Through transposon mutagenesis and DNA sequence analysis, the main disease determinant of the entomopathogenic bacterium Yersinia entomophaga MH96 was localized to an ϳ32-kb pathogenicity island (PAI) designated PAI Ye96 . Residing within PAI Ye96 are seven open reading frames that encode an insecticidal toxin complex (TC), comprising not only the readily recognized toxin complex A (TCA), TCB, and TCC components but also two chitinase proteins that form a composite TC molecule. The central TC gene-associated region (ϳ19 kb) of PAI Ye96 was deleted from the Y. entomophaga MH96 genome, and a subsequent bioassay of the ⌬TC derivative toward Costelytra zealandica larvae showed it to be innocuous. Virulence of the ⌬TC mutant strain could be restored by the introduction of a clone containing the entire PAI Ye96 TC gene region. As much as 0.5 mg of the TC is released per 100 ml of Luria-Bertani broth at 25°C, while at 30 or 37°C, no TC could be detected in the culture supernatant. Filter-sterilized culture supernatants derived from Y. entomophaga MH96, but not from the ⌬TC strain grown at temperatures of 25°C or less, were able to cause mortality. The 50% lethal doses (LD 50 s) of the TC toward diamondback moth Plutella xylostella and C. zealandica larvae were defined as 30 ng and 50 ng, respectively, at 5 days after ingestion. Histological analysis of the effect of the TC toward P. xylostella larva showed that within 48 h after ingestion of the TC, there was a general dissolution of the larval midgut.Toxin complexes (TCs) active on insects were first identified in the nematode-associated bacterium Photorhabdus luminescens and termed TCs, as three proteins combined to form a complex with insecticidal activity (5). TC toxins were subsequently identified in the genome of Serratia entomophila where they reside in the designated gene order sepA, sepB, and sepC (33); this toxin complex ABC designation defines the revised nomenclature of the TC proteins (25). The TC toxins derived from P. luminescens reside as multiple but dissimilar orthologues throughout the P. luminescens T011 genome (22), and different insecticidal activities may be attributed to a different TC cluster (32). The S. entomophila sepABC genes are plasmid borne, and their translated products are host specific, only causing amber disease in larvae of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) (33). TC-like toxins have since been identified in the genome of Xenorhabdus nematophilus (60), Pseudomonas syringae pv. tomato DC3000 (9), and some Yersinia species. The toxin complex A (TCA)-like (tcaB) gene of Yersinia pestis CO92 contains a frameshift mutation, and the toxin complex B (TCB)-like (tcaC) gene contains an internal deletion (51), indicative of a loss of function, while the corresponding TCA-like and TCBlike orthologues in Y. pestis KIM and 91001 do not (18, 62). Tennant et al. (66) showed that mutations in each of the Yersinia enterocolitica biotype 1A T83 genes, TCA-like (tcbA), TCB-like (tcaC), and TCC-like (tccC) gen...

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.

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.

A highly virulent strain, AGR96X, exhibiting specific pathogenicity against larvae of the New Zealand grass grub (; Coleoptera: Scarabaeidae) and the New Zealand manuka beetle ( and ; Coleoptera: Scarabaeidae), was isolated from a diseased grass grub larva. A 12-day median lethal dose of 4.89 × 10 ± 0.92 × 10 cells per grass grub larva was defined for AGR96X, and death occurred within 5 to 12 days following the ingestion of a high bacterial dose. During the infection period, the bacterium rapidly multiplied within the insect host and invaded the hemocoel, leading to a mean bacterial load of 8.2 × 10 cells per larva at 6 days postingestion. Genome sequencing of strain AGR96X revealed the presence of a variant of the antifeeding prophage (Afp), a tailocin designated AfpX. Unlike Afp, AfpX contains two Afp16 tail-length termination protein orthologs and two putative toxin components. A 37-kb DNA fragment encoding the AfpX-associated region was cloned, transformed into, and fed to and larvae, causing mortality. In addition, the deletion of the putative chaperone component abolished the virulence of AGR96X. Unlike Afp, the AfpX tailocin could be induced by mitomycin C. Transmission electron microscopy analysis revealed the presence of Afp-like particles of various lengths, and when the purified AfpX tailocin was fed to grass grub or manuka beetle larvae, they underwent phenotypic changes similar to those of larvae fed AGR96X. strain AGR96X shows dual activity against larvae of endemic New Zealand pasture pests, the grass grub () and the manuka beetle ( spp.). Unlike , the causal agent of amber disease, which takes 3 to 4 months to kill grass grub larvae, AGR96X causes mortality within 5 to 12 days of ingestion and invades the insect hemocoel. AGR96X produces a unique variant of the antifeeding prophage (Afp), a cell-free phage-like entity that is proposed to deliver protein toxins to the grass grub target site, causing a cessation of feeding activity. Unlike other Afp variants, AGR96X Afp, named AfpX, contains two tail-length termination proteins, resulting in greater variability in the AfpX length. AfpX shows dual activity against both grass grub and manuka beetle larvae. AGR96X is a viable alternative to for pest control in New Zealand pasture systems.

