Characterization of a cytotoxic pilin subunit of Xenorhabdus nematophila

https://doi.org/10.1016/j.bbrc.2003.12.187Get rights and content

Abstract

Xenorhabdus nematophila is an insect pathogenic bacterium, known to produce protein toxins that kill the larval host. We have described a cytotoxic pilin subunit of X. nematophila, which is expressed on the cell surface and also secreted in the extracellular medium associated with outer membrane vesicles. A 17 kDa pilin subunit was isolated and purified from X. nematophila cell surface. The protein showed cytotoxicity to larval hemocytes of Helicoverpa armigera in an in vitro assay, causing agglutination of the cells, and releasing cytoplasmic enzyme lactate dehydrogenase in the medium. The pilin protein was able to bind to the surface of larval hemocytes. The binding and cytotoxicity of the purified 17 kDa protein to hemocytes was inhibited by antiserum raised against the pilin protein. The study demonstrates for the first time a cytotoxic structural subunit of pilin from an entomopathogenic bacterium X. nematophila that is excreted in the extracellular medium with outer membrane vesicles.

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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