Thermal stable and proteinase-K resistant insecticidal toxins produced by Photorhabdus luminescens

Photorhabdus is lives in a mutualistic association with nematodes from the family Heterorhabditis. Bacteria of the Photorhabdus can survive independently and cause toxicity in a larger variety of insects. In the present study, insecticidal activity of non-portentous heat-stable metabolites of Photorhabdus luminescens was evaluated against Galleria mellonella. For this purpose, the culture extract of P. luminescens was injected into the G. mellonella larvae, which killed almost 90% of larvae within 48 h. The extract showed 100% insecticidal activity after heat treatment of 70 C for 30 min and even 60% and 40% activity lasted at 80 C and 90 C respectively. The extract also showed a high degree of thermal stability and was 100% actives after 60 min at 70 C. In addition, insecticidal activity was preserved up to 100% after all proteinase-K treatments (0 ?g/mL to 50 ?g/mL). The results revealed that the extracts were non-portentous and showed high thermal resistance and stability. Keywords: Photorhabdus, insecticidal activity, toxins, heat stable non-proteinaceous

Beauveria bassiana Ribotoxin (BbRib) Induces Silkworm Cell Apoptosis via Activating Ros Stress Response

The BbRib gene participates in the infection process of Beauveria bassiana (B. bassiana). It also helps pathogenic fungi to escape and defeat the insect host immune defense system by regulating the innate immune response. However, model insects are rarely used to study the mechanism of fungal ribosomal toxin protein. In this study, BbRib protein was produced by prokaryotic expression and injected into silkworm (Bombyx mori) larvae. The physiological and biochemical indexes of silkworm were monitored, and the pathological effects of BbRib protein on immune tissues of silkworm were examined by Hematoxylin and Eosin (HE) staining. BbRib protein can significantly affect the growth and development of the silkworm, causing poisoning, destroying the midgut and fat body and producing physiological changes. The ROS stress response in the adipose tissue and cells of the silkworm was activated to induce apoptosis. These results indicated that the BbRib gene not only participates in the infection process of B. bassiana, it also helps the pathogenic fungi escape the immune system by regulating the innate immune system of the silkworm, allowing it to break through the silkworm’s immune defense. This study reveals the potential molecular mechanism of BbRib protein to insect toxicity, and provides a theoretical basis and material basis for the development and use of novel insecticidal toxins.

Transition Phase Regulator AbrB Positively Regulates the sip1Ab1 Gene Expression in Bacillus thuringiensis

Bacillus thuringiensis Sip proteins are secreted insecticidal toxins that are toxic to coleopteran pests. In this study, we investigated the transcriptional mechanism of the sip gene and showed strong evidence that Sip1Ab1 is secreted in the transition phase and that AbrB, a transition phase regulator that is usually a repressor, positively and directly regulates sip1Ab1 .

Bacillus thuringiensis Bioinsecticides Induce Developmental Defects in Non-Target Drosophila melanogaster Larvae

Bioinsecticides made from the bacterium Bacillus thuringiensis (Bt) are the bestselling bioinsecticide worldwide. Among Bt bioinsecticides, those based on the strain Bt subsp. kurstaki (Btk) are widely used in farming to specifically control pest lepidopteran larvae. Although there is much evidence of the lack of acute lethality of Btk products for non-target animals, only scarce data are available on their potential non-lethal developmental adverse effects. Using a concentration that could be reached in the field upon sprayings, we show that Btk products impair growth and developmental time of the non-target dipteran Drosophila melanogaster. We demonstrate that these effects are mediated by the synergy between Btk bacteria and Btk insecticidal toxins. We further show that Btk bioinsecticides trigger intestinal cell death and alter protein digestion without modifying the food intake and feeding behavior of the larvae. Interestingly, these harmful effects can be mitigated by a protein-rich diet or by adding the probiotic bacterium Lactobacillus plantarum into the food. Finally, we unravel two new cellular mechanisms allowing the larval midgut to maintain its integrity upon Btk aggression: First the flattening of surviving enterocytes and second, the generation of new immature cells arising from the adult midgut precursor cells. Together, these mechanisms participate to quickly fill in the holes left by the dying enterocytes.

Rap-Phr Systems from Plasmids pAW63 and pHT8-1 Act Together To Regulate Sporulation in the Bacillus thuringiensis Serovar kurstaki HD73 Strain

ABSTRACT Bacillus thuringiensis is a Gram-positive spore-forming bacterium pathogenic to various insect species. This property is due to the Cry toxins encoded by plasmid genes and mostly produced during sporulation. B. thuringiensis contains a remarkable number of extrachromosomal DNA molecules and a great number of plasmid rap-phr genes. Rap-Phr quorum-sensing systems regulate different bacterial processes, notably the commitment to sporulation in Bacillus species. Rap proteins are quorum sensors acting as phosphatases on Spo0F, an intermediate of the sporulation phosphorelay, and are inhibited by Phr peptides that function as signaling molecules. In this study, we characterize the Rap63-Phr63 system encoded by the pAW63 plasmid from the B. thuringiensis serovar kurstaki HD73 strain. Rap63 has moderate activity on sporulation and is inhibited by the Phr63 peptide. The rap63-phr63 genes are cotranscribed, and the phr63 gene is also transcribed from a σH-specific promoter. We show that Rap63-Phr63 regulates sporulation together with the Rap8-Phr8 system harbored by plasmid pHT8_1 of the HD73 strain. Interestingly, the deletion of both phr63 and phr8 genes in the same strain has a greater negative effect on sporulation than the sum of the loss of each phr gene. Despite the similarities in the Phr8 and Phr63 sequences, there is no cross talk between the two systems. Our results suggest a synergism of these two Rap-Phr systems in the regulation of the sporulation of B. thuringiensis at the end of the infectious cycle in insects, thus pointing out the roles of the plasmids in the fitness of the bacterium. IMPORTANCE The life cycle of Bacillus thuringiensis in insect larvae is regulated by quorum-sensing systems of the RNPP family. After the toxemia caused by Cry insecticidal toxins, the sequential activation of these systems allows the bacterium to trigger first a state of virulence (regulated by PlcR-PapR) and then a necrotrophic lifestyle (regulated by NprR-NprX); ultimately, sporulation is controlled by the Rap-Phr systems. Our study describes a new rap-phr operon carried by a B. thuringiensis plasmid and shows that the Rap protein has a moderate effect on sporulation. However, this system, in combination with another plasmidic rap-phr operon, provides effective control of sporulation when the bacteria develop in the cadavers of infected insect larvae. Overall, this study highlights the important adaptive role of the plasmid Rap-Phr systems in the developmental fate of B. thuringiensis and its survival within its ecological niche.

