An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits the expression of an antibacterial peptide, cecropin, of the beet armyworm, Spodoptera exigua

doi:10.1016/j.jinsphys.2004.03.005

Journal of Insect Physiology

Volume 50, Issue 6, June 2004, Pages 489-496

https://doi.org/10.1016/j.jinsphys.2004.03.005 Get rights and content

Abstract

An entomopathogenic bacterium, Xenorhabdus nematophila, is known to depress hemocyte nodule formation of target insects by inhibiting eicosanoid biosynthesis. This study analyzed the inhibitory effect of X. nematophila on the humoral immunity of the target insects and tested its association with the host eicosanoid pathway. Plasma collected from the fifth instar larvae of Spodoptera exigua, when they were injected with X. nematophila, did not show antibacterial activity against Escherichia coli by a growth inhibition zone assay. In comparison, heat-killed X. nematophila induced significant antibacterial activity in the plasma. The antibacterial humoral activity was further demonstrated by examining a specific potent antibacterial peptide, cecropin. Two cecropin genes (‘A’ and ‘B’) were partially cloned from the fifth instar larvae of S. exigua by conserved degenerate primers using nested reverse transcriptase-polymerase chain reaction (RT-PCR). They showed high homologies with known cecropins from other lepidopteran species. Northern analysis using the cecropin probe showed that the injection of the heat-killed X. nematophila induced significant expression of a cecropin mRNA transcript (≈1.1 kb), but the larvae injected with the live bacteria did not show the corresponding transcript. Injection of arachidonic acid did not rescue the inhibition of X. nematophila based on either antibacterial activity or cecropin gene expression. The addition of dexamethasone, a specific phospholipase A₂ inhibitor, did not inhibit antibacterial activity or cecropin gene expression when the larvae were injected with heat-killed X. nematophila. These results suggest that X. nematophila inhibits the antibacterial humoral immune reaction as well as the cellular immune reaction in S. exigua and that the inhibition of X. nematophila on the expression of the antibacterial peptide is not associated with inhibition of the eicosanoid pathway.

Introduction

An entomopathogenic bacterium, Xenorhabdus nematophila, is the intestinal symbiont of the entomopathogenic nematode, Steinernema carpocapsae (Akhurst, 1980). The bacteria are released into the hemocoel of the target insect after infection by S. carpocapsae and play significant roles in the pathogenicity of the bacteria–nematode complex by inducing host insect immunodepression (Kaya and Gaugler, 1993).

In response to the bacterial and nematode infection, the insects have the potential to express the defense reactions of both the cellular and humoral immune systems (Dunphy and Thurston, 1990). The beet armyworm, Spodoptera exigua, can form ≈30 hemocyte nodules when they were infected with heat-killed X. nematophila (Park and Kim, 2000). Lysozymes of S. exigua are significantly activated by the injection of bacterial lipopolysaccharides (Bae and Kim, 2003). However, live X. nematophila can significantly inhibit the hemocytic nodule formation by interrupting the eicosanoid-mediating pathway (Park and Kim, 2000, Park et al., 2003, Park et al., 2003). X. nematophila inhibits phospholipase A₂ (PLA₂) because arachidonic acid, the catalytic product of PLA₂, reverses the inhibitory action of the bacteria (Park and Kim, 2000), and the hemocytic PLA₂ cannot release arachidonic acid in the presence of the bacteria or the bacterial extract (Park, 2003). In addition, the PLA₂ that is inhibited by X. nematophila in S. exigua shows susceptibility to p-bromophenacyl bromide, a secretory PLA₂ inhibitor in vertebrates (Park and Kim, 2003).

X. nematophila appears to inhibit humoral immune reactions. Activation of prophenoloxidase in the target insects is inhibited by the infection of X. nematophila (de Silva et al., 2000, Park and Kim, 2003). Phenoloxidase activity is dependent on the PLA₂ activity in S. exigua (Park and Kim, 2003), which suggests that the inhibitory action of X. nematophila against PLA₂ can lower prophenoloxidase activation. Induction of lysozyme, an antibacterial peptide, was inhibited by X. nematophila in the host of S. exigua (Park et al., in press). Cecropins, the primary antibacterial peptides induced in Galleria mellonella immunized by another entomopathogenic bacteria, Photorhabdus luminescens, are inhibited by a specific cecropin-degrading proteinase secreted by the bacteria (Jarosz, 1998). Caldas et al. (2002) purified a secretory proteinase from X. nematophila and showed that the proteinase inhibited the activity of a commercial cecropin A.

This study assesses the inhibitory effect of X. nematophila on humoral antibacterial activity and a specific antibacterial gene expressed by S. exigua. We also test whether the PLA₂-inhibitory effect of X. nematophila is linked to its inhibitory effect on the humoral antibacterial activity.

Section snippets

Insect and bacterial culture

The larvae, S. exigua, were reared on an artificial diet (Gho et al., 1990) at 25±1 °C, a photoperiod of 16:8 (L:D) h, and RH 60±5%. Adults were fed 10% sucrose solution. X. nematophila was isolated from S. carpocapsae collected in Korea (Park et al., 1999). The bacteria were cultured in tryptic soy agar medium (TSA) (Difco, USA) at 28 °C. The bacterial colonies used in this study were regarded as the phase I variant because they showed red color on NBTA (nutrient agar+0.0025% bromothymol

X. nematophila depresses the induction of humoral antibacterial activity

The effect of X. nematophila on the humoral immune reactions was analyzed by the growth inhibition zone assay. Plasma from the larvae injected with heat-killed X. nematophila had significantly high antibacterial activity against Gram-negative Es. coli, but not against Gram-positive En. faecalis (Fig. 1). In comparison, the plasma from larvae infected with live X. nematophila did not show significant antibacterial activity against either bacteria.

The plasma of larvae injected with heat-killed X.

Discussion

To express potent pathogenicity, X. nematophila should overcome the immune reactions expressed by the target insect hosts. Our early report (Park and Kim, 2000) showed that X. nematophila could inhibit a cellular immune reaction, hemocyte nodulation, of the infected hemolymph. Here, we showed that the injection of X. nematophila could depress the humoral antibacterial activity of S. exigua because the heat-killed bacteria significantly induced antibacterial activity of the target insects. Thus,

Acknowledgements

We thank Youngim Song for supplying materials and encouragement. We also appreciate Neil Basio for his kind English corrections. This study was supported by the Special Grants Research Program of the Korea Ministry of Agriculture, Forestry and Fisheries.

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An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits the expression of an antibacterial peptide, cecropin, of the beet armyworm, Spodoptera exigua

Abstract

Introduction

Section snippets

Insect and bacterial culture

X. nematophila depresses the induction of humoral antibacterial activity

Discussion

Acknowledgements

Current Opinion in Immunology

Insect Biochemistry and Molecular Biology

Comparative Biochemistry and Physiology

Current Opinion in Immunology

Comparative Biochemistry and Physiology

Analytical Biochemistry

Developmental and Comparative Immunology

Current Opinion in Microbiology

Current Opinion in Immunology

Comparative Biochemistry and Physiology

FEBS Letters

Journal of Insect Physiology

Journal of Asia-Pacific Entomology

Korean Journal of Applied Entomology

Biochemical and Biophysical Research Communications

Journal of Biological Chemistry

Morphological and functional dimorphism in Xenorhabdus spp., bacteria symbiotically associated with the insect pathogenic nematodes Neoaplectana and Heterorhabditis

Journal of General Microbiology