The importance of being adhesive

Phytonematodes cause annual crop losses of $100 billion per year. Pasteuria penetrans is a bacterium that has potential to be developed into a biological control agent as an alternative to nematicides. This poster is an overview of a collagen-like gene that is responsible for adhesion of the bacterium to the nematode's cuticle as the first step in the infection process.Non peer reviewedFinal Accepted Versio

The Molecular Basis of Pasteuria-nematode Interactions Using Closely Related Bacillus spp.

Phytonematodes are known to cause substantial losses in crop yields across the world. Since the middle of the last century, these pests have been adequately controlled by chemical nematicides. However, due to increasing public health concern, strict regulations in the EU and elsewhere have significantly reduced the usage of these environmentally not-so-safe chemicals. This has led us to look for reliable biological alternatives. The Pasteuria group of Gram-positive endospore-forming bacteria (phylum: Firmicutes) often associated with nematode-suppressive soils are potentially reliable nematode biocontrol agents. However, the highly specific interaction of Pasteuria to their nematode hosts poses a challenge to the management of heterogeneous populations of nematodes in the field; the mechanism behind this specificity remains unclear. One of the fundamental basis of host specificity is the attachment of Pasteuria endospores to the cuticle of their host nematodes which is the first and essential step in the infection process. Thus, understanding the molecular mechanisms that govern the attachment process is important in identifying suitable populations of Pasteuria for effective broad-range management of plant parasitic nematodes in soil. Previous studies suggest the presence of immunogenic collagen-like fibres and carbohydrates on the endospore coat of Pasteuria that may have a role in the initial interaction of the endospores with their nematode hosts. Published work on phylogeny relates Pasteuria to Bacillus spp. most of which have well annotated and characterized genomes while the genome of Pasteuria remains to be sequenced completely. In this thesis, I attempt to explore the endospore biology of obligate and fastidious Pasteuria spp. using the wide knowledgebase of well studied Bacillus endospores. The primary aim was to characterize the immunogeneic determinants that are possibly responsible for the attachment of Pasteuria endospores to the host nematode cuticle by a combination of computational and lab-based approaches. To approve the suggested phylogenetic closeness of Pasteuria to Bacillus, the first part of the study focused on phylogeny reconstruction of Pasteuria spp. amongst Bacillus spp. and other members of the phylum Firmicutes. This was followed by in silico studies to identify candidate collagen-like genes in P. penetrans; the putative functional proteins encoded by these candidate genes were then comparatively characterized with collagens from other organisms including the members of the genus Bacillus. The surface associated collagen-like proteins and other possible immunogens on the endospores of Pasteuria were characterized by protein immunoblotting, lectin blotting and immunofluorescence microscopy and comparisons were made with B. thuringiensis endospores. Lastly, endospore attachment assays were done to test the hypothesis that collagens and carbohydrates play a role in Pasteuria endospore attachment. The results of the computational analyses suggest a family of collagen coding putative genes in the Pasteuria genome, all of which are predicted to have varied biochemical properties and are seemingly of diverse evolutionary origin. The Western blot and microscopic analyses show that the endospores of P. penetrans and B. thuringiensis share some common immunodominant surface epitopes. The attachment assays confirm the involvement of collagens and at least one carbohydrate (N-acetylglucosamine) in the endospore attachment. However, the results also indicate possible involvement of other adhesins in the process; to support this, at the end of the thesis, I propose a new ‘Multitype Adhesin Model’ for initial interaction of Pasteuria endospores with the cuticle of their host nematodes. The outcomes of this project will help in identifying the molecular basis of the complex Pasteuria-nematode interaction. This will provide a basis to develop environmentally benign nematode bio-management strategies

Exploring Bacillus thuringiensis as a model for endospore adhesion and its potential to investigate adhesins in Pasteuria penetrans

© 2022 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology. https://creativecommons.org/licenses/by/4.0/Aims: Phytonematodes are a constraint on crop production and have been controlled using nematicides; these are highly toxic and legislation in Europe and elsewhere is prohibiting their use and alternatives are being sought. Pasteuria penetrans is a hyperparasitic bacterium that form endospores and have potential to control root‐knot nematodes (Meloidogyne spp.), but their attachment to the nematode cuticle is host‐specific. Understanding host specificity has relied upon endospore inhibition bioassays using immunological and biochemical approaches. Phylogenetic analysis of survey sequences has shown P. penetrans to be closely related to Bacillus and to have a diverse range of collagen‐like fibres which we hypothesise to be involved in the endospore adhesion. However, due to the obligately hyperparasitic nature of Pasteuria species, identifying and characterizing these collagenous‐like proteins through gain of function has proved difficult and new approaches are required.  Methods and Results: Using antibodies raised to synthetic peptides based on Pasteuria collagen‐like genes we show similarities between P. penetrans and the more easily cultured bacterium Bacillus thuringiensis and suggest it be used as a gain of function platform/model. Using immunological approaches similar proteins between P. penetrans and B. thuringiensis are identified and characterized, one >250 kDa and another ~72 kDa are glycosylated with N‐acetylglucosamine and both of which are digested if treated with collagenase. These treatments also affected endospore attachment and suggest these proteins are involved in adhesion of endospores to nematode cuticle.  Conclusion: There are conserved similarities in the collagen‐like proteins present on the surface of endospores of both P. penetrans and B. thuringiensis. Significance and Impact of Study: As B. thuringiensis is relatively easy to culture and can be transformed, it could be developed as a platform for studying the role of the collagen‐like adhesins from Pasteuria in endospore adhesion.Peer reviewedFinal Published versio

