A wide range of pathogenic bacteria produce phospholipases, and the putative role of PLA in virulence has been studied in some of these pathogens. Outer membrane-associated PLAs (OMPLAs) were first identified in E. coli  and orthologs were subsequently reported in numerous gram-negative bacteria, including H. pylori (PldA) . The OMPLAs have been well-characterized and are thought to enhance bacterial growth, colonization, and survival. In addition to modulation of the bacterial membrane, some OMPLAs were shown to have contact-dependent hemolytic/cytolytic activities . Another group of PLAs (e.g., YplA , ExoU , PlaA , and SlaA ) is secreted from bacterial cells. Purified ExoU and SlaA [38, 39] recombinant proteins do not show cytotoxic activity when added exogenously, and there is little information on the cytotoxicity of other secretory PLAs.
To our knowledge, ShlA is the only previously reported hemolysin from S. marcescens. Although, a ΔshlAB mutant showed hemolytic activity on blood agar plates, it did not exhibit contact-dependent hemolytic activity (Fig. 1C). Therefore, we performed functional cloning, which identified PhlA as an S. marcescens candidate hemolytic factor (Fig. 2A).
In the experiments reported here, we described the hemolytic and cytotoxic activities of S. marcescens PhlA. PhlA itself did not directly induce the destabilization of target cell membranes, but the LPL produced from PL by PhlA phospholipase activity showed hemolytic and cytolytic activities. Therefore, PhlA and ShlA have different hemolytic mechanisms. In addition, ShlA was expressed at lower temperature, but its expression decreased at 37°C . In contrast, PhlA was expressed at 37°C, although its temperature regulation has not been elaborated. Also, PhlA hydrolyzed phosphoethanolamine (Fig. 3C), which is required for ShlA activity , implying that PhlA production could potentially regulate ShlA activity.
Tsubokura et al.  reported PL-dependent hemolytic activity in a Y. enterocolitica culture filtrate. Schmiel et al.  independently identified this hemolysin as a lecithin-dependent phospholipase A (YplA). However, there were no data on whether YplA also had cytotoxic activity in the presence of PL, similar to that reported here for S. marcescens PhlA.
PhlA cleaved the ester bond of PL at the sn-1 site, and produced fatty acids and LPL from several PLs; e.g., PC, PS, PE, and CL (Fig. 2C). LPL production by PL cleavage might explain why PL addition was required for PhlA hemolytic activity of (Fig. 4A), since LPL may act as a surfactant and induce hemolysis. We detected PhlA hemolytic activity on human blood agar, but not on sheep or horse blood agar (Fig. 1A). However, sheep and horse RBC were lysed with purified PhlA in the presence of PL. This difference may be explained if PLs are released from human RBCs during the preparation of blood agar, and then become substrates for added or secreted PhlA resulting in the production of LPL. In agreement with this possibility, we observed hemolysis around bacterial colonies by addition of egg yolk lecithin to sheep and horse blood agar plates (date not shown).
Our results on the mechanism of PhlA cytotoxic activity allowed us to quantitate cytotoxic activity in a liquid assay. Numerous reports have shown that bacterial phospholipases contribute to pathogenesis by directly hydrolyzing host membrane phospholipids and modulation of the host immune system via the production of lipid second messengers (5, 6, 31). Although PhlA did not produce direct cytotoxicity on cultured cells, the pathogenetic role of indirect cytotoxicity via LPL production should be investigated.
It has been reported that Pseudomonas aeruginosa ExoU inhibited neutrophil function in the lungs of infected mice  and group A Streptococcus (GAS) SlaA contributed to colonization of the upper respiratory tract . Furthermore, a PhlA-like phospholipase, Y. enterocolitica YplA, has been shown to play a role in bacterial colonization of the intestinal tract and increasing the pathological changes resulting from the host inflammatory response in the mouse model . The high degree of homology between YplA and PhlA suggests that PhlA may also play a role in S. marcescens colonization, since S. marcescens is thought to be a commensal in the intestinal tract where PLs are supplied by the host diet. The pathogenic role of PhlA remains to be elaborated.