-
Parasites & Vectors Oct 2023Nematodes of the genus Heterorhabditis are important biocontrol agents as they form a lethal combination with their symbiotic Photorhabdus bacteria against agricultural...
Taxonomic and molecular characterization of a new entomopathogenic nematode species, Heterorhabditis casmirica n. sp., and whole genome sequencing of its associated bacterial symbiont.
BACKGROUND
Nematodes of the genus Heterorhabditis are important biocontrol agents as they form a lethal combination with their symbiotic Photorhabdus bacteria against agricultural insect pests. This study describes a new species of Heterorhabditis.
METHODS
Six Heterorhabditis nematode populations were recovered from agricultural soils in Jammu and Kashmir, India. An initial examination using mitochondrial and nuclear genes showed that they belong to a new species. To describe this new species, a variety of analyses were conducted, including reconstructing phylogenetic relationships based on multiple genes, characterizing the nematodes at the morphological and morphometric levels, performing self-crossing and cross-hybridization experiments, and isolating and characterizing their symbiotic bacteria.
RESULTS
The newly discovered species, Heterorhabditis casmirica n. sp., shares 94% mitochondrial cytochrome C oxidase subunit I gene (COI) sequence identity with Heterorhabditis bacteriophora and Heterorhabditis ruandica, and 93% with Heterorhabditis zacatecana. Morphologically, it differs from H. bacteriophora in its infective juvenile phasmids (present vs. inconspicuous) and bacterial pouch visibility in the ventricular portion of the intestine (invisible vs. visible); genital papilla 1 (GP1) position (at manubrium level vs. more anterior), and in its b ratio (body length/neck length), c ratio (tail length/bulb width), and D% [(excretory pore/neck length) × 100]. Other morphological differences include anterior end to the nerve ring distance (77-100 vs. 121-130 μm), V% [(anterior end of vulva/body length) × 100] (46-57 vs. 41-47) in hermaphroditic females; rectum size (slightly longer than the anal body diameter vs. about three times longer), phasmids (smaller vs. inconspicuous), body length (0.13-2.0 vs. 0.32-0.39 mm), body diameter (73-150 vs. 160-220 μm), anterior end to the excretory pore distance (135-157 vs. 174-214 μm), and demanian ratios in amphimictic females. Morphological differences with H. ruandica and H. zacatecana were also observed. Furthermore, H. casmirica n. sp. did not mate or produce fertile progeny with other Heterorhabditis nematodes reported from India. It was also discovered that H. casmirica n. sp. is associated with Photorhabdus luminescence subsp. clarkei symbiotic bacteria.
CONCLUSIONS
The discovery of H. casmirica n. sp. provides novel insights into the diversity and evolution of Heterorhabditis nematodes and their symbiotic bacteria. This new species adds to the catalog of entomopathogenic nematodes in India.
Topics: Female; Animals; Rhabditoidea; Phylogeny; Nematoda; Photorhabdus; Whole Genome Sequencing
PubMed: 37880744
DOI: 10.1186/s13071-023-05990-z -
Pathogens (Basel, Switzerland) Aug 2023Vector-borne diseases pose a severe threat to human and animal health. L. (Diptera: Culicidae) is a widespread mosquito species and serves as a vector for the...
Vector-borne diseases pose a severe threat to human and animal health. L. (Diptera: Culicidae) is a widespread mosquito species and serves as a vector for the transmission of infectious diseases such as West Nile disease and Lymphatic Filariasis. Synthetic insecticides have been the prime control method for many years to suppress populations. However, recently, the use of insecticides has begun to be questioned due to the detrimental impact on human health and the natural environment. Therefore, many authorities urge the development of eco-friendly control methods that are nontoxic to humans. The bacterial associates [ and spp. (Enterobacterales: Morganellaceae)] of entomopathogenic nematodes (EPNs) ( spp. and spp.) (Rhabditida: Heterorhabditidae and Steinernematidae) are one of the green approaches to combat a variety of insect pests. In the present study, the mosquitocidal activity of the cell-free supernatants and cell suspension (4 × 10 cells mL) of four different symbiotic bacteria (, , , and subsp. ) was assessed against different development stages of (The 1st/2nd and 3rd/4th instar larvae and pupa) under laboratory conditions. The bacterial symbionts were able to kill all the development stages with varying levels of mortality. The 1st/2nd instar larvae exhibited the highest susceptibility to the cell-free supernatants and cell suspensions of symbiotic bacteria and the efficacy of the cell-free supernatants and cell suspensions gradually declined with increasing phases of growth. The highest effectiveness was achieved by the KCS-4S strain inducing 95% mortality to the 1st/2nd instar larvae. The results indicate that tested bacterial symbionts have great potential as an eco-friendly alternative to insecticides.
PubMed: 37764903
DOI: 10.3390/pathogens12091095 -
Antibiotics (Basel, Switzerland) Sep 2023Anti-microbial peptides provide a powerful toolkit for combating multidrug resistance. Combating eukaryotic pathogens is complicated because the intracellular drug...
Anti-microbial peptides provide a powerful toolkit for combating multidrug resistance. Combating eukaryotic pathogens is complicated because the intracellular drug targets in the eukaryotic pathogen are frequently homologs of cellular structures of vital importance in the host organism. The entomopathogenic bacteria (EPB), symbionts of entomopathogenic-nematode species, release a series of non-ribosomal templated anti-microbial peptides. Some may be potential drug candidates. The ability of an entomopathogenic-nematode/entomopathogenic bacterium symbiotic complex to survive in a given polyxenic milieu is a coevolutionary product. This explains that those gene complexes that are responsible for the biosynthesis of different non-ribosomal templated anti-microbial protective peptides (including those that are potently capable of inactivating the protist mammalian pathogen and the gallinaceous bird pathogen ) are co-regulated. Our approach is based on comparative anti-microbial bioassays of the culture media of the wild-type and regulatory mutant strains. We concluded that and are excellent sources of non-ribosomal templated anti-microbial peptides that are efficient antagonists of the mentioned pathogens. Data on selective cytotoxicity of different cell-free culture media encourage us to forecast that the recently discovered "easy-PACId" research strategy is suitable for constructing entomopathogenic-bacterium (EPB) strains producing and releasing single, harmless, non-ribosomal templated anti-microbial peptides with considerable drug, (probiotic)-candidate potential.
PubMed: 37760758
DOI: 10.3390/antibiotics12091462 -
MicroPublication Biology 2023The fruit fly is an excellent model for dissecting the molecular and functional bases of bacterial pathogenicity and host antibacterial immune response. The...
The fruit fly is an excellent model for dissecting the molecular and functional bases of bacterial pathogenicity and host antibacterial immune response. The Gram-negative bacterium is an insect-specific pathogen that forms a mutualistic relationship with the entomopathogenic nematode . Here we find that oral infection of larvae with moderately reduces their survival ability while the bacteria replicate efficiently in the infected insects. This information will contribute towards understanding host gut immunity against potent bacterial pathogens.
PubMed: 37711508
DOI: 10.17912/micropub.biology.000938 -
Bio-protocol Jul 2023The easyPACId (easy Promoter Activation and Compound Identification) approach is focused on the targeted activation of natural product biosynthetic gene clusters (BGCs)...
The easyPACId (easy Promoter Activation and Compound Identification) approach is focused on the targeted activation of natural product biosynthetic gene clusters (BGCs) encoding non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), NRPS-PKS hybrids, or other BGC classes. It was applied to entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus by exchanging the natural promoter of desired BGCs against the L-arabinose inducible PBAD promoter in ∆hfq mutants of the respective strains. The crude (culture) extracts of the cultivated easyPACId mutants are enriched with the single compound or compound class and can be tested directly against various target organisms without further purification of the produced natural products. Furthermore, isolation and identification of compounds from these mutants is simplified due to the reduced background in the ∆hfq strains. The approach avoids problems often encountered in heterologous expression hosts, chemical synthesis, or tedious extraction of desired compounds from wild-type crude extracts. This protocol describes easyPACId for Xenorhabdus and Photorhabdus, but it was also successfully adapted to Pseudomonas entomophila and might be suitable for other proteobacteria that carry hfq.
PubMed: 37449040
DOI: 10.21769/BioProtoc.4709 -
Heliyon Jun 2023The Rcs sensor system, comprising the RcsB/RcsC/RcsD and RcsF proteins, is used by bacteria of the order to withstand envelope damage. In non-stress conditions, Rcs is...
The Rcs sensor system, comprising the RcsB/RcsC/RcsD and RcsF proteins, is used by bacteria of the order to withstand envelope damage. In non-stress conditions, Rcs is repressed by IgaA, a membrane protein with three cytoplasmic regions (cyt-1, cyt-2 and cyt-3). How the Rcs-IgaA axis evolved within has not been yet explored. Here, we report phylogenetic data supporting co-evolution of IgaA with RcsC/RcsD. Functional exchange assays showed that IgaA from and , but not from or the endosymbionts and , repress the Rcs system of . IgaA from , however, repress only partially the Rcs system despite being produced at high levels in the complementation assay. The modelled structures of these IgaA variants uncovered one periplasmic and two cytoplasmic conserved β-rich architectures forming partially closed small β-barrel (SBB) domains. Conserved residues map in a connector linking cytoplasmic SSB-1 and SBB-2 domains (E180-R265); a region of cyt-1 facing cyt-2 (R188-E194-D309 and T191-H326); and between cyt-2 and cyt-3 (H293-E328-R686). These structures validated early studies in that assigned a role in function to R188, T191 and G262, and in addition revealed a previously unnoticed "hybrid" SBB-2 domain to which cyt-1 and cyt-2 contribute. IgaA variants not functional or partially functional in lack H192-P249 and R255-D313 interactions. Among these variants, only IgaA from conserves the helix α6 in SSB-1 that is present in IgaA from and . RcsF and RcsD, which interact directly with IgaA, failed to show structural features linked to specific IgaA variants. Altogether, our data provide new insights into IgaA by mapping residues selected differently during evolution and involved in function. Our data also infer contrasting lifestyles of bacteria as source of variability in the IgaA-RcsD/IgaA-RcsF interactions.
PubMed: 37303533
DOI: 10.1016/j.heliyon.2023.e16661 -
Heliyon May 2023Significant progress has been made in recent years on exploring immunometabolism, a field that integrates two processes essential for maintaining tissue and organismal...
Significant progress has been made in recent years on exploring immunometabolism, a field that integrates two processes essential for maintaining tissue and organismal homeostasis, immunity and metabolism. The nematode parasite , its mutualistic bacteria , and the fruit fly constitute a unique system to investigate the molecular basis of host immunometabolic response to nematode-bacterial complexes. In this study, we explored the contribution of the two major immune signaling pathways, Toll and Imd, to sugar metabolism in larvae during infection with nematodes. We infected Toll or Imd signaling loss-of-function mutant larvae with nematodes and assessed larval survival ability, feeding rate, and sugar metabolism. We found no significant differences in the survival ability or levels of sugar metabolites in any of the mutant larvae when responding to infection. However, we found that the Imd mutant larvae have higher feeding rate than controls during the early stages of infection. In addition, feeding rates are lower in Imd mutants relative to the control larvae as the infection progresses. We further showed that and gene expression increases in Imd mutants compared to controls early in the infection, but their expression levels decrease at later times. These findings indicate that Imd signaling activity regulates the feeding rate and and expression in larvae infected with . Results from this study facilitate our understanding of the link between host innate immunity and sugar metabolism in the context of infectious diseases caused by parasitic nematodes.
PubMed: 37251825
DOI: 10.1016/j.heliyon.2023.e16139 -
Acta Crystallographica. Section D,... Jun 2023Gram-negative bacteria such as Aeromonas and Yersinia spp. have developed mechanisms to inhibit the immune defense of their host. Effector proteins are directly injected...
