Yersinia Entomophaga Research Articles

Yersinia entomophaga Tc toxin is released by T10SS-dependent lysis of specialized cell subpopulations.

Disease-causing bacteria secrete numerous toxins to invade and subjugate their hosts. Unlike many smaller toxins, the secretion machinery of most large toxins remains enigmatic. By combining genomic editing, proteomic profiling and cryo-electron tomography of the insect pathogen Yersinia entomophaga, we demonstrate that a specialized subset of these cells produces a complex toxin cocktail, including the nearly ribosome-sized Tc toxin YenTc, which is subsequently exported by controlled cell lysis using a transcriptionally coupled, pH-dependent type 10 secretion system (T10SS). Our results dissect the Tc toxin export process by a T10SS, identifying that T10SSs operate via a previously unknown lytic mode of action and establishing them as crucial players in the size-insensitive release of cytoplasmically folded toxins. With T10SSs directly embedded in Tc toxin operons of major pathogens, we anticipate that our findings may model an important aspect of pathogenesis in bacteria with substantial impact on agriculture and healthcare.

Phylogenetic Relatedness and Genome Structure of Yersinia ruckeri Revealed by Whole Genome Sequencing and a Comparative Analysis.

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), a serious infection that affects global aquaculture with high economic impact. The present study used whole genome sequences to perform a comparative analysis on 10 Y. ruckeri strains and to explore their genetic relatedness to other members of the genus. Y. ruckeri, Yersinia entomophaga, and Yersinia nurmii formed a species complex that constitutes the most basal lineage of the genus. The results showed that the taxonomy of Y. ruckeri strains is better defined by using a core genome alignment and phylogenetic analysis. The distribution of accessory genes in all Yersinia species revealed the presence of 303 distinctive genes in Y. ruckeri. Of these, 169 genes were distributed in 17 genomic islands potentially involved in the pathogenesis of ERM via (1) encoding virulence factors such as Afp18, Yrp1, phage proteins and (2) improving the metabolic capabilities by enhancing utilization and metabolism of iron, amino acids (specifically, arginine and histidine), and carbohydrates. The genome of Y. ruckeri is highly conserved regarding gene structure, gene layout and functional categorization of genes. It contains various components of mobile genetic elements but lacks the CRISPR-Cas system and possesses a stable set of virulence genes possibly playing a critical role in pathogenicity. Distinct virulence plasmids were exclusively restricted to a specific clonal group of Y. ruckeri (CG4), possibly indicating a selective advantage. Phylogenetic analysis of Y. ruckeri genomes revealed the co-presence of multiple genetically distant lineages of Y. ruckeri strains circulating in Germany. Our results also suggest a possible dissemination of a specific group of strains in the United States, Peru, Germany, and Denmark. In conclusion, this study provides new insights into the taxonomy and evolution of Y. ruckeri and contributes to a better understanding of the pathogenicity of ERM in aquaculture. The genomic analysis presented here offers a framework for the development of more efficient control strategies for this pathogen.

Assessment of toxicity and persistence of Yersinia entomophaga and its Yen-Tc associated toxin.

The insect-pathogenic bacterium Yersinia entomophaga MH96 is currently under development as a microbial pesticide active against various pasture and crop pests such as the diamondback moth Plutella xylostella and the cotton bollworm Helicoverpa armigeria. To enable nonrestricted field trials of Y. entomophaga MH96, information on the persistence and nontarget effects of the bacterium and its Yen-Tc proteinaceous toxin are required. The Y. entomophaga Yen-Tc associated toxin was found to have limited persistence on foliage and is inactivated by UV light. The Yen-Tc was rapidly degraded in ovine or bovine rumen fluid or the intestinal fluid of H. armigera. In H. armigera an intestinal protein of >50 kDa was found to cleave the Yen-Tc bond. Assessment of Y. entomophaga persistence on foliage and in soil found that after 42 days the bacterium could not be detected in soil at 20% soil moisture content but persisted for 72 days at 30-40% soil moisture. Nontarget effects of Y. entomophaga towards earthworms found that the bacterium afforded no adverse effects on worm growth or behavior. A summary of historic Yen-Tc and Y. entomophaga persistence and toxicity data is presented. The bacterium Y. entomophaga and its Yen-Tc associated toxin have limited persistence in the environment, with the Yen-Tc being susceptible to UV inactivation and proteolytic degradation, and the bacterium persisting longer in soil of a high moisture content. © 2020 Society of Chemical Industry.

