Xenorhabdus nematophila (Enterobacteriacea) Secretes a Cation-selective Calcium-independent Porin Which Causes Vacuolation of the Rough Endoplasmic Reticulum and Cell Lysis (original) (raw)
Related papers
The Cytotoxic Fimbrial Structural Subunit of Xenorhabdus nematophila Is a PoreForming Toxin
Journal of Bacteriology, 2006
We have purified a fimbrial shaft protein (MrxA) of Xenorhabdus nematophila. The soluble monomeric protein lysed larval hemocytes of Helicoverpa armigera. Osmotic protection of the cells with polyethylene glycol suggested that the 17-kDa MrxA subunit makes pores in the target cell membrane. The internal diameter of the pores was estimated to be >2.9 nm. Electron microscopy confirmed the formation of pores by the fimbrial subunit. MrxA protein oligomerized in the presence of liposomes. Electrophysiological studies demonstrated that MrxA formed large, voltage-gated passive-diffusion channels in lipid bilayers.
Applied and Environmental Microbiology, 2001
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. . 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.
Applied and Environmental Microbiology, 2001
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.
Insecticidal Toxin Complex Proteins from Xenorhabdus nematophilus: STRUCTURE AND PORE FORMATION
Journal of Biological Chemistry, 2011
Toxin complexes from Xenorhabdus and Photorhabdus spp. bacteria represent novel insecticidal proteins. We purified a native toxin complex (toxin complex 1) from Xenorhabdus nematophilus. The toxin complex is composed of three different proteins, XptA2, XptB1, and XptC1, representing products from class A, B, and C toxin complex genes, respectively. We showed that recombinant XptA2 and co-produced recombinant XptB1 and XptC1 bind together with a 4:1:1 stoichiometry. XptA2 forms a tetramer of ϳ1,120 kDa that bound to solubilized insect brush border membranes and induced pore formation in black lipid membranes. Co-expressed XptB1 and XptC1 form a tight 1:1 binary complex where XptC1 is C-terminally truncated, resulting in a 77-kDa protein. The ϳ30-kDa C-terminally cleaved portion of XptC1 apparently only loosely associates with this binary complex. XptA2 had only modest oral toxicity against lepidopteran insects but as a complex with co-produced XptB1 and XptC1 had high levels of insecticidal activity. Addition of co-expressed class B (TcdB2) and class C (TccC3) proteins from Photorhabdus luminescens to the Xenorhabdus XptA2 protein resulted in formation of a hybrid toxin complex protein with the same 4:1:1 stoichiometry as the native Xenorhabdus toxin complex 1. This hybrid toxin complex, like the native toxin complex, was highly active against insects.
Insecticidal Toxin Complex Proteins from Xenorhabdus nematophilus
Journal of Biological Chemistry, 2011
Toxin complexes from Xenorhabdus and Photorhabdus spp. bacteria represent novel insecticidal proteins. We purified a native toxin complex (toxin complex 1) from Xenorhabdus nematophilus. The toxin complex is composed of three different proteins, XptA2, XptB1, and XptC1, representing products from class A, B, and C toxin complex genes, respectively. We showed that recombinant XptA2 and co-produced recombinant XptB1 and XptC1 bind together with a 4:1:1 stoichiometry. XptA2 forms a tetramer of ϳ1,120 kDa that bound to solubilized insect brush border membranes and induced pore formation in black lipid membranes. Co-expressed XptB1 and XptC1 form a tight 1:1 binary complex where XptC1 is C-terminally truncated, resulting in a 77-kDa protein. The ϳ30-kDa C-terminally cleaved portion of XptC1 apparently only loosely associates with this binary complex. XptA2 had only modest oral toxicity against lepidopteran insects but as a complex with co-produced XptB1 and XptC1 had high levels of insecticidal activity. Addition of co-expressed class B (TcdB2) and class C (TccC3) proteins from Photorhabdus luminescens to the Xenorhabdus XptA2 protein resulted in formation of a hybrid toxin complex protein with the same 4:1:1 stoichiometry as the native Xenorhabdus toxin complex 1. This hybrid toxin complex, like the native toxin complex, was highly active against insects.
