Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics

Biology Direct Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics Dapeng Zhang 0 Robson F de Souza 0 1 Vivek Anantharaman 0 Lakshminarayan M Iyer 0 L Aravind 0 0 National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health , Bethesda, MD 20894 , USA 1 Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo , Sao Paulo , Brazil Background: Proteinaceous toxins are observed across all levels of inter-organismal and intra-genomic conflicts. These include recently discovered prokaryotic polymorphic toxin systems implicated in intra-specific conflicts. They are characterized by a remarkable diversity of C-terminal toxin domains generated by recombination with standalone toxin-coding cassettes. Prior analysis revealed a striking diversity of nuclease and deaminase domains among the toxin modules. We systematically investigated polymorphic toxin systems using comparative genomics, sequence and structure analysis. Results: Polymorphic toxin systems are distributed across all major bacterial lineages and are delivered by at least eight distinct secretory systems. In addition to type-II, these include type-V, VI, VII (ESX), and the poorly characterized Photorhabdus virulence cassettes (PVC), PrsW-dependent and MuF phage-capsid-like systems. We present evidence that trafficking of these toxins is often accompanied by autoproteolytic processing catalyzed by HINT, ZU5, PrsW, caspase-like, papain-like, and a novel metallopeptidase associated with the PVC system. We identified over 150 distinct toxin domains in these systems. These span an extraordinary catalytic spectrum to include 23 distinct clades of peptidases, numerous previously unrecognized versions of nucleases and deaminases, ADP-ribosyltransferases, ADP ribosyl cyclases, RelA/SpoT-like nucleotidyltransferases, glycosyltranferases and other enzymes predicted to modify lipids and carbohydrates, and a pore-forming toxin domain. Several of these toxin domains are shared with host-directed effectors of pathogenic bacteria. Over 90 families of immunity proteins might neutralize anywhere between a single to at least 27 distinct types of toxin domains. In some organisms multiple tandem immunity genes or immunity protein domains are organized into polyimmunity loci or polyimmunity proteins. Gene-neighborhood-analysis of polymorphic toxin systems predicts the presence of novel trafficking-related components, and also the organizational logic that allows toxin diversification through recombination. Domain architecture and protein-length analysis revealed that these toxins might be deployed as secreted factors, through directed injection, or via inter-cellular contact facilitated by filamentous structures formed by RHS/YD, filamentous hemagglutinin and other repeats. Phyletic pattern and life-style analysis indicate that polymorphic toxins and polyimmunity loci participate in cooperative behavior and facultative 'cheating' in several ecosystems such as the human oral cavity and soil. Multiple domains from these systems have also been repeatedly transferred to eukaryotes and their viruses, such as the nucleo-cytoplasmic large DNA viruses. Conclusions: Along with a comprehensive inventory of toxins and immunity proteins, we present several testable predictions regarding active sites and catalytic mechanisms of toxins, their processing and trafficking and their role - Background Production and deployment of chemical armaments is one of the most common strategies in inter-organismal conflict. Such molecules, namely toxins or antibiotics, are observed at practically every level of biological organization ranging from multicellular organisms like animals and plants, through bacteria, all the way down to intra-genomic selfish elements [1-4]. These molecules span an entire biochemical spectrum from diffusible small molecules (e.g. antibiotics) to some of the largest proteins in the biological world (secreted bacterial toxins) [5,6]. Beyond their natural roles, these molecules have considerable significance as biotechnological reagents, biodefense agents, therapeutic targets, and therapeutics against numerous disease-causing agents [1,2,4,6,7]. Traditional toxicology has now been joined by genomics and sequence analysis in uncovering the enormous biochemical diversity across life forms of such molecules and of the systems that synthesize and traffic them. This diversity is seen both in the structure and action of systems involved in synthesis of diffusible antibiotics and proteinaceous toxins [5,6]. It is becoming increasingly clear that proteinaceous toxins are a common feature of biological conflicts at every organizational level [7]: 1) In antagonistic interactions between different multicellular eukaryotes, such as the castor bean ricin, Aspergillus sarcin and various snake venom proteins [2,3,8,9]. 2) Action by multicellular organisms against their pathogens (e.g. anti-microbial peptide toxins and defensive RNases such as RNaseA and RNase L [10-13]). 3) Action of pathogenic and symbiotic bacteria directed against their hosts (e.g. the cholera toxin and the shiga toxin [4,14]). 4) Interspecific conflict in bacteria [15]. 5) Conflict between bacterial sibling strains of the same species, namely contact dependent inhibition systems and related secreted toxins [16-19]. 6) Inter-genomic conflicts between cellular genomes and selfish replicons residing in the same cell (e.g. classical bacteriocins and plasmid addiction toxins [20]). 7) Intra-genomic conflicts between selfish elements and the host genome (restriction-modification systems [21] and genomic toxin-antitoxin systems [22-24]). Studies in the past decade are pointing to certain unifying themes across the proteinaceous toxins deployed in each of these distinct types of biological conflict. The most prominent theme is the use of enzymatic toxins that disrupt the flow of biological information by targeting nucleic acids and proteins [7]. Thus, several toxin domains are nucleases targeting genomic DNA, tRNAs and rRNAs, nucleic acid base glycosylases, nucleic acidmodifying enzymes, peptidases that cleave key protein targets, and protein-modifying enzymes that alter the properties of proteins, such as components of the translation apparatus [4,6,7,17,18,25]. A secondary theme seen across toxins from phylogenetically diverse sources is the presence of domains that disrupt cellular integrity by forming pores in cellular membranes [26,27]. Genomic analysis has also revealed that the richest source of proteinaceous toxins is the bacterial superkingdom, wherein several systems involved in most of the levels of biological conflict enumerated above are encountered [4,6,17,18,21,22,25]. It is also becoming apparent that inter- and intra- specific and inter- and intra- genomic conflicts in prokaryotes ha (...truncated)