The smcL gene of Listeria ivanovii encodes a sphingomyelinase C that mediates bacterial escape from the phagocytic vacuole

The ruminant pathogen Listeria ivanovii differs from Listeria monocytogenes in that it causes strong, bizonal haemolysis and a characteristic shovel-s haped co-operative haemolytic ('CAMP-like') reaction with Rhodococcus equi. We cloned the gene responsible for the differential haemolytic properties of L. ivanovii, smcL. It encodes a sphingomyelinase C (SMase) highly simila r (> 50% identity) to the SMases from Staphylococcus aureus (p-toxin), Baci llus cereus and Leptospira interrogans. smcL was transcribed monocistronica lly and was expressed independently of PrfA. Low-stringency Southern blots demonstrated that, within the genus Listeria, smcL was present only in L. i vanovii. We constructed an smcL knock-out mutant. Its phenotype on blood ag ar was identical to that of L. monocytogenes (i.e. weak haemolysis and no s hovel-shaped CAMP-like reaction with R. equi). This mutant was less virulen t for mice, and its intracellular proliferation was impaired in the bovine epithelial-like cell line MDBK. The role of SmcL in intracellular survival was investigated using an L. monocytogenes mutant lacking the membrane-dama ging determinants hly, plcA and plcB, being thus unable to grow intracellul arly. Complementation of this mutant with smcL on a plasmid was sufficient to promote bacterial intracellular proliferation in MDBK cells. Transmissio n electron microscopy showed that SmcL mediates the disruption of the phago cytic vacuole and the release of bacteria into the cytosol. Therefore, L. i vanovii possesses a third phospholipase with membrane-damaging activity tha t, together with PlcA and PlcB, may act in concert with the pore-forming to xin Hly to mediate efficient escape from the vacuolar compartment. The 5' e nd of smcL is contiguous with the internalin locus i-inlFE, which is also s pecific to L. ivanovii and is required for full virulence in mice. Thus, sm cL forms part of a novel virulence gene cluster in Listeria that is species specific.