Two distinct hemolytic activities in Xenorhabdus nematophila are active against immunocompetent insect cells

Xenorhabdus spp. and Photorhabdus spp. are major insect bacterial pathogens symbiotically associated with nematodes. These bacteria are transported by their nematode hosts into the hemocoel of the insect prey, where they proliferate within hemolymph. In this work we report that wild strains belonging to different species of both genera are able to produce hemolysin activity on blood agar plates. Using a hemocyte monolayer bioassay, cytolytic activity against immunocompetent cells from the hemolymph of Spodoptera littoralis (Lepidoptera: Noctuidae) was found only in supernatants of Xenorhabdus; none was detected in supernatants of various strains of Photorhabdus. During in vitro bacterial growth of Xenorhabdus nematophila F1, two successive bursts of cytolytic activity were detected. The first extracellular cytolytic activity occurred when bacterial cells reached the stationary phase. It also displayed a hemolytic activity on sheep red blood cells, and it was heat labile. Among insect hemocyte types, granulocytes were the preferred target. Lysis of hemocytes by necrosis was preceded by a dramatic vacuolization of the cells. In contrast the second burst of cytolytic activity occurred late during stationary phase and caused hemolysis of rabbit red blood cells, and insect plasmatocytes were the preferred target. This second activity is heat resistant and produced shrinkage and necrosis of hemocytes. Insertional inactivation of flhD gene in X. nematophila leads to the loss of hemolysis activity on sheep red blood cells and an attenuated virulence phenotype in S. littoralis (A. Givaudan and A. Lanois, J. Bacteriol. 182:107-115, 2000). This mutant was unable to produce the early cytolytic activity, but it always displayed the late cytolytic effect, preferably active on plasmatocytes. Thus, X. nematophila produced two independent cytolytic activities against different insect cell targets known for their major role in cellular immunity.