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 prol iferate within hemolymph, In this work we report that wild strains belongin g to different species of both genera are able to produce hemolysin activit y on blood agar plates. Using a hemocyte monolayer bioassay, cytolytic acti vity against immunocompetent cells from the hemolymph of Spodoptera littora lis (Lepidoptera: Noctuidae) was found only in supernatants of Xenorhabdus; none was detected in supernatants of various strains of Photorhabdus, Duri ng in vitro bacterial growth of Xenorhabdus nematophila F1, two successive bursts of cytolytic activity were detected. The first extracellular cytolyt ic activity occurred when bacterial cells reached the stationary phase. It also displayed a hemolytic activity on sheep red blood cells, and it was he at labile, Among insect hemocyte types, granulocytes were the preferred tar get. Lysis of hemocytes by necrosis was preceded by a dramatic vacuolizatio n of the cells. In contrast the second burst of cytolytic activity occurred late during stationary phase and caused hemolysis of rabbit red blood cell s, and insect plasmatocytes were the preferred target. This second activity is heat resistant and produced shrinkage and necrosis of hemocytes, insert ional inactivation of flhD gene in X. nematophila leads to the loss of hemo lysis activity on sheep red blood cells and an attenuated virulence phenoty pe 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 plasmat ocytes. Thus, X. nematophila produced two independent cytolytic activities against different insect cell targets known for their major role in cellula r immunity.