No abstract

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 ϳ4 ؋ 10 7 CFU per larva of a Y. entomophaga toxin complex (Yen-TC) deletion derivative, the Y. entomophaga ⌬TC 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 ϳ3 Y. entomophaga or Y. entomophaga ⌬TC 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 ϳ3 ؋ 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. The Gram-negative bacterium Yersinia entomophaga was isolated from the cadaver of a Costelytra zealandica (Coleoptera: Scarabaeidae) larva, and has proven consistently pathogenic by per os challenge to this host, as well as to a wide range of coleopteran, lepidopteran, and orthopteran species (1). A 6-day median lethal dose (LD 50 ) was determined as 2.9 ϫ 10 4 Y. entomophaga cells per C. zealandica larva. Following per os challenge with a bacterial dose of ϳ1 ϫ 10 7 Y. entomophaga cells per larva, the larvae typically vomited, expelled frass pellets, changed color from a healthy gray to amber and then a moribund brown, and exhibited reduced feeding activity. By 48 h postingestion, Y. entomophaga cells could be visualized within the hemocoelic cavity of the larvae, after which time death ensued (2).The main virulence determinant of Y. entomophaga is an insect-active toxin complex (TC) derivative termed "Yen-TC" (3). TCs were first identified in the genome of Photorhabdus luminescens (4) and have since been identified in other bacterial genera, including members of the genus Yersinia (5). Typically, TCs are comprised of three proteins, TC-A, TC-B, and TC-C, which combine to form the insect-active complex (6). Yen-TC is comprised of seven subunit proteins: two TC-A-like proteins (YenA1 and YenA2), a TC-B-like protein (YenB), two TC-C-like proteins (YenC1 and YenC2), and two chitinases (Chi1 and Chi2), which combine to form the insect-active TC. Recent structural analysis has revealed that both the Yen-TC and the P. lumine...

The effect of supplementation with substances having antioxidant properties on the adaptive responses of human skin fibroblasts to UV-induced oxidative stress was studied in vitro. UVR was found to induce a substantial oxidative stress in fibroblasts, resulting in an increased release of superoxide anions and an increase in lipid peroxidation (shown by an elevated malonaldehyde content). Sub-lethal doses of UVR were also found to induce adaptive responses in the fibroblast antioxidant defences, with a transient rise in catalase and superoxide dismutase activities followed by a slower, large increase in cellular glutathione content. Supplementation of the fibroblasts with the antioxidants, Trolox (a water soluble analogue of alpha-tocopherol), ascorbic acid or beta-carotene, had differential effects on these responses. Trolox supplementation reduced the UVR-induced cellular oxidative stress and adaptive response in a predictable concentration-dependent manner. This was in contrast to ascorbic acid which increased superoxide release from fibroblasts. At low doses, ascorbate supplements also reduced the magnitude of the adaptive increases in catalase and superoxide dismutase activities and increase in glutathione content. Beta-carotene had a similar effect to ascorbic acid, reducing the extent of the adaptations to UVR at lower doses while simultaneously increasing superoxide release and malonaldehyde content. These in vitro data indicate that only the vitamin E analogue suppressed UVR-induced oxidative stress in a predictable manner and suggest that common dietary antioxidants may not be equally effective in reducing the potential deleterious effects of UVR-induced oxidative stress in skin.

No abstract