Killing activity and rescue function of genome-wide toxin–antitoxin loci of Mycobacterium tuberculosis

Toxin–antitoxin (TA) loci are typically two-component systems that encode a stable toxin, which binds an essential host target leading to cell growth arrest and/or cell death, and an unstable antitoxin, which prevents the cytotoxic activity of the toxin. The ubiquitous presence of these loci in bacterial genomes, along with their demonstrated toxicity not only in the native but also in heterologous systems, has provided the possibility of their use in wide-spectrum antibacterials. Mycobacterium tuberculosis contains nearly 40 TA loci, most of which are yet to be characterized. Here we report the heterologous toxicity of these TA loci in Escherichia coli and show that only a few of the M. tuberculosis -encoded toxins can inhibit E. coli growth and have a killing effect. This killing effect can be suppressed by coexpression of the cognate antitoxin. This work has identified functional TA pairs for sequences that are presently unannotated in the mycobacterial genome. These toxins need to be further tested for their activity in the native host and other organism backgrounds and growth environments for utilization of their antibacterial potential.     

Potentiation and cellular phenotypes of the insecticidal Toxin complexes of Photorhabdus bacteria

The toxin complex (tc) genes of bacteria comprise a large and growing family whose mode of action remains obscure. In the insect pathogen Photorhabdus, tc genes encode high molecular weight insecticidal toxins with oral activity against caterpillar pests. One protein, TcdA, has recently been expressed in transgenic plants and shown to confer insect resistance. These toxins therefore represent alternatives to toxins from Bacillus thuringiensis (Bt) for deployment in transgenic crops. Levels of TcdA expression in transgenic plants were, however, low and the full toxicity associated with the native toxin was not reconstituted. Here we show that increased activity of the toxin TcdA1 requires potentiation by either of two pairs of gene products, TcdB1 and TccC1 or TcdB2 and TccC3. Moreover, these same pairs of proteins can also cross-potentiate a second toxin, TcaA1B1. To elucidate the likely functional domains present in these large proteins, we expressed fragments of each 'toxin' or 'potentiator' gene within mammalian cells. Several domains produced abnormal cellular morphologies leading to cell death, while others showed specific phenotypes such as nuclear translocation. Our results prove that the Tc toxins are complex proteins with multiple functional domains. They also show that both toxin genes and their potentiator pairs will need to be expressed to reconstitute full activity in insect-resistant transgenic plants. Moreover, they suggest that the same potentiator pair will be able to cross-potentiate more than one toxin in a single plant.     

Localization of Ptr ToxA Produced by Pyrenophora tritici-repentis Reveals Protein Import into Wheat Mesophyll Cells

The plant pathogenic fungus Pyrenophora tritici-repentis secretes host-selective toxins (HSTs) that function as pathogenicity factors. Unlike most HSTs that are products of enzymatic pathways, at least two toxins produced by P. tritici-repentis are proteins and, thus, products of single genes. Sensitivity to these toxins in the host is conferred by a single gene for each toxin. To study the site of action of Ptr ToxA (ToxA), toxin-sensitive and -insensitive wheat (Triticum aestivum) cultivars were treated with ToxA followed by proteinase K. ToxA was resistant to protease, but only in sensitive leaves, suggesting that ToxA is either protected from the protease by association with a receptor or internalized. Immunolocalization and green fluorescent protein tagged ToxA localization demonstrate that ToxA is internalized in sensitive wheat cultivars only. Once internalized, ToxA localizes to cytoplasmic compartments and to chloroplasts. Intracellular expression of ToxA by biolistic bombardment into both toxin-sensitive and -insensitive cells results in cell death, suggesting that the ToxA internal site of action is present in both cell types. However, because ToxA is internalized only in sensitive cultivars, toxin sensitivity, and therefore the ToxA sensitivity gene, are most likely related to protein import. The results of this study show that the ToxA protein is capable of crossing the plant plasma membrane from the apoplastic space to the interior of the plant cell in the absence of a pathogen.     

