Two Distinct Hemolytic Activities in Xenorhabdus nematophila Are Active against Immunocompetent Insect Cells

Abstract

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.

Xenorhabdus nematophila (Enterobacteriacea) Secretes a Cation-selective Calcium-independent Porin Which Causes Vacuolation of the Rough Endoplasmic Reticulum and Cell Lysis

“…nematophila (strain F1, phase variant I, laboratory collection) were grown in Luria Bertani broth at 28°C. In these conditions the maximum production of flhD-dependent cytotoxic activity (C1) (10) was reached in 20-hold cultures. A purification of the factor responsible for this activity was achieved from this C1 culture supernatant.…”

“…Some of these factors are cytotoxic in vitro for insect immunocompetent cells and also have hemolytic activity on mammal red blood cells. Interestingly, all these cytotoxic and hemolytic activities are absent in P. luminescens broth growth (10), suggesting differences in the mode of virulence of these two related entomopathogenic bacteria species. We know that these toxins have very little homology with known sequences and represent a new class of toxins (8).…”

“…Phagocytosis is mainly achieved by macrophage-like cells that belong to the morphotype "granular hemocyte 1" (GH1) (12). As GH1 are one of the targets of the cytotoxic activities evidenced in X. nematophila broth growth (10), these activities appear to be good candidates for supporting, at least in part, the immunosuppressive effect. In the present work we report the purification of the flhDC-controlled lysin.…”

“…In X. nematophila the existence of toxins active both on red blood cells and on hemocytes allows us to study the mode of action of these molecules on mammal cells and on insect cells. Two different hemolytic activities were identified that appear sequentially in the course of bacterial growth (10). The earliest hemolytic activity (activity C1) appears in broth growth when bacteria culture reaches the stationary phase.…”

“…Other insecticidal toxins active after injection are also produced by P. luminescens (7). Genes coding for similar entomotoxins were cloned in Xenorhabdus nematophila (8), and various cytotoxic factors were identified in bacterial broth growth of this species (9,10). Some of these factors are cytotoxic in vitro for insect immunocompetent cells and also have hemolytic activity on mammal red blood cells.…”