PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C FROM LISTERIA-MONOCYTOGENES CONTRIBUTES TO INTRACELLULAR SURVIVAL AND GROWTH OF LISTERIA-INNOCUA

Listeria monocytogenes is a facultative intracellular organism that is capable of replicating within macrophage and macrophage-like cells. T he species secretes a phosphatidylinositol-specific phospholipase C (P I-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 whe n introduced into Listeria innocua, a species that does not grow intra cellularly or contain plcA, transformation with the recombinant pWS2-2 plasmid was performed. Phospholipase C activity in Listeria innocua/p WS2-2 was confirmed on a brain heart infusion-phosphatidylinositol aga r plate, whereas wild-type L. innocua did not produce PI-PLC activity. Intracellular growth of L. innocua/pWS2-2 was subsequently measured i n the macrophage-like cell line J774 by Giemsa staining and viable cou nt determinations at specific time points following infection. The J77 4 cells infected with wild-type L. innocua showed a falling viable cou nt through 8 h postinfection. Although J774 cells infected with L. inn ocua/pWS2-2 also initially displayed reduced viable counts, the viable count rose after 6 h postinfection and increased further at 8 h posti nfection before a subsequent decline again at 16 h postinfection. Giem sa staining revealed fewer than 6 bacteria in individual macrophage ce lls 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 afte r 6 h; moreover, electron micrographs showed that the L. innocua/ pWS2 -2 cells were replicating inside the phagosome of the host cell. Furth ermore, Thoria Sol labeling demonstrated that lysosomes had fused with these phagosomes, and acridine orange staining revealed that the comp artments were acidified. These results demonstrate that L. innocua cel ls transformed with the plasmid-borne plcA gene, and expressing functi onal PI-PLC, are able to grow intracellularly in what appear to be pha golysosomes, although between 3 and 6 h is needed for this to manifest itself. Intracellular growth specifically in L. innocua may be a seco ndary function associated,vith the plcA gene product. The addition of this one gene, plcA, to a species of Listeria that in the wild-type st ate does not replicate intracellularly apparently can now allow some o f the bacteria to transiently multiply inside the phagosomes of host m acrophage cells.