Viral induced yeast apoptosis

In an analogous system to mammals, induction of an apoptotic cell death programme (PCD) in yeast is not only restricted to various exogenous factors and stimuli, but can also be triggered by viral killer toxins and viral pathogens. In yeast, toxin secreting killer strains are frequently infected with double-stranded (ds)RNA viruses that are responsible for killer phenotype expression and toxin secretion in the infected host. In most cases, the viral toxins are either pore-forming proteins (such as K1, K2, and zygocin) that kill non-infected and sensitive yeast cells by disrupting cytoplasmic membrane function, or protein toxins (such as K28) that act in the nucleus by blocking DNA synthesis and subsequently causing a G1/S cell cycle arrest. Interestingly, while all these virus toxins cause necrotic cell death at high concentration, they trigger caspase- and ROS-mediated apoptosis at low-to-moderate concentration, indicating that even low toxin doses are deadly by triggering PCD in enemy cells. Remarkably, viral toxins are not solely responsible for cell death induction in vivo, as killer viruses themselves were shown to trigger apoptosis in non-infected yeast. Thus, as killer virus-infected and toxin secreting yeasts are effectively protected and immune to their own toxin, killer yeasts bear the intrinsic potential to dominate over time in their natural habitat.     

Cytolytic toxins as triggers of plant immune response

NEP1-like proteins (NLPs) are secreted proteins from fungi, oomycetes and bacteria, triggering immune responses and cell death in dicotyledonous plants. It has been unclear for a long time, whether NLPs are toxins or triggers of plant immunity. In a recent study we report that NLPs are toxins that exert cytolytic activity on dicotyledonous plants. Mutational analysis revealed a causal link between membrane damaging, cell death inducing and virulence promoting properties of NLPs. Interestingly, also induction of immune responses by NLPs required the same protein fold, providing evidence for damage-induced immunity in plants. Structural similarity to pore forming toxins from marine invertebrates allows the proposal of a model for the mode of NLP interaction with the host''s membrane.Key words: toxin, immunity, virulence, crystal structure, plant immunity, pathogen     

Detection of glycosylated and deglycosylated extensin precursors by indirect competitive ELISA

As part of their defense mechanism against herbivores or phytophagous insects, many plant tissues contain lectins. Some of these lectins are potent toxins which kill animal cells by arresting protein synthesis. An attractive strategy for developing specifically cytotoxic chemotherapeutic agents is to link cell type-specific monoclonal antibodies to potent toxins. The plant protein ricin has emerged as the toxin of choice for such constructs.     

Type II heat-labile enterotoxins from 50 diverse Escherichia coli isolates belong almost exclusively to the LT-IIc family and may be prophage encoded

Some enterotoxigenic Escherichia coli (ETEC) produce a type II heat-labile enterotoxin (LT-II) that activates adenylate cyclase in susceptible cells but is not neutralized by antisera against cholera toxin or type I heat-labile enterotoxin (LT-I). LT-I variants encoded by plasmids in ETEC from humans and pigs have amino acid sequences that are ≥ 95% identical. In contrast, LT-II toxins are chromosomally encoded and are much more diverse. Early studies characterized LT-IIa and LT-IIb variants, but a novel LT-IIc was reported recently. Here we characterized the LT-II encoding loci from 48 additional ETEC isolates. Two encoded LT-IIa, none encoded LT-IIb, and 46 encoded highly related variants of LT-IIc. Phylogenetic analysis indicated that the predicted LT-IIc toxins encoded by these loci could be assigned to 6 subgroups. The loci corresponding to individual toxins within each subgroup had DNA sequences that were more than 99% identical. The LT-IIc subgroups appear to have arisen by multiple recombinational events between progenitor loci encoding LT-IIc1- and LT-IIc3-like variants. All loci from representative isolates encoding the LT-IIa, LT-IIb, and each subgroup of LT-IIc enterotoxins are preceded by highly-related genes that are between 80 and 93% identical to predicted phage lysozyme genes. DNA sequences immediately following the B genes differ considerably between toxin subgroups, but all are most closely related to genomic sequences found in predicted prophages. Together these data suggest that the LT-II loci are inserted into lambdoid type prophages that may or may not be infectious. These findings raise the possibility that production of LT-II enterotoxins by ETEC may be determined by phage conversion and may be activated by induction of prophage, in a manner similar to control of production of Shiga-like toxins by converting phages in isolates of enterohemmorhagic E. coli.     

Against the mainstream: the membrane‐associated type I toxin BsrG from Bacillus subtilis interferes with cell envelope biosynthesis without increasing membrane permeability

Toxin‐antitoxin loci, which encode a toxic protein alongside with either RNA or