Understanding nematode suppressive soils: molecular interactions between Pasteuria endospores and the nematode surface coat

K. G. Davies, A. Srivastava, K. Kumar, and S. Mohan, ‘Understanding nematode suppressive soils: molecular interactions between Pasteuria endospores and the nematode surface coat’, Aspects of Applied Biology 130, 4th Symposium of Potato Cyst Nematode Management (including other nematode parasites of potatoes), pp. 143-147, September 2015.The knowledge that the plant-parasitic nematode hyperparasite Pasteuria penetrans is important in nematode suppressive soils has long been recognised. The ability to mass produce this organism in vitro circumvents one of its major constraints. However, successful biological control can only be established if the strains that are deployed can attach to and infect pest nematodes. Currently, in respect to the Pasteuria that infects root-knot nematodes (Meloidogyne spp), it is thought that collagen-like fibres on the surface of the endospore are interacting with a receptor on the nematode cuticle and that mucin-like molecules play an important role in modulating this process. Here we report that an antibody raised to whole endospores of P. penetrans also recognises extracts from endospores of Bacillus thuringiensis (Bt), suggesting that Bt can be used as a model for Pasteuria endospores. Bioinformatics shows that mucin-like genes identified in C. elegans are present in Globodera pallida.Final Accepted Versio

Exploiting genomics to improve the biological control potential of Pasteuria spp., an organisms with potential to control plant-parasitic nematodes

The Pasteuria group of Gram positive bacteria are invertebrate parasites with the potential to be developed into biological control agents of plant-parasitic nematodes. A key step in the infection process is the attachment of endospores to the cuticle of plant-parasitic nematodes, possibly through a Velcro-like attachment system involving the collagen-like fibres of the exosporium (Davies, 2009). Phylogenetically these bacteria are members of the Firmicutes and closely related to the members of genus Bacillus. Some of the genes involved in the construction of the endospore and in particular the exosporium in Bacillus spp. have already been identified. The Pasteuria sequences in the public databases and the complete genomes of Bacillus spp. were investigated for the genes linked with the endospore and associated exosporium. On the basis of our in silico studies we report the presence of genes putatively similar to bclA, exsJ and vrrB in Pasteuria.Non peer reviewedFinal Accepted Versio

Exploring the mechanisms of host-specificity of a hyperparasitic bacterium (Pasteuria spp.) with potential to control tropical root-knot nematodes (Meloidogyne spp.): insights from Caenorhabditis elegans

Plant-parasitic nematodes are important economic pests of a range of tropical crops. Strategies for managing these pests have relied on a range of approaches, including crop rotation, the utilization of genetic resistance, cultural techniques, and since the 1950’s the use of nematicides. Although nematicides have been hugely successful in controlling nematodes, their toxicity to humans, domestic animals, beneficial organisms, and the environment has raised concerns regarding their use. Alternatives are therefore being sought. The Pasteuria group of bacteria that form endospores has generated much interest among companies wanting to develop microbial biocontrol products. A major challenge in developing these bacteria as biocontrol agents is their host-specificity; one population of the bacterium can attach to and infect one population of plant-parasitic nematode but not another of the same species. Here we will review the mechanism by which infection is initiated with the adhesion of endospores to the nematode cuticle. To understand the genetics of the molecular processes between Pasteuria endospores and the nematode cuticle, the review focuses on the nature of the bacterial adhesins and how they interact with the nematode cuticle receptors by exploiting new insights gained from studies of bacterial infections of Carnorhabditis elegans. A new Velcro-like multiple adhesin model is proposed in which the cuticle surface coat, which has an important role in endospore adhesion, is a complex extracellular matrix containing glycans originating in seam cells. The genes associated with these seam cells appear to have a dual role by retaining some characteristics of stem cells

Evidence for diversifying selection of genetic regions of encoding putative collagen-like host-adhesive fibers in Pasteuria penetrans