Insects
Xenorhabdus nematophila is a Gram-negative bacterium symbiont of the entomopathogen nematode Steinernema carpocapsae whose immunosuppressive properties over host’s immune response have been thoroughly investigated. In particular, live X. nematophila actively impairs phagocytosis in host’s hemocytes through the secretion of inhibitors of eicosanoids synthesis. In this article we have investigated the cell surface structural features of X. nematophila responsible for the elusion from phagocytosis. To this end we have studied the uptake of heat-killed (hk), fluorescein isothiocyanate (FITC)-labeled X. nematophila by phagocytes from both a host insect and a mammalian species. In vitro dead X. nematophila passively resists engulfment by insect hemocytes without impairing the phagocytosis machinery whereas, unexpectedly, in vivo a significant phagocytosis of dead X. nematophila was observed. X. nematophila in vivo phagocytosis was increased by the co-injection of the specific inhibitor of...
Journal of Biological Chemistry, 2007
Xenorhabdus nematophila, a member of the Enterobacteriaceae, kills many species of insects by strongly depressing the immune system and colonizing the entire body. A peptide cytotoxin has been purified from X. nematophila broth growth, and the cytolytic effect on insect immunocytes and hemolytic effect on mammalian red blood cells of this toxin have been described (Ribeiro, C., Vignes, M., and Brehélin, M. (2003) J. Biol. Chem. 278, 3030-3039). We show here that this toxin, Xenorhabdus ␣-xenorhabdolysin (Xax), triggers apoptosis in both insect and mammalian cells. We also report the cloning and sequencing of two genes, xaxAB, encoding this toxin in X. nematophila. The expression of both genes in recombinant Escherichia coli led to the production of active cytotoxin/hemolysin. However, hemolytic activity was observed only if the two peptides were added in the appropriate order. Furthermore, we report here that inactivation of xaxAB genes in X. nematophila abolished the major cytotoxic activity present in broth growth, called C1. We also show that these genes are present in various entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus, in Pseudomonas entomophila, in the human pathogens Yersinia enterocolitica and Proteus mirabilis, and in the plant pathogen Pseudomonas syringae. This toxin cannot be classified in any known family of cytotoxins on the basis of amino acid sequences, locus organization, and activity features. It is, therefore, probably the prototype of a new family of binary toxins.
Xenorhabdus nematophila and Photorhabdus luminescens are entomopathogenic symbionts that produce several toxic proteins that can interfere with the immune system of insects. Here, we showed that outer membrane proteins (OMPs) could be involved as virulence factors during bacterial symbiont pathogenesis. Purified OMPs from bacterial culture were injected fifth instar larvae of Spodoptera exigua Hübner. Larvae were surveyed for fluctuations in total haemocyte counts (THC), granulocyte percentage (cellular defence), protease, phospholipase A2 (PLA2), and phenoloxidase (PO) activities (humoral defence) at specific time intervals. Changes in the expression of the three antimicrobial peptides (AMPs), cecropin, attacin, and spodoptericin, were also measured. Larvae treated with both types of OMPs had more haemocytes than did the negative controls. OMPs of X. nematophila caused more haemocyte destruction than did the OMPs of P. luminescens. The OMPs of both bacterial species initially ...
FEBS Letters, 1995
Membrane potential measurements using a fluorescent dye indicated that two specific toxins active against Spodoptera frugiperda larvae (CryIC and CryID) cause immediate permeability changes in midgut epithelial brush border membrane vesicles (BBMV). The initial response and the sustained permeability change are cationic, not very K ÷ selective, and occur at in vivo lethal doses (nM). The toxin response has a different ion selectivity and is more sensitive to Ba 2+ than the intrinsic cation permeability of BBMV. Experiments incorporating BBMV into planar fipid bilayers (PLB) demonstrated that these vesicles contain cation channels (31, 47 and 76 pS). A 2-40 fold conductance increase was induced by nM concentrations of toxin in PLB containing BBMV. Cationic single channel transitions of 50, 106, 360 and 752 pS were resolved. Thus, Bacillus thuringiensis ~endotoxins induce an increase in cation membrane permeability involving ion channels in BBMV-containing functional receptors.