Potential for a biopesticide bait to control black beetle, Heteronychus arator (Coleoptera: Scarabaeidae).

Yersinia entomophaga is an entomopathogenic bacterium that is active against scarab beetles, among other insects. In New Zealand, the African black beetle, Heteronychus arator (Coleoptera: Scarabaeidae), is a major pest of pastures and arable crops but very few control options exist and no insecticides are registered for use in established pastures. In laboratory bioassays, H. arator adults were susceptible to a bait containing Y. entomophaga at low doses. This bait was more effective against H. arator adults during spring than autumn in small-scale field plots (320 mm diameter). A large-scale field trial (40 × 40 m plots) reduced adult numbers substantially: approximately twice as many beetles were captured in pitfall traps from untreated plots compared with plots treated with the Y. entomophaga bait at 70 kg ha-1 . This single bait application in spring also reduced subsequent larval populations in summer. Heteronychus arator is a difficult pest to manage using chemical insecticides. This biopesticide with Y. entomophaga as the active ingredient offers a new solution for New Zealand pastures, with potential for application to other crops affected by H. arator and for control of other pests. © 2020 Society of Chemical Industry.

Development of a Yersinia entomophaga bait for control of larvae of the porina moth (Wiseana spp.), a pest of New Zealand improved grassland systems.

Porina is the common name for moths and larvae of the genus Wiseana (Lepidoptera: Hepialidae), some of which are significant pasture pests in New Zealand. Because of environmental concerns and the non-target effects of insecticide control measures, biological alternatives for the control of insect pests such as porina are required. Using a food preference assay and time-lapse photography, a range of low-cost food ingredients were assessed for their palatability to porina larvae. Lead candidates were combined into extruded bait variants, allowing assessment of their palatability to porina larvae. A composite bait consisting of palatable ingredients was developed, into which the porina-active entomopathogen Yersinia entomophaga was incorporated. A 7 day minimum median lethal dose of approximately 6.0× ±1× 106 Y. entomophaga cells per 0.02 g of bait was defined. Field trials showed that the mean change in larval density over time differed between treatments, with Y. entomophaga bait applied at 87 kg ha-1 resulting in a mean 65% reduction in larval density relative to the control plots, and diflubenzuron treatment resulting in a mean 77% reduction relative to the control plots. The mean dry matter yields over the course of the trial were highest for diflubenzuron (5029 kg ha-1 ), followed by the Y. entomophaga (4783 kg ha-1 ) and control (4673 kg ha-1 ) treatments. The bacterium Y. entomophaga applied as a composite bait offers an environmentally sustainable approach for porina pest control. © 2019 Society of Chemical Industry.

Cryo-EM structures of the pore-forming A subunit from the Yersinia entomophaga ABC toxin

ABC toxins are pore-forming virulence factors produced by pathogenic bacteria. YenTcA is the pore-forming and membrane binding A subunit of the ABC toxin YenTc, produced by the insect pathogen Yersinia entomophaga. Here we present cryo-EM structures of YenTcA, purified from the native source. The soluble pre-pore structure, determined at an average resolution of 4.4 Å, reveals a pentameric assembly that in contrast to other characterised ABC toxins is formed by two TcA-like proteins (YenA1 and YenA2) and decorated by two endochitinases (Chi1 and Chi2). We also identify conformational changes that accompany membrane pore formation by visualising YenTcA inserted into liposomes. A clear outward rotation of the Chi1 subunits allows for access of the protruding translocation pore to the membrane. Our results highlight structural and functional diversity within the ABC toxin subfamily, explaining how different ABC toxins are capable of recognising diverse hosts.