© FEMS 2018. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.Pasteuria spp. belong to a group of genetically diverse endospore-forming bacteria (phylum: Firmicutes) that are known to parasitize plant-parasitic nematodes and water fleas (Daphnia spp.). Collagen-like fibres form the nap on the surface of endospores and the genes encoding these sequences have been hypothesised to be involved in the adhesion of the endospores of Pasteuria spp. to their hosts. We report a group of 17 unique collagen-like genes putatively encoded by Pasteuria penetrans (strain: Res148) that formed five different phylogenetic clusters and suggest that collagen-like proteins are an important source of genetic diversity in animal pathogenic Firmicutes including Pasteuria. Additionally, and unexpectedly, we identified a putative collagen-like sequence which had a very different sequence structure to the other collagen-like proteins but was similar to the protein sequences in Megaviruses that are involved in host-parasite interactions. We, therefore, suggest that these diverse endospore surface proteins in Pasteuria are involved in biological functions, such as cellular adhesion; however, they are not of monophyletic origin and were possibly obtained de novo by mutation or possibly through selection acting upon several historic horizontal gene transfer events.Peer reviewedFinal Published versio

Characterization of the putative antigenic determinants on Pasteuria endospore surface using Bacillus thuringiensis as a comparative tool

Arohi Srivastava, Sharad Mohan, Keith Davies, ‘Characterization of the putative antigenic determinants on Pasteuria endospore surface using Bacillus thuringiensis as a comparative tool’, poster presented at the European Society of Nematologists 32nd Symposium, Braga, Portugal, 28 August -2 September, 2016.Biological control agents are emerging as potential environmentally benign means of combating phytonematodes across the world. Some species of Pasteuria are endospore forming bacterial hyperparasites of nematodes associated with nematode suppressive soils. The prospective use of these bacteria in nematode management is well acknowledged by the scientific community. However, the mechanism by which Pasteuria endospores attach to the nematode cuticle and infect their nematode hosts is yet to be understood. Previously the involvement of parasporal collagen-like fibres present on the exosporium of Pasteuria endospores have been suggested as specificity determinants. Collagen-like fibres similar to Pasteuria parasporal fibres are known to exist and are well characterized on the exosporium of other closely related animal pathogenic Bacillus spp. Using comparative studies we explore the surface architecture and antigenic properties of Pasteuria endospores. Protein extracts from endospores of Bacillus thuringiensis and Pasteuria were compared using Western blot analysis. Antibodies raised against whole Pasteuria endospores and to synthetic peptides of Pasteuria collagen-like proteins showed cross-reactivity to endospore extracts B. thuringiensis. Further antigenic similarities in their exosporium have been confirmed by immunofluorescence microscopic studies where endospores from both the genera were labelled equally with Pasteuria ¬–specific antibodies. To further understand the determinants involved in the Pasteuria-nematode attachment process, Pasteuria endospores were treated with collagenase, β-N acetylglucosaminidase and anti-collagen antibodies and in vitro attachment assays were performed. We observed a marked decrease in the number of endospores attached to the nematode juvenilesNon peer reviewe

Exploring the endospore coat of Pasteuria penetrans, the bacterial hyperparasite of plant-parasitic nematodes, using Bacillus spp.

Arohi Srivastava, Tim H. Mauchline, Keith Davies, ‘Exploring the endospore coat of Pasteuria penetrans, the bacterial hyperparasite of plant-parasitic nematodes, using Bacillus spp.’, poster presented at the 7th European Spores Conference, Royal Holloway University of London, UK, 18-20 April, 2016.Pasteuria penetrans, previously named as Bacillus penetrans, belong to a group of Gram positive endospore forming bacteria (phylum: Firmicutes; family: Pasteuriaceae) that are hyperparasites of plant-parasitic nematodes. These bacteria could provide an environmentally benign and sustainable control strategy for economically important plant-parasitic nematodes as an alternative to nematicides. Earlier studies based on 16S rRNA, spoOA and other housekeeping genes suggest a close relatedness of Pasteuria to Bacillus. They are obligate parasites, but recently they have been mass produced by proprietary fermentation technology owned by Syngenta. The attachment of Pasteuria endospores to the cuticle of their host nematode is a crucial step in the infection process. The molecular structure of the Pasteuria endospore is little studied, although collagen-like fibres present on the spore surface are thought to be involved in spore attachment to its host. Some 4000+ genome survey sequences of Pasteuria are available in public databases, but there is not as yet a complete annotated genome. In the current study, we utilize well characterized endospore forming Bacillus spp. as comparative tools to investigate the surface of Pasteuria endospore. Sequenced genomes of animal pathogenic and non-pathogenic Bacillus spp. were used to identify selected genes related to endospore and exosporium formation and function. Synteny across the selected genes was explored using a web-based synteny explorer software SyntTax (Oberto, 2013). The genome survey sequences (GSS database, NCBI) of Pasteuria were interrogated for the presence of sequences homologous to selected Bacillus genes. In silico studies identified the presence of at least 10 collagen-like coding genes in the Pasteuria genome. Antibodies raised against Pasteuria endospores show cross-reactivity to proteins extracted from endospores of Bacillus thuringiensis following Western blot analyses. This indicates similarities in the endospore protein profile of Bacillus and Pasteuria. Likewise, subsequent immunofluorescence microscopic comparisons of Pasteuria and Bacillus thuringiensis endospores coats showed similarities in their antigenic determinants and further characterisation of these determinants suggested the involvement of carbohydrate moieties.Non peer reviewe