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Journal of Nanobiotechnology Dec 2021Shrimp aquaculture has suffered huge economic losses over the past decade due to the outbreak of acute hepatopancreatic necrosis disease (AHPND), which is mainly caused...
BACKGROUND
Shrimp aquaculture has suffered huge economic losses over the past decade due to the outbreak of acute hepatopancreatic necrosis disease (AHPND), which is mainly caused by the bacteria Vibrio parahaemolyticus (V. parahaemolyticus) with the virulence pVA1 plasmid, which encodes a secretory photorhabdus insect-related (Pir) toxin composed of PirA and PirB proteins. The Pir toxin mainly attacks the hepatopancreas, a major metabolic organ in shrimp, thereby causing necrosis and loss of function. The pandemic of antibiotic-resistant strains makes the impact worse.
METHODS
Mild pyrolysis of a mixture of polysaccharide dextran 70 and the crosslinker 1,8-diaminooctane at 180 ℃ for 3 h to form carbonized nanogels (DAO/DEX-CNGs) through controlled cross-linking and carbonization. The multifunctional therapeutic CNGs inherit nanogel-like structures and functional groups from their precursor molecules.
RESULTS
DAO/DEX-CNGs manifest broad-spectrum antibacterial activity against Vibrio parahaemolyticus responsible for AHPND and even multiple drug-resistant strains. The polymer-like structures and functional groups on graphitic-carbon within the CNGs exhibit multiple treatment effects, including disruption of bacterial membranes, elevating bacterial oxidative stress, and neutralization of PirAB toxins. The inhibition of Vibrio in the midgut of infected shrimp, protection of hepatopancreas tissue from Pir toxin, and suppressing overstimulation of the immune system in severe V. parahaemolyticus infection, revealing that CNGs can effectively guard shrimp from Vibrio invasion. Moreover, shrimps fed with DAO/DEX-CNGs were carefully examined, such as the expression of the immune-related genes, hepatopancreas biopsy, and intestinal microbiota. Few adverse effects on shrimps were observed.
CONCLUSION
Our work proposes brand-new applications of multifunctional carbon-based nanomaterials as efficient anti-Vibrio agents in the aquatic industry that hold great potential as feed additives to reduce antibiotic overuse in aquaculture.
Topics: Animals; Anti-Infective Agents; Artemia; Biocompatible Materials; Carbon; Dextrans; Gram-Negative Bacteria; Gram-Positive Bacteria; Hepatopancreas; Nanogels; Toxins, Biological; Vibrio Infections; Vibrio parahaemolyticus
PubMed: 34952588
DOI: 10.1186/s12951-021-01194-8 -
Microbiology Spectrum Dec 2021There is great need for therapeutics against multidrug-resistant, Gram-negative bacterial pathogens. Recently, darobactin A, a novel bicyclic heptapeptide that...
There is great need for therapeutics against multidrug-resistant, Gram-negative bacterial pathogens. Recently, darobactin A, a novel bicyclic heptapeptide that selectively kills Gram-negative bacteria by targeting the outer membrane protein BamA, was discovered. Its efficacy was proven in animal infection models of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, thus promoting darobactin A as a promising lead compound. Originally discovered from members of the nematode-symbiotic genus , the biosynthetic gene cluster (BGC) encoding the synthesis of darobactin A can also be found in other members of the class . Therein, the precursor peptides DarB to -F, which differ in their core sequence from darobactin A, were identified . Even though production of these analogs was not observed in the putative producer strains, we were able to generate them by mutasynthetic derivatization of a heterologous expression system. The analogs generated were isolated and tested for their bioactivity. The most potent compound, darobactin B, was used for cocrystallization with the target BamA, revealing a binding site identical to that of darobactin A. Despite its potency, darobactin B did not exhibit cytotoxicity, and it was slightly more active against Acinetobacter baumannii isolates than darobactin A. Furthermore, we evaluated the plasma protein binding of darobactin A and B, indicating their different pharmacokinetic properties. This is the first report on new members of this new antibiotic class, which is likely to expand to several promising therapeutic candidates. Therapeutic options to combat Gram-negative bacterial pathogens are dwindling with increasing antibiotic resistance. This study presents a proof of concept for the heterologous-expression approach to expand on the novel antibiotic class of darobactins and to generate analogs with different activities and pharmacokinetic properties. In combination with the structural data of the target BamA, this approach may contribute to structure-activity relationship (SAR) data to optimize inhibitors of this essential outer membrane protein of Gram-negative pathogens.