First isolation of Yersinia entomophaga in human urinary tract.

Yersinia entomophaga is an insect pathogen first isolated from larvae of Coleoptera in New Zealand in 2011. We report here the first isolation of Y. entomophaga from human urine. Using whole-genome sequencing, we confirmed the presence of specific chromosomal virulence genes and identified a plasmid harbouring a quinolone resistance gene.

Purification of the Yersinia entomophaga Yen-TC Toxin Complex Using Size Exclusion Chromatography.

The Yersinia entomophaga toxin complex (Yen-TC) is the bacterium's main virulence determinant. Because of its high insect activity, methods were developed to allow the routine isolation and purification of Yen-TC from an overnight bacterial culture using size exclusion chromatography. Here we outline an overnight purification procedure using a 100-ml culture volume, where approximately 2 mg of Yen-TC, with an approximate purity of 95-98 %, can be routinely obtained.

Temperature-Dependent Galleria mellonella Mortality as a Result of Yersinia entomophaga Infection.

The bacterium Yersinia entomophaga is pathogenic to a range of insect species, with death typically occurring within 2 to 5 days of ingestion. Per os challenge of larvae of the greater wax moth (Galleria mellonella) confirmed that Y. entomophaga was virulent when fed to larvae held at 25°C but was avirulent when fed to larvae maintained at 37°C. At 25°C, a dose of ~4 × 10(7) CFU per larva of a Y. entomophaga toxin complex (Yen-TC) deletion derivative, the Y. entomophaga ΔTC variant, resulted in 27% mortality. This low level of activity was restored to near-wild-type levels by augmentation of the diet with a sublethal dose of purified Yen-TC. Intrahemocoelic injection of ~3 Y. entomophaga or Y. entomophaga ΔTC cells per larva gave a 4-day median lethal dose, with similar levels of mortality observed at both 25 and 37°C. Following intrahemocoelic injection of a Yen-TC YenA1 green fluorescent protein fusion strain into larvae maintained at 25°C, the bacteria did not fluoresce until the population density reached 2 × 10(7) CFU ml(-1) of hemolymph. The observed cells also took an irregular form. When the larvae were maintained at 37°C, the cells were small and the observed fluorescence was sporadic and weak, being more consistent at a population density of ~3 × 10(9) CFU ml(-1) of hemolymph. These findings provide further understanding of the pathobiology of Y. entomophaga in insects, showing that the bacterium gains direct access to the hemocoelic cavity, from where it rapidly multiplies to cause disease.

Assessing the potential of <i>Yersinia entomophaga</i> to control plantain moth in a laboratory assay

The recent increased use of the highquality droughttolerant forage plant plantain (Plantago lanceolata) (Plantaginaceae) has been associated with outbreaks of native geometrid caterpillars such as Scopula rubraria The outbreaks most often occur under warm climatic conditions and within plantain monocrops Pest populations can reach 11500 larvae/m2 Stands can be severely damaged with 90 of plants destroyed and stand life expectancy reduced by 23 years This study assessed the efficacy of the insecticidal bacterium Yersinia entomophaga against S rubraria under laboratory conditions using a leaf history of S rubraria and the pathobiology of Y entomophaga suggest the bacterium could be developed as a biopesticide for use against S rubraria

Pathology of Yersinia entomophaga MH96 towards Costelytra zealandica (Coleoptera; Scarabaeidae) larvae

