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Insects Jun 2018Many Gram-negative bacteria have evolved insect pathogenic lifestyles. In all cases, the ability to cause disease in insects involves specific bacterial proteins... (Review)
Review
Many Gram-negative bacteria have evolved insect pathogenic lifestyles. In all cases, the ability to cause disease in insects involves specific bacterial proteins exported either to the surface, the extracellular environment, or the cytoplasm of the host cell. They also have several distinct mechanisms for secreting such proteins. In this review, we summarize the major protein secretion systems and discuss examples of secreted proteins that contribute to the virulence of a variety of Gram-negative entomopathogenic bacteria, including , , , , and species. We also briefly summarize two classes of exported protein complexes, the PVC-like elements, and the Tc toxin complexes that were first described in entomopathogenic bacteria.
PubMed: 29921761
DOI: 10.3390/insects9020068 -
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 -
Research in Microbiology 2021Understanding the mode of action of pathogenic bacteria through in vitro studies can provide additional insight into their infection strategies. Here we have...
Understanding the mode of action of pathogenic bacteria through in vitro studies can provide additional insight into their infection strategies. Here we have characterized the effect of Photorhabdus luminescens and Photorhabdus asymbiotica on two distinct insect cell lines. We report that insect cell survival and metabolism as well as bacterial proliferation differ between infection with two Photorhabdus species. These findings reinforce the notion that P. luminescens and P. asymbiotica deploy diverse tactics to infect insect cells. This knowledge might lead to better appreciation of the interaction between pathogenic bacteria and different types of insect cells.
Topics: Animals; Bacterial Proteins; Cell Line; In Vitro Techniques; Insecta; Photorhabdus; Virulence
PubMed: 33794299
DOI: 10.1016/j.resmic.2021.103832 -
Structure (London, England : 1993) May 2023Modification of the polyketide anthraquinone AQ-256 in the entomopathogenic Photorhabdus luminescens involves several O-methylations, but the biosynthetic gene cluster...
Modification of the polyketide anthraquinone AQ-256 in the entomopathogenic Photorhabdus luminescens involves several O-methylations, but the biosynthetic gene cluster antA-I lacks corresponding tailoring enzymes. We here describe the identification of five putative, highly homologous O-methyltransferases encoded in the genome of P. luminescens. Activity assays in vitro and deletion experiments in vivo revealed that three of them account for anthraquinone tailoring by producing three monomethylated and two dimethylated species of AQ-256. X-ray structures of all five enzymes indicate high structural and mechanistic similarity. As confirmed by structure-based mutagenesis, a conserved histidine at the active site likely functions as a general base for substrate deprotonation and subsequent methyl transfer in all enzymes. Eight complex structures with AQ-256 as well as mono- and dimethylated derivatives confirm the substrate specificity patterns found in vitro and visualize how single amino acid differences in the active-site pockets impact substrate orientation and govern site-specific methylation.
Topics: Methyltransferases; Methylation; Photorhabdus; Catalytic Domain; Anthraquinones
PubMed: 36963398
DOI: 10.1016/j.str.2023.03.001 -
The Science of the Total Environment Jun 2023Larvae of the greater wax moth Galleria mellonella are common pests of beehives and commercial apiaries, and in more applied settings, these insects act as alternative...
Larvae of the greater wax moth Galleria mellonella are common pests of beehives and commercial apiaries, and in more applied settings, these insects act as alternative in vivo bioassays to rodents for studying microbial virulence, antibiotic development, and toxicology. In the current study, our aim was to assess the putative adverse effects of background gamma radiation levels on G. mellonella. To achieve this, we exposed larvae to low (0.014 mGy/h), medium (0.056 mGy/h), and high (1.33 mGy/h) doses of caesium-137 and measured larval pupation events, weight, faecal discharge, susceptibility to bacterial and fungal challenges, immune cell counts, activity, and viability (i.e., haemocyte encapsulation) and melanisation levels. The effects of low and medium levels of radiation were distinguishable from the highest dose rates used - the latter insects weighed the least and pupated earlier. In general, radiation exposure modulated cellular and humoral immunity over time, with larvae showing heightened encapsulation/melanisation levels at the higher dose rates but were more susceptible to bacterial (Photorhabdus luminescens) infection. There were few signs of radiation impacts after 7 days exposure, whereas marked changes were recorded between 14 and 28 days. Our data suggest that G. mellonella demonstrates plasticity at the whole organism and cellular levels when irradiated and offers insight into how such animals may cope in radiologically contaminated environments (e.g. Chornobyl Exclusion Zone).
Topics: Animals; Moths; Larva; Gamma Rays; Anti-Bacterial Agents; Virulence
PubMed: 36906041
DOI: 10.1016/j.scitotenv.2023.162742 -
Applied and Environmental Microbiology Oct 2016Insect larvae killed by entomopathogenic nematodes are thought to contain bacterial communities dominated by a single bacterial genus, that of the nematode's bacterial...