Topics: Acinetobacter baumannii; Animals; Anti-Bacterial Agents; Anti-Infective Agents; Bacterial Outer Membrane Proteins; Cell Line; Drug Resistance, Multiple, Bacterial; Escherichia coli; Escherichia coli Proteins; Gram-Negative Bacteria; Humans; Klebsiella pneumoniae; Microbial Sensitivity Tests; Multigene Family; Phenylpropionates; Pseudomonas aeruginosa; Structure-Activity Relationship
PubMed: 34937193
DOI: 10.1128/spectrum.01535-21 -
Plant Methods Dec 2021The bacterial leaf pathogen Pseudomonas syringae pv tomato (Pst) is the most popular model pathogen for plant pathology research. Previous methods to study the plant-Pst...
BACKGROUND
The bacterial leaf pathogen Pseudomonas syringae pv tomato (Pst) is the most popular model pathogen for plant pathology research. Previous methods to study the plant-Pst interactions rely on destructive quantification of Pst colonisation, which can be labour- and time-consuming and does not allow for spatial-temporal monitoring of the bacterial colonisation. Here, we describe a rapid and non-destructive method to quantify and visualise spatial-temporal colonisation by Pst in intact leaves of Arabidopsis and tomato.
RESULTS
The method presented here uses a bioluminescent Pst DC3000 strain that constitutively expresses the luxCDABE operon from Photorhabdus luminescens (Pst::LUX) and requires a common gel documentation (Gel Doc) system with a sensitive CCD/CMOS camera and imaging software (Photoshop or Image J). By capturing bright field and bioluminescence images from Pst::LUX-infected leaves, we imaged the spatiotemporal dynamics of Pst infection. Analysis of bioluminescence from live Pst bacteria over a 5-day time course after spray inoculation of Arabidopsis revealed transition of the bacterial presence from the older leaves to the younger leaves and apical meristem. Colonisation by Pst:LUX bioluminescence was obtained from digital photos by calculating relative bioluminescence values, which is adjusted for bioluminescence intensity and normalised by leaf surface. This method detected statistically significant differences in Pst::LUX colonisation between Arabidopsis genotypes varying in basal resistance, as well as statistically significant reductions in Pst::LUX colonisation by resistance-inducing treatments in both Arabidopsis and tomato. Comparison of relative bioluminescence values to conventional colony counting on selective agar medium revealed a statistically significant correlation, which was reproducible between different Gel Doc systems.
CONCLUSIONS
We present a non-destructive method to quantify colonisation by bioluminescent Pst::LUX in plants. Using a common Gel Doc system and imaging software, our method requires less time and labour than conventional methods that are based on destructive sampling of infected leaf material. Furthermore, in contrast to conventional strategies, our method provides additional information about the spatial-temporal patterns of Pst colonisation.
PubMed: 34903271
DOI: 10.1186/s13007-021-00826-2 -
ACS Omega Dec 2021Individual bacteria communicate by the release and interpretation of small molecules, a phenomenon known as quorum sensing (QS). We hypothesized that QS compounds...
Individual bacteria communicate by the release and interpretation of small molecules, a phenomenon known as quorum sensing (QS). We hypothesized that QS compounds extruded by could be interpreted by -a form of interspecies communication. We interrogate the structure-activity relationship within the recently discovered pyrone QS network and reveal the exquisite structural features required for targeted phenotypic behavior. The interruption of QS is an exciting, nonbiocidal approach to tackling infection, and understanding its nuances can only be achieved by studies such as this.
PubMed: 34901665
DOI: 10.1021/acsomega.1c05508 -
Virulence Dec 2021bacteria secrete a repertoire of protein toxins that can kill the host insect. Among them, toxin complex (Tc) proteins have gained significant attention due to their...
bacteria secrete a repertoire of protein toxins that can kill the host insect. Among them, toxin complex (Tc) proteins have gained significant attention due to their wider conservation across the different bacterial genera. In our laboratory, a C-terminal domain of TcaB protein was characterized from bacterium that conferred the potent oral insecticidal effect on . However, the role of insect gut receptors in the TcaB intoxication process was yet to be investigated. In the current study, we examined the transcription of candidate midgut receptors in TcaB-infected larvae and subsequently cloned a cadherin-like gene, , from was highly transcribed in the fourth-instar larval stage and specifically in the midgut tissues. Our ligand blot and binding ELISA assays indicated that TcaB binds to the truncated peptides from the GmCAD transmembrane-proximal region with greater affinity than that from the transmembrane-distal region. Oral administration of bacterially expressed dsRNA in severely attenuated the expression of target mRNA, which in turn alleviated the negative effect of TcaB on insect survival (TcaB-induced mortality in CAD dsRNA pretreated larvae reduced by 72-83% compared to control), implying the association of GmCAD in the TcaB intoxication process. Present findings form a basis of future research related to the insect gut receptor interactions with toxins.
Topics: Animals; Insecta; Larva; Moths; Photorhabdus
PubMed: 34882066
DOI: 10.1080/21505594.2021.2006996 -
Nature Communications Dec 2021Bacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins...
Bacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, comprising a C-terminal toxin domain fused to a N-terminal domain that adapts to the delivery apparatus. Polymorphic toxins include bacteriocins, contact-dependent growth inhibition systems, and specialized Hcp, VgrG, PAAR or Rhs Type VI secretion (T6SS) components. We recently described and characterized Tre23, a toxin domain fused to a T6SS-associated Rhs protein in Photorhabdus laumondii, Rhs1. Here, we show that Rhs1 forms a complex with the T6SS spike protein VgrG and the EagR chaperone. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs1 complex formation requires the VgrG C-terminal β-helix and the Rhs1 N-terminal region. We then report the cryo-electron-microscopy structure of the Rhs1-EagR complex, demonstrating that the Rhs1 central region forms a β-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs1 protein through aspartyl autoproteolysis. We propose a model for Rhs1 loading on the T6SS, transport and delivery into the target cell.
Topics: Adaptation, Physiological; Bacterial Proteins; Bacterial Toxins; Bacteriocins; Contact Inhibition; Cryoelectron Microscopy; Mass Spectrometry; Models, Molecular; Photorhabdus; Type VI Secretion Systems
PubMed: 34853317
DOI: 10.1038/s41467-021-27388-0 -
Insects Nov 2021The European grapevine moth (EGVM) (Lepidoptera: Tortricidae) is a relevant pest in the Palearctic region vineyards and is present in the Americas. Their management...
The European grapevine moth (EGVM) (Lepidoptera: Tortricidae) is a relevant pest in the Palearctic region vineyards and is present in the Americas. Their management using biological control agents and environmentally friendly biotechnical tools would reduce intensive pesticide use. The entomopathogenic nematodes (EPNs) in the families Steinernematidae and Heterorhabditidae are well-known virulent agents against arthropod pests thanks to symbiotic bacteria in the genera and (respectively) that produce natural products with insecticidal potential. Novel technological advances allow field applications of EPNs and those bioactive compounds as powerful bio-tools against aerial insect pests. This study aimed to determine the viability of four EPN species (, , , and ) as biological control agents against EGVM larval instars (L1, L3, and L5) and pupae. Additionally, the bioactive compounds from their four symbiotic bacteria (, , , and subsp. respectively) were tested as unfiltered ferment (UF) and cell-free supernatant (CFS) against the EGVM larval instars L1 and L3. All of the EPN species showed the capability of killing EGVM during the larval and pupal stages, particularly (mortalities of ~50% for L1 and >75% for L3 and L5 in only two days), followed by efficacy by . Similarly, the bacterial bioactive compounds produced higher larval mortality at three days against L1 (>90%) than L3 (~50%), making the application of UF more virulent than the application of CFS. Our findings indicate that both steinernematid species and their symbiotic bacterial bioactive compounds could be considered for a novel agro-technological approach to control in vineyards. Further research into co-formulation with adjuvants is required to expand their viability when implemented for aboveground grapevine application.
PubMed: 34821833
DOI: 10.3390/insects12111033 -
Genetics Jan 2022Entomopathogenic nematodes (EPNs), including Heterorhabditis and Steinernema, are parasitic to insects and contain mutualistically symbiotic bacteria in their intestines...
The entomopathogenic nematode Steinernema hermaphroditum is a self-fertilizing hermaphrodite and a genetically tractable system for the study of parasitic and mutualistic symbiosis.
Entomopathogenic nematodes (EPNs), including Heterorhabditis and Steinernema, are parasitic to insects and contain mutualistically symbiotic bacteria in their intestines (Photorhabdus and Xenorhabdus, respectively) and therefore offer opportunities to study both mutualistic and parasitic symbiosis. The establishment of genetic tools in EPNs has been impeded by limited genetic tractability, inconsistent growth in vitro, variable cryopreservation, and low mating efficiency. We obtained the recently described Steinernema hermaphroditum strain CS34 and optimized its in vitro growth, with a rapid generation time on a lawn of its native symbiotic bacteria Xenorhabdus griffiniae. We developed a simple and efficient cryopreservation method. Previously, S. hermaphroditum isolated from insect hosts was described as producing hermaphrodites in the first generation. We discovered that CS34, when grown in vitro, produced consecutive generations of autonomously reproducing hermaphrodites accompanied by rare males. We performed mutagenesis screens in S. hermaphroditum that produced mutant lines with visible and heritable phenotypes. Genetic analysis of the mutants demonstrated that this species reproduces by self-fertilization rather than parthenogenesis and that its sex is determined chromosomally. Genetic mapping has thus far identified markers on the X chromosome and three of four autosomes. We report that S. hermaphroditum CS34 is the first consistently hermaphroditic EPN and is suitable for genetic model development to study naturally occurring mutualistic symbiosis and insect parasitism.
Topics: Symbiosis
PubMed: 34791196
DOI: 10.1093/genetics/iyab170 -
Journal of Nematology 2021Species of the nematode genus are important biological control agents against agricultural pests. The taxonomy of this group is still unclear as it currently relies on...
Species of the nematode genus are important biological control agents against agricultural pests. The taxonomy of this group is still unclear as it currently relies on phylogenetic reconstructions based on a few genetic markers with little resolutive power, specially of closely related species. To fill this knowledge gap, we sequenced several phylogenetically relevant genetic loci and used them to reconstruct phylogenetic trees, to calculate sequence similarity scores, and to determine signatures of species- and population-specific genetic polymorphism. In addition, we revisited the current literature related to the description, synonymisation, and declaration as of species to compile taxonomically relevant morphological and morphometric characters, characterized new nematode isolates at the morphological and morphometrical level, and conducted self-crossing and cross-hybridization experiments. The results of this study show that the sequences of the mitochondrial cytochrome C oxidase subunit I (COI) gene provide better phylogenetic resolutive power than the sequences of nuclear rRNA genes and that this gene marker can phylogenetically resolve closely related species and even populations of the same species with high precision. Using this gene marker, we found two new species, n. sp. and n. sp. A detailed characterization of these species at the morphological and morphometric levels and nematode reproduction assays revealed that the threshold for species delimitation in this genus, using sequences, is 97% to 98%. Our study illustrates the importance of rigorous morphological and morphometric characterization and multi-locus sequencing for the description of new species within the genus , serves to clarify the phylogenetic relationships of this important group of biological control agents, and can inform future species descriptions to advance our efforts towards developing more tools for sustainable and environmentally friendly agriculture.
PubMed: 34790901
DOI: 10.21307/jofnem-2021-089 -
International Journal of Molecular... Nov 2021Entomopathogenic nematodes (Rhabditida: and ) are a group of organisms capable of infecting larvae of insects living in soil, including representatives of the family ....
Entomopathogenic nematodes (Rhabditida: and ) are a group of organisms capable of infecting larvae of insects living in soil, including representatives of the family . Their insecticidal activity is related to the presence of symbiotic bacteria spp. or spp. in the alimentary tract, which are released into the insect body, leading to its death caused by bacterial toxins and septicemia. Although the antibacterial activities of symbionts of entomopathogenic nematodes have been well described, there is insufficient knowledge of the interactions between these bacteria and microorganisms that naturally inhabit the alimentary tract of insects infested by nematodes. In this study, 900 bacterial strains isolated from midgut samples of larvae were tested for their antagonistic activity against the selected five and species. Cross-streak tests showed significant antibacterial activity of 20 isolates. These bacteria were identified as , , , , sp., , , sp., and sp. Since some representatives of the intestinal microbiota of are able to inhibit the growth of and bacteria in vitro, it can be assumed that this type of bacterial interaction may occur at certain stages of insect infection by or nematodes.
Topics: Animals; Bacterial Infections; Bacterial Toxins; Coleoptera; Gastrointestinal Microbiome; Larva; Photorhabdus; Symbiosis; Xenorhabdus
PubMed: 34769435
DOI: 10.3390/ijms222112005