innocua cells transformed with the plasmid-borne plcA gene, and expressing functional PI-PLC, are able to grow intracellularly in what appear to be phagolysosomes, although between 3 and 6 h is needed for this to manifest itself. Intracellular growth specifically in L. innocua may be a secondary function associated with the plcA gene product. The addition of this one gene, plcA, to a species of Listeria that in the wild-type state does not replicate intracellularly apparently can now allow some of the bacteria to transiently multiply inside the phagosomes of host macrophage cells.
authors.affiliation: Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut für Biowissenschaften, Universität Würzburg, Germany.
authors.forename: W R
authors.initials: WR
authors.lastname: Schwan
authors.forename: A
authors.initials: A
authors.lastname: Demuth
authors.forename: M
authors.initials: M
authors.lastname: Kuhn
authors.forename: W
authors.initials: W
authors.lastname: Goebel
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D066298:In Vitro Techniques; D008087:Listeria / Q000201:enzymology; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000254:growth & development; D008247:Lysosomes / Q000382:microbiology; D008264:Macrophages / Q000382:microbiology; D008969:Molecular Sequence Data; D010588:Phagosomes / Q000382:microbiology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D017931:DNA Primers; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase