Biochemical and Biophysical Research Communications
Characterization of a cytotoxic pilin subunit of Xenorhabdus nematophila
Section snippets
Materials and methods
Bacteria and growth conditions. The strain used in this study X. nematophila 19061 was obtained from ATCC (Rockville, MD). The X. nematophila culture was streaked on nutrient agar supplemented with 0.004% (wt/vol.) triphenyl tetrazolium chloride and 0.025% (wt/vol.) bromothymol blue (NBTA) [2]. Broth cultures were grown from a single blue colony in LB medium at 28 °C with shaking at 150 rpm. E. coli K-12 was used as a reference strain.
Preparation and purification of outer membrane vesicles from
Purification and Identification of pilin subunit from X. nematophila
The 17 kDa pilin protein purified by ammonium sulphate precipitation and sucrose density gradient centrifugation migrated as a single band at 17 kDa in the SDS–PAGE (Fig. 1A, lane 3). The N-terminal sequence of the protein was found to be APTQGDGTVK, which was identical to the N-terminal sequence of the 17 kDa band present in the OMV preparation (Fig. 1A, lane1) and was 70% homologous to the PapA protein, the structural subunit of the P pilus of E. coli. The purified pilin subunit formed fibrous
Discussion
A 17 kDa protein constituting the structural subunit of pilin with cytotoxicity to insect larval hemocytes has been purified from the surface of X. nematophila cells. Hemocytes are the primary immunocompetent cells in insects, and interference in their normal functioning is known to affect insect viability adversely. Since X. nematophila whole cells have been shown to cause larval hemocyte clumping in an earlier study [20], we examined the activity of the pilin subunit in hemocyte agglutination
Acknowledgements
This work was funded by ICGEB, New Delhi and Ministry of Environment and Forest, Government of India. P.K. is a recipient of Senior Research Fellowship from CSIR, Government of India. The immunofluorescence experiments were carried out with the support of Dr. C. Chitnis. H. armigera insect larvae were kindly provided by Dr. G.P. Gupta, IARI.
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Novel insecticidal chitinase from the insect pathogen Xenorhabdus nematophila
2020, International Journal of Biological MacromoleculesCitation Excerpt :In earlier studies, we demonstrated oral insecticidal activity in the outer membrane vesicles (OMV) excreted by X. nematophila in the extracellular medium [70]. Biochemical analysis of the OMVs revealed presence of a number of proteins with accessory functions namely, XnGroEL, a pilin subunit and a chitinase, in addition to the typical outer membrane proteins [46,50,71]. Chitinases are usually involved in cuticle replacement, insect growth and metamorphosis during the development of insects.
Role of Endosymbionts in Insect–Parasitic Nematode Interactions
2018, Trends in ParasitologyCitation Excerpt :Similarly, Xenorhabdus bacteria use specific morphological features, such as fimbriae, to agglutinate insect hemocytes, lipopolysaccharide, which confers adverse effects on hemocyte function, or at different phases, they secrete insecticidal molecules that possess hemolytic activity against plasmatocytes and granulocytes (Figure 3) [78–80]. Some of these molecules have been previously characterized and include a cation-selective calcium-independent porin that targets the plasma membrane of insect hemocytes, a pilin subunit secreted through outer membrane vesicles with binding, cytotoxic, and agglutinating properties against lepidopteran larval hemocytes, certain formyl peptides that interfere with hemocyte adhesion, a pore-forming fimbrial shaft protein that lyses larval hemocytes, a toxin that triggers apoptosis in insect cells, and the metabolite benzylideneacetone that prevents hemocyte aggregation and spreading [81–87]. This metabolite also participates in the inhibition of phospholipase A2 (PLA2), which catalyzes the biosynthesis of eicosanoids, which regulate insect immune mechanisms, including cellular responses (Figure 3) [88–92].
Response of larval Ephestia kuehniella (Lepidoptera: Pyralidae) to individual Bacillus thuringiensis kurstaki toxins mixed with Xenorhabdus nematophila
2013, Journal of Invertebrate PathologyCitation Excerpt :These findings corroborate with our results but could not explain the lower pathogenicity of Xenorhabdus supernatant. Two other molecules secreted by X. nematophila were also described to be active in the insect midgut: A24 tox, a secreted protein which ceases larvae feeding; its main site of action is the insect midgut (Brown et al., 2004, 2006) and a 17-kDa pilin subunit of Xenorhabdus was shown to break down the midgut epithelial lining and basement membrane and to cause sloughing of cell debris into the midgut lumen (Khandelwal et al., 2004). Otherwise the toxicity of cells could be attributed to other factors and depends on larvae.
An insecticidal GroEL protein with chitin binding activity from Xenorhabdus nematophila
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