The entomopathogenic bacterium Xenorhabdus nematophila secretes at least eight bacterial metabolites that play crucial roles suppressing target insect immune responses by inhibiting eicosanoid biosynthesis. We analyzed sequential changes in bacterial metabolite production during bacterial growth and analyzed their individual immunosuppressive activities against the insect host, Spodoptera exigua. X. nematophila exhibited a typical bacterial growth pattern in both insect host and culture medium, and eight metabolites were secreted at different time points. At the early growth phase (6-12 h), Ac-FGV and PHPP were detected in significant amounts in the culture broth. At this early phase, both Ac-FGV (18 μg/ml) and oxindole (110 μg/ml) levels significantly inhibited phenoloxidase and phospholipase A(2) activities in S. exigua hemolymph. At the late growth phase (12-36 h), all eight metabolites were detected at significant levels (10-140 μg/ml) in the culture broth and were sufficient to induce hemocyte toxicity. These results suggest that X. nematophila sequentially produces immunosuppressive metabolites that might sequentially and cooperatively inhibit different steps of insect immune responses.
A phase variation has been reported in an entomopathogenic bacterium, Xenorhabdus nematophila. Compared with a wild-type primary form, a secondary form usually loses several physiological and biochemical characters. This study showed that the phase variation of X. nematophila caused a significant alteration in its immunosuppressive activity and subsequent entomopathogenicity. A secondary form of X. nematophila was detected in laboratory colonies and exhibited significant differences in dye absorption and entomopathogenicity. In addition, the secondary form was different in its production of eicosanoid-biosynthesis inhibitors (EBIs) compared with the primary form of X. nematophila. Production of oxindole and phydroxypropionic acid was significantly reduced in the culture broth of the secondary form of X. nematophila. The reduced EBI production resulted in significant suppression in the inhibitory effects on cellular nodule formation and phenoloxidase activity. Culture broth of the primary form of X. nematophila enhanced the pathogenicity of Bacillus thuringiensis ( Bt) significantly more than the culture broth of the secondary form. Furthermore, this study developed a highly efficient "Dual Bt-Plus" to control both lepidopteran insect pests Plutella xylostella and Spodoptera exigua, by mixing two effective Bt strains along with the addition of potent bacterial metabolites or 100-fold concentrated X. nematophila culture broth.
Obtaining single–molecular–level fingerprints of biomolecules and electron–transfer dynamic imaging in living cells are critically demanded in postgenomic life sciences and medicine. However, the possible solution called plasmonic resonance energy transfer (PRET) spectroscopy remains challenging due to the fixed scattering spectrum of a plasmonic nanoparticle and limited multiplexing. Here, multiplexed metasurfaces‐driven PRET hyperspectral imaging, to probe biological light–matter interactions, is reported. Pixelated metasurfaces with engineered scattering spectra are first designed over the entire visible range by the precision nanoengineering of gap plasmon and grating effects of metasurface clusters. Pixelated metasurfaces are created and their full dark‐field coloration is optically characterized with visible color palettes and high‐resolution color printings of the art pieces. Furthermore, three different biomolecules (i.e., chlorophyll a, chlorophyll b, and cytochrome c) are applied on metasurfaces for color palettes to obtain selective molecular fingerprint imaging due to the unique biological light–matter interactions with application‐specific biomedical metasurfaces. This metasurface‐driven PRET hyperspectral imaging will open up a new path for multiplexed real‐time molecular sensing and imaging methods.
While breathing, alveoli are exposed to external irritants, which contribute to the pathogenesis of lung disease. Therefore, in situ monitoring of alveolar responses to stimuli of toxicants under in vivo environments is important to understand lung disease. For this purpose, 3D cell cultures are recently employed for examining cellular responses of pulmonary systems exposed to irritants; however, most of them have used ex situ assays requiring cell lysis and fluorescent labeling. Here, an alveoli‐like multifunctional scaffold is demonstrated for optical and electrochemical monitoring of cellular responses of pneumocytes. Porous foam with dimensions like the alveoli structure is used as a backbone for the scaffold, wherein electroactive metal–organic framework crystals, optically active gold nanoparticles, and biocompatible hyaluronic acid are integrated. The fabricated multifunctional scaffold allows for label‐free detection and real‐time monitoring of oxidative stress released in pneumocytes under toxic‐conditions via redox‐active amperometry and nanospectroscopy. Moreover, cellular behavior can be statistically classified based on fingerprint Raman signals collected from the cells on the scaffold. The developed scaffold is expected to serve as a promising platform to investigate cellular responses and disease pathogenesis, owing to its versatility in monitoring electrical and optical signals from cells in situ in the 3D microenvironments.
A microbial insecticide "Bt-Plus" has been developed to enhance an insecticidal efficacy of an entomopathogenic bacterium, Bacillus thuringiensis (Bt). However, its wettable powder formulation is not preferred by farmers and industry producers due to relatively high cost. This study aimed to develop a soluble concentrate formulation of Bt-Plus. To this end, an optimal mixture ratio of two bacterial culture broths was determined to be 5:4 (v/v) of Bt and Xenorhabdus nematophila (Xn) along with 10% ethanol preservative. In addition, Bt broth was concentrated by 10 times to apply the mixture at 1,000 times fold dilution. The resulting liquid formulation was sprayed on cabbage crop field infested by late instar larvae of the diamondback moth, Plutella xylostella. The field assay showed about 77% control efficacy at 7 days after treatment, which was comparable to those of current commercial biopesticides targeting P. xylostella. For storage test in both low and room temperatures, the liquid formation showed a relatively stable control efficacy at least for a month. To develop a quality control technique to exhibit a stable control efficacy of Bt-Plus, Bt spore density (5 × 10 11 spores/mL) and eight active component concentrations of Xn bacterial metabolites in the formulation products have been proposed in this study.
To enhance Bacillus thuringiensis (Bt) efficacy, four Cry toxins were purified from four different Bt strains and assessed in their combined efficacy. The Cry mixtures significantly expanded their target insect spectra. Bacterial culture broth of Xenorhabdus nematophila (Xn) significantly suppressed insect cellular immune response and increased Cry toxicity. The addition of Xn culture broth to Cry mixture significantly enhanced Bt efficacy in target insect spectrum and insecticidal activity.
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