Gram-negative bacteria such as Aeromonas and Yersinia spp. have developed mechanisms to inhibit the immune defense of their host. Effector proteins are directly injected into the host cytoplasm from the bacterial cytosol via type III secretion systems (T3SSs), where they modulate the cytoskeleton and signaling of the cell. Assembly of, and secretion via, T3SSs is tightly regulated by a number of bacterial proteins, including SctX (AscX in Aeromonas), the secretion of which is essential for T3SS function. Here, crystal structures of AscX in complex with SctY chaperones from Yersinia or Photorhabdus spp. carrying homologous T3SSs are described. There are crystal pathologies in all cases, with one crystal form diffracting anisotropically and the other two exhibiting strong pseudotranslation. The new structures reveal that the positioning of the substrate is very similar on different chaperones. However, the two C-terminal SctX helices that cap the N-terminal tetratricopeptide repeat of SctY shift and tilt depending on the identity of the chaperone. Moreover, the C-terminus of the α3 helix of AscX exhibits an unprecedented kink in two of the structures. In previous structures, the C-terminus of SctX protrudes beyond the chaperone as a straight helix: a conformation that is required for binding to the nonameric export gate SctV but that is unfavorable for binary SctX-SctY complexes due to the hydrophobicity of helix α3 of SctX. A kink in helix α3 may allow the chaperone to shield the hydrophobic C-terminus of SctX in solution.
Topics: Protein Binding; Molecular Chaperones; Bacterial Proteins; Yersinia; Hydrophobic and Hydrophilic Interactions
PubMed: 37204817
DOI: 10.1107/S2059798323003248 -
PLoS Pathogens May 2023Photorhabdus insect-related toxins A and B (PirA and PirB) were first recognized as insecticidal toxins from Photorhabdus luminescens. However, subsequent studies showed... (Review)
Review
Photorhabdus insect-related toxins A and B (PirA and PirB) were first recognized as insecticidal toxins from Photorhabdus luminescens. However, subsequent studies showed that their homologs from Vibrio parahaemolyticus also play critical roles in the pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimps. Based on the structural features of the PirA/PirB toxins, it was suggested that they might function in the same way as a Bacillus thuringiensis Cry pore-forming toxin. However, unlike Cry toxins, studies on the PirA/PirB toxins are still scarce, and their cytotoxic mechanism remains to be clarified. In this review, based on our studies of V. parahaemolyticus PirAvp/PirBvp, we summarize the current understanding of the gene locations, expression control, activation, and cytotoxic mechanism of this type of toxin. Given the important role these toxins play in aquatic disease and their potential use in pest control applications, we also suggest further topics for research. We hope the information presented here will be helpful for future PirA/PirB studies.
Topics: Animals; Photorhabdus; Penaeidae; Bacterial Proteins; Bacterial Toxins; Insecta; Vibrio parahaemolyticus
PubMed: 37141203
DOI: 10.1371/journal.ppat.1011330 -
Pathogens (Basel, Switzerland) Mar 2023Symbiotic bacteria form a mutualistic relationship with nematodes and are pathogenic to many insect pests. They kill insects using various strategies to evade or...
Symbiotic bacteria form a mutualistic relationship with nematodes and are pathogenic to many insect pests. They kill insects using various strategies to evade or suppress their humoral and cellular immunity. Here we evaluate the toxic effects of these bacteria and their secondary metabolites on the survival and phenoloxidase (PO) activation of larvae using biochemical and molecular methods. The results show H06 and All treatments caused significant reductions in the number of larvae in a dose-dependent manner. Secondly, the immune system recognizes symbiotic bacteria at early and late stages of infection via the induction of C-type lectin. Live symbiotic bacteria significantly inhibit PO activity in whereas heat-treated bacteria strongly increase PO activity. Additionally, expression levels of four proPhenoloxidase genes following treatment with H06 and All were compared. We found that the expression levels of all proPhenoloxidase genes were significantly down-regulated at all-time points. Similarly, treatments of larvae with metabolites benzylideneacetone and oxindole significantly down-regulated the expression of the PPO gene and inhibited PO activity. However, the addition of arachidonic acid to metabolite-treated larvae restored the expression level of the PPO gene and increased PO activity. Our results provide new insight into the roles of symbiotic bacteria in countering the insect phenoloxidase activation system.
PubMed: 37111392
DOI: 10.3390/pathogens12040506