UNLABELLED
Insect larvae killed by entomopathogenic nematodes are thought to contain bacterial communities dominated by a single bacterial genus, that of the nematode's bacterial symbiont. In this study, we used next-generation sequencing to profile bacterial community dynamics in greater wax moth (Galleria mellonella) larvae cadavers killed by Heterorhabditis nematodes and their Photorhabdus symbionts. We found that, although Photorhabdus strains did initially displace an Enterococcus-dominated community present in uninfected G. mellonella insect larvae, the cadaver community was not static. Twelve days postinfection, Photorhabdus shared the cadaver with Stenotrophomonas species. Consistent with this result, Stenotrophomonas strains isolated from infected cadavers were resistant to Photorhabdus-mediated toxicity in solid coculture assays. We isolated and characterized a Photorhabdus-produced antibiotic from G. mellonella cadavers, produced it synthetically, and demonstrated that both the natural and synthetic compounds decreased G. mellonella-associated Enterococcus growth, but not Stenotrophomonas growth, in vitro Finally, we showed that the Stenotrophomonas strains described here negatively affected Photorhabdus growth in vitro Our results add an important dimension to a broader understanding of Heterorhabditis-Photorhabdus biology and also demonstrate that interspecific bacterial competition likely characterizes even a theoretically monoxenic environment, such as a Heterorhabditis-Photorhabdus-parasitized insect cadaver.
IMPORTANCE
Understanding, and eventually manipulating, both human and environmental health depends on a complete accounting of the forces that act on and shape microbial communities. One of these underlying forces is hypothesized to be resource competition. A resource that has received little attention in the general microbiological literature, but likely has ecological and evolutionary importance, is dead/decaying multicellular organisms. Metazoan cadavers, including those of insects, are ephemeral and nutrient-rich environments, where resource competition might shape interspecific macrobiotic and microbiotic interactions. This study is the first to use a next-generation sequencing approach to study the community dynamics of bacteria within a model insect cadaver system: insect larvae parasitized by entomopathogenic nematodes and their bacterial symbionts. By integrating bioinformatic, biochemical, and classic in vitro microbiological approaches, we have provided mechanistic insight into how antibiotic-mediated bacterial interactions may shape community dynamics within insect cadavers.
Topics: Animals; Anti-Bacterial Agents; Cadaver; Larva; Microbiota; Moths; Photorhabdus; RNA, Bacterial; RNA, Ribosomal, 16S; Rhabditida; Sequence Analysis, DNA; Stenotrophomonas; Stilbenes
PubMed: 27451445
DOI: 10.1128/AEM.01211-16 -
Gene Aug 2021The genome sequences of entomopathogenic nematode (EPN) bacteria and their functional analyses can lead to the genetic engineering of the bacteria for use as biocontrol...
Genome assembly and annotation of Photorhabdus heterorhabditis strain ETL reveals genetic features involved in pathogenicity with its associated entomopathogenic nematode and anti-host effectors with biocontrol potential applications.
The genome sequences of entomopathogenic nematode (EPN) bacteria and their functional analyses can lead to the genetic engineering of the bacteria for use as biocontrol agents. The bacterial symbiont Photorhabdus heterorhabditis strain ETL isolated from an insect pathogenic nematode, Heterorhabditis zealandica strain ETL, collected in the northernmost region of South Africa was studied to reveal information that can be useful in the design of improvement strategies for both effective and liquid production method of EPN-based pesticides. The strain ETL genome was found closely related to the type strain genome of P. australis DSM 17,609 (~60 to 99.9% CDSs similarity), but closely related to the not yet genome-sequenced type strain, P. heterorhabditis. It has a genome size of 4,866,148 bp and G + C content of 42.4% similar to other Photorhabdus. It contains 4,351 protein coding genes (CDSs) of which, at least 84% are shared with the de facto type strain P. luminescens subsp. laumondii TTO1, and has 318 unknown CDSs and the genome has a higher degree of plasticity allowing it to adapt to different environmental conditions, and to be virulent against various insects; observed through genes acquired through horizontal gene transfer mechanisms, clustered regularly interspaced short palindromic repeats, non-determined polyketide- and non-ribosomal peptide- synthase gene clusters, and many genes associated with uncharacterized proteins; which also justify the strain ETL's genes differences (quantity and quality) compared to P. luminescens subsp. laumondii TTO1. The protein coding sequences contained genes with both bio-engineering and EPNs mass production importance, of which numerous are uncharacterized.
Topics: Animals; Base Sequence; Biological Control Agents; Genes, Bacterial; Genome, Bacterial; Host-Pathogen Interactions; Molecular Sequence Annotation; Photorhabdus; Phylogeny; Strongyloidea; Virulence
PubMed: 34147570
DOI: 10.1016/j.gene.2021.145780 -
Chembiochem : a European Journal of... May 2021The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising...
The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881-1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.
Topics: Bacterial Proteins; Drug Design; Escherichia coli; Multigene Family; Peptides, Cyclic; Photorhabdus; Proteasome Inhibitors; Structure-Activity Relationship
PubMed: 33452852
DOI: 10.1002/cbic.202100014 -
BMC Genomics Nov 2022Nematodes are a major group of soil inhabiting organisms. Heterorhabditis nematodes are insect-pathogenic nematodes and live in a close symbiotic association with...
BACKGROUND
Nematodes are a major group of soil inhabiting organisms. Heterorhabditis nematodes are insect-pathogenic nematodes and live in a close symbiotic association with Photorhabdus bacteria. Heterorhabditis-Photorhabdus pair offers a powerful and genetically tractable model to study animal-microbe symbiosis. It is possible to generate symbiont bacteria free (axenic) stages in Heterorhabditis. Here, we compared the transcriptome of symbiotic early-adult stage Heterorhabditis nematodes with axenic early-adult nematodes to determine the nematode genes and pathways involved in symbiosis with Photorhabdus bacteria.
RESULTS
A de-novo reference transcriptome assembly of 95.7 Mb was created for H. bacteriophora by using all the reads. The assembly contained 46,599 transcripts with N50 value of 2,681 bp and the average transcript length was 2,054 bp. The differentially expressed transcripts were identified by mapping reads from symbiotic and axenic nematodes to the reference assembly. A total of 754 differentially expressed transcripts were identified in symbiotic nematodes as compared to the axenic nematodes. The ribosomal pathway was identified as the most affected among the differentially expressed transcripts. Additionally, 12,151 transcripts were unique to symbiotic nematodes. Endocytosis, cAMP signalling and focal adhesion were the top three enriched pathways in symbiotic nematodes, while a large number of transcripts coding for various responses against bacteria, such as bacterial recognition, canonical immune signalling pathways, and antimicrobial effectors could also be identified.
CONCLUSIONS
The symbiotic Heterorhabditis nematodes respond to the presence of symbiotic bacteria by expressing various transcripts involved in a multi-layered immune response which might represent non-systemic and evolved localized responses to maintain mutualistic bacteria at non-threatening levels. Subject to further functional validation of the identified transcripts, our findings suggest that Heterorhabditis nematode immune system plays a critical role in maintenance of symbiosis with Photorhabdus bacteria.
Topics: Animals; Photorhabdus; Rhabditoidea; Symbiosis; Sequence Analysis, RNA; RNA
PubMed: 36344922
DOI: 10.1186/s12864-022-08952-4 -
PloS One 2018Entomopathogenic nematodes (EPNs) that are symbiotically associated with Xenorhabdus and Photorhabdus bacteria can kill target insects via direct infection and toxin...
Identification of entomopathogenic nematodes and symbiotic bacteria from Nam Nao National Park in Thailand and larvicidal activity of symbiotic bacteria against Aedes aegypti and Aedes albopictus.
Entomopathogenic nematodes (EPNs) that are symbiotically associated with Xenorhabdus and Photorhabdus bacteria can kill target insects via direct infection and toxin action. There are limited reports identifying such organisms in the National Park of Thailand. Therefore, the objectives of this study were to identify EPNs and symbiotic bacteria from Nam Nao National Park, Phetchabun Province, Thailand and to evaluate the larvicidal activity of bacteria against Aedes aegypti and Ae. albopictus. A total of 12 EPN isolates belonging to Steinernema and Heterorhabditis were obtained form 940 soil samples between February 2014 and July 2016. EPNs were molecularly identified as S. websteri (10 isolates) and H. baujardi (2 isolates). Symbiotic bacteria were isolated from EPNs and molecularly identified as P. luminescens subsp. akhurstii (13 isolates), X. stockiae (11 isolates), X. vietnamensis (2 isolates) and X. japonica (1 isolate). For the bioassay, bacterial suspensions were evaluated for toxicity against third to early fourth instar larvae of Aedes spp. The larvae of both Aedes species were orally susceptible to symbiotic bacteria. The highest larval mortality of Ae. aegypti was 99% after exposure to X. stockiae (bNN112.3_TH) at 96 h, and the highest mortality of Ae. albopictus was 98% after exposure to P. luminescens subsp. akhurstii (bNN121.4_TH) at 96 h. In contrast to the control groups (Escherichia coli and distilled water), the mortality rate of both mosquito larvae ranged between 0 and 7% at 72 h. Here, we report the first observation of X. vietnamensis in Thailand. Additionally, we report the first observation of P. luminescens subsp. akhurstii associated with H. baujardi in Thailand. X. stockiae has potential to be a biocontrol agent for mosquitoes. This investigation provides a survey of the basic diversity of EPNs and symbiotic bacteria in the National Park of Thailand, and it is a bacterial resource for further studies of bioactive compounds.
Topics: Aedes; Animals; Larva; Nematoda; Parks, Recreational; Photorhabdus; Phylogeny; Soil; Symbiosis; Thailand; Xenorhabdus
PubMed: 29641570
DOI: 10.1371/journal.pone.0195681