The bacterium Yersinia entomophaga was isolated from larvae of the New Zealand grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae), found in soil. Following ingestion of a lethal dose of bacteria, larvae of C. zealandica reduced feeding activity and movement. After approximately 4h infected larvae convulsed and regurgitated dark digestive fluid and expelled frass pellets leaving the midgut empty and the larva amber in appearance. In the initial stages of infection, ingested bacteria were mostly contained within the peritrophic membrane and expelled with the gut fluid or transferred into the hind gut. While few Y. entomophaga were associated with the midgut epithelial cells, by 24h cells were swelling and bursting with vesicles being expelled into the midgut lumen. By 48h, bacteria had entered the haemocoel and the midgut cells had further deteriorated. After 72h, the cellular remnants were totally detached from the basal membrane the infected insects were filled with bacteria and moribund or dead with septicaemia. Mortality was directly proportional to dose and time after infection. By applying a range of doses, the LD50 was determined as 2.9×104Y. entomophaga per C. zealandica larva, with an LT50 of 2.94days for doses of>1×105 per larva. Ingestion of low doses of bacteria did not inhibit feeding activity but led more slowly to death. By time of death, Y. entomophaga had multiplied, approximately 500 fold, in the cadavers of the infected larvae.

Laboratory bioassays of new synthetic and microbial insecticides to control Eucalyptus tortoise beetle <i>Paropsis charybdis</i>

Paropsis charybdis or eucalyptus tortoise beetle is (one of) the most significant pests of eucalypts in New Zealand It severely defoliates Eucalyptus nitens and Eucalyptus globulus plantations leaving heavilydamaged trees with reduced growth and poor form Laboratory bioassays of new synthetic and microbial insecticides were undertaken to identify management options compatible with biological control The current insecticide used for operational control alphacypermethrin was a positive control Of the products tested spinetoram (Sparta) gave the most promising results (100 mortality after 14 days) for control of adult P charybdis Spinetoram and spinosad (Success Naturalyte) the microbial control agents Beauveria bassiana (F305 ex Botanigard) and Bacillus thuringiensis var tenebrionis and the enterobacterium Yersinia entomophaga all resulted in high mortality of larval P charybdis after 4 days Targeting larvae alone may not be economically viable leaving spinetoram as the only possible additional insecticide for managing P charybdis Potential barriers of cost application and nontarget impacts are yet to be addressed

Histopathological effects of the Yen-Tc toxin complex from Yersinia entomophaga MH96 (Enterobacteriaceae) on the Costelytra zealandica (Coleoptera: Scarabaeidae) larval midgut.

Yersinia entomophaga MH96, which was originally isolated from the New Zealand grass grub, Costelytra zealandica, produces an orally active proteinaceous toxin complex (Yen-Tc), and this toxin is responsible for mortality in a range of insect species, mainly within the Coleoptera and Lepidoptera. The genes encoding Yen-Tc are members of the toxin complex (Tc) family, with orthologs identified in several other bacterial species. As the mechanism of Yen-Tc activity remains unknown, a histopathological examination of C. zealandica larvae was undertaken in conjunction with cultured cells to identify the effects of Yen-Tc and to distinguish the contributions that its individual subunit components make upon intoxication. A progressive series of events that led to the deterioration of the midgut epithelium was observed. Additionally, experiments using a cell culture assay system were carried out to determine the cellular effects of intoxication on cells after topical application and the transient expression of Yen-Tc and its individual components. While observations were broadly consistent with those previously reported for other Tc family members, some differences were noted. In particular, the distinct stepwise disintegration of the midgut shared features associated with both apoptosis and necrotic programmed cell death pathways. Second, we observed, for the first time, a contribution of toxicity from two chitinases associated with the Yen-Tc complex. Our findings were suggestive of the activities encoded within the subunit components of Yen-Tc targeting different sites along putative programmed cell death pathways. Given the observed broad host range for Yen-Tc, these targeted loci are likely to be widely shared among insects.

Survival of <i>Yersinia entomophaga</i> MH96 in a pasture ecosystem and effects on pest and nontarget invertebrate populations

Yersinia entomophaga MH96 (Ye MH96) has shown activity against the pasture pest porina (Wiseana spp larvae) in laboratory bioassays In this field trial Ye MH96 was applied to pasture as three separate formulations The presence of viable Ye MH96 was detected in soil for 112 days following application but the number of bacteria decreased rapidly following heavy rainfall 4 days after treatment Porina numbers were reduced by all formulations of Ye MH96 at 5 and 17 weeks (3440 and 6172) and by fenitrothion an insecticide used to control porina (93 and 96 respectively) Other nontarget Lepidoptera were also reduced by Ye MH96 No effects of Ye MH96 were observed on the earthworms Aporrectodea caliginosa and Lumbricus rubellus the pasture pest Listronotus bonariensis or pasture inhabiting Staphylinidae The survival of Ye MH96 for long enough to affect the target insect despite the heavy rain suggests that a microbial alternative to conventional insecticide management of porina may be possible