Structural and Functional Insights into the C-terminal Fragment of Insecticidal Vip3A Toxin of Bacillus thuringiensis

The vegetative insecticidal proteins (Vips) secreted by Bacillus thuringiensis are regarded as the new generation of insecticidal toxins because they have different insecticidal properties compared with commonly applied insecticidal crystal proteins (Cry toxins). Vip3A toxin, representing the vast majority of Vips, has been used commercially in transgenic crops and bio-insecticides. However, the lack of both structural information on Vip3A and a clear understanding of its insecticidal mechanism at the molecular level limits its further development and broader application. Here we present the first crystal structure of the C-terminal fragment of Vip3A toxin (Vip3Aa11200–789). Since all members of this insecticidal protein family are highly conserved, the structure of Vip3A provides unique insight into the general domain architecture and protein fold of the Vip3A family of insecticidal toxins. Our structural analysis reveals a four-domain organization, featuring a potential membrane insertion region, a receptor binding domain, and two potential glycan binding domains of Vip3A. In addition, cytotoxicity assays and insect bioassays show that the purified C-terminal fragment of Vip3Aa toxin alone have no insecticidal activity. Taken together, these findings provide insights into the mode of action of the Vip3A family of insecticidal toxins and will boost the development of Vip3A into more efficient bio-insecticides.

Xenorhabdus bovienii strain jolietti uses a type 6 secretion system to kill closely related Xenorhabdus strains

ABSTRACT Xenorhabdus bovienii strain jolietti (XBJ) is a Gram-negative bacterium that interacts with several organisms as a part of its life cycle. It is a beneficial symbiont of nematodes, a potent pathogen of a wide range of soil-dwelling insects and also has the ability to kill soil- and insect-associated microbes. Entomopathogenic Steinernema nematodes vector XBJ into insects, releasing the bacteria into the insect body cavity. There, XBJ produce a variety of insecticidal toxins and antimicrobials. XBJ's genome also encodes two separate Type Six Secretion Systems (T6SSs), structures that allow bacteria to inject specific proteins directly into other cells, but their roles in the XBJ life cycle are mostly unknown. To probe the function of these T6SSs, we generated mutant strains lacking the key structural protein Hcp from each T6SS and assessed phenotypes related to different parts of XBJ's life cycle. Here we demonstrate that one of the T6SSs is more highly expressed in in vitro growth conditions and has antibacterial activity against other Xenorhabdus strains, and that the two T6SSs have a redundant role in biofilm formation.

Bacillus thuringiensis bioinsecticides induce developmental defects in non-target Drosophila melanogaster larvae

Bioinsecticides made from the bacterium Bacillus thuringiensis (Bt) are the best-selling bioinsecticide worldwide. Among Bt bioinsecticides, those based on the strain Bt var. kurstaki (Btk) are widely used in farming to specifically control pest lepidopteran larvae. Although there is much evidence of the lack of acute lethality of Btk products for non-target animals, only scarce data are available on their potential non-lethal developmental adverse effects. Using doses that could be reached in the field upon sprayings, we have shown that Btk products impair growth and developmental time of the non-target dipteran Drosophila melanogaster. These effects are mediated by the synergy between Btk bacteria and Btk insecticidal toxins, which induces a significant apoptosis of larval enterocytes, resulting in a decreased intestinal capacity to digest proteins. The harmful effects can be mitigated by a protein-rich diet or by adding the probiotic bacterium Lactobacillus plantarum into the food. Finally, we showed that the larval midgut maintain its integrity upon Btk aggression thanks to both the flattening of surviving enterocytes and the generation of new immature cells arising from the adult midgut precursor cells.

Structural insights into the insecticidal Vip3A toxin of Bacillus thuringiensis

The vegetative insecticidal proteins (Vips) secreted by Bacillus thuringiensis are regarded as the new generation of insecticidal toxins because they have different insecticidal properties compared with commonly applied insecticidal crystal proteins (Cry toxins). Vip3A toxin, representing the vast majority of Vips, has been used commercially in transgenic crops and bio-insecticides. However, the lack of both structural information of Vip3A and a clear understanding of its insecticidal mechanism at the molecular level, limits its further development and broader application. Here we present the first crystal structure of the Vip3A toxin in an activated form. Since all members of this insecticidal protein family are highly conserved, the structure of Vip3A provides unique insight into the general domain architecture and protein fold of the Vip3 family of insecticidal toxins. Our structural analysis reveals a four-domain organization, featuring a potential membrane insertion region, a receptor binding domain, and two glycan binding domains of activated Vip3A. We further identify the specific glycan moieties recognized by Vip3A through a glycan array screen. Taken together, these findings provide insights into the mode of action of Vip3 family of insecticidal toxins, and will boost the development of Vip3 into more efficient bio-insecticides.