Immunosuppressive Activities of Novel PLA2 Inhibitors from Xenorhabdus hominickii, an Entomopathogenic Bacterium

Abstract

Eicosanoids mediate both cellular and humoral immune responses in insects. Phospholipase A2 (PLA2) catalyzes the first committed step in eicosanoid biosynthesis. It is a common pathogenic target of two entomopathogenic bacteria, Xenorhabdus and Photorhabdus. The objective of this study was to identify novel PLA2 inhibitors from X. hominickii and determine their immunosuppressive activities. To identify novel PLA2 inhibitors, stepwise fractionation of X. hominickii culture broth and subsequent enzyme assays were performed. Eight purified fractions of bacterial metabolites were obtained. Gas chromatography and mass spectrometry (GC-MS) analysis predicted that the main components in these eight fractions were 2-cyanobenzoic acid, dibutylamine, 2-ethyl 1-hexanol, phthalimide (PM), dioctyl terephthalate, docosane, bis (2-ethylhexyl) phthalate, and 3-ethoxy-4-methoxyphenol (EMP). Their synthetic compounds inhibited the activity of PLA2 in hemocytes of a lepidopteran insect, Spodoptera exigua, in a dose-dependent manner. They also showed significant inhibitory activities against immune responses such as prophenoloxidase activation and hemocytic nodulation of S. exigua larvae, with PM and EMP exhibiting the most potent inhibitory activities. These immunosuppressive activities were specific through PLA2 inhibition because an addition of arachidonic acid, a catalytic product of PLA2, significantly rescued such suppressed immune responses. The two most potent compounds (PM and EMP) showed significant insecticidal activities after oral administration. When the compounds were mixed with Bacillus thuringiensis (Bt), they markedly increased Bt pathogenicity. This study identified eight PLA2 inhibitors from bacterial metabolites of X. hominickii and demonstrated their potential as novel insecticides.

Immune mediation of HMG-like DSP1 via Toll-Spätzle pathway and its specific inhibition by salicylic acid analogs

“…To look for effective inhibitor(s) of Se-DSP1, bacterial culture broth of X . hominickii , an insect pathogen known to induce immunosuppression [ 7 ], was fractionated ( S4 Fig ) and the resulting fractions were assessed for their binding affinities for rSe-DSP1 ( Fig 9A ). Four organic extracts were assessed.…”

“…GC-MS analysis of potent bacterial fractions followed the method described by Mollah et al [ 7 ]. Briefly, an MS (5977A Network, Agilent Technologies; Santa Clara, CA, USA) was coupled with GC (7890B, Agilent Technologies) equipped with a non-polar column (HP5 MS column, Agilent Technologies).…”

“…For successful pathogenicity, X . hominickii needs to produce and release secondary metabolites such as phenylethylamides to inhibit phospholipase A 2 (PLA 2 ) and shutdown eicosanoid biosynthesis [ 7 , 8 ]. PLA 2 catalyzes hydrolysis of phospholipids at sn-2 ester bond to release arachidonic acid.…”

Salicylic Acid, a Plant Hormone, Suppresses Phytophagous Insect Immune Response by Interrupting HMG-Like DSP1

“…For a pathogenicity test, Bacillus thuringiensis subsp. aizawai (Bt) and Xenorhabdus hominickii ANU101 (Xh) were cultured with the method described previously (Mollah et al, 2020a ).…”

“…Total RNA extraction, complementary DNA (cDNA) preparation, and quantitative reverse transcription (RT-q)PCR were performed following the methods described previously (Mollah et al, 2020a , b ). The synthesized first-stranded cDNA was used as a template for PCR amplification with 35 cycles of 94°C for 30 s, 58.8°C for 30 s, and 72°C for 30 s after an initial heat treatment at 94°C for 2 min with the gene-specific primers listed in Supplementary Table S2 .…”