3D structure of the Yersinia entomophaga toxin complex and implications for insecticidal activity

Toxin complex (Tc) proteins are a class of bacterial protein toxins that form large, multisubunit complexes. Comprising TcA, B, and C components, they are of great interest because many exhibit potent insecticidal activity. Here we report the structure of a novel Tc, Yen-Tc, isolated from the bacterium Yersinia entomophaga MH96, which differs from the majority of bacterially derived Tcs in that it exhibits oral activity toward a broad range of insect pests, including the diamondback moth (Plutella xylostella). We have determined the structure of the Yen-Tc using single particle electron microscopy and studied its mechanism of toxicity by comparative analyses of two variants of the complex exhibiting different toxicity profiles. We show that the A subunits form the basis of a fivefold symmetric assembly that differs substantially in structure and subunit arrangement from its most well characterized homologue, the Xenorhabdus nematophila toxin XptA1. Histopathological and quantitative dose response analyses identify the B and C subunits, which map to a single, surface-accessible region of the structure, as the sole determinants of toxicity. Finally, we show that the assembled Yen-Tc has endochitinase activity and attribute this to putative chitinase subunits that decorate the surface of the TcA scaffold, an observation that may explain the oral toxicity associated with the complex.

Structural Analysis of Chi1 Chitinase from Yen-Tc: The Multisubunit Insecticidal ABC Toxin Complex of Yersinia entomophaga

Yersinia entomophaga MH96 is a native New Zealand soil bacterium that secretes a large ABC-type protein toxin complex, Yen-Tc, similar to those produced by nematode-associated bacteria such as Photorhabdus luminescens. Y. entomophaga displays an exceptionally virulent pathogenic phenotype in sensitive insect species, causing death within 72 h of infection. Because of this phenotype, there is intrinsic interest in the mechanism of action of Yen-Tc, and it also has the potential to function as a novel class of biopesticide. We have identified genes that encode chitinases as part of the toxin complex loci in Y. entomophaga MH96, P. luminescens, Photorhabdus asymbiotica and Xenorhabdus nematophila. Furthermore, we have shown that the secreted toxin complex from Y. entomophaga MH96 includes two chitinases as an integral part of the complex, a feature not described previously in other ABC toxins and possibly related to the severe disease caused by this bacterium. We present here the structure of the Y. entomophaga MH96 Chi1 chitinase, determined by X-ray crystallography to 1.74 Å resolution, and show that a ring of five symmetrically arranged lobes on the surface of the Yen-Tc toxin complex structure, as determined by single-particle electron microscopy, provides a good fit to the Chi1 monomer. We also confirm that the isolated chitinases display endochitinase activity, as does the complete toxin complex.

Effect of the bacterium <i>Yersinia entomophaga</i> on adult bronze beetle

The entomopathogenic bacterium Yersinia entomophaga MH96 (Ye MH96) was produced by aerobic fermentation and applied in laboratory and field bioassays to control bronze beetle (Eucolaspis sp) In the laboratory bacteria were applied to apple leaves at a range of cell densities using a Potter Tower After 6 days of exposure the LC50 of field collected beetles was calculated as approximately 50106 cells/cm2 of leaf surface with high concentrations killing all treated adults Time to death was inversely correlated with dose rate with the LT50 at the highest dose being 36 days Marked apple shoots were sprayed in the field each shoot enclosed in a sleeve cage and healthy beetles added After 7 days beetle mortality averaged 42 following a single application and 66 after multiple treatments Leaf damage was reduced by bacterial application The results show that Ye MH96 has potential for development as a biopesticide against this intractable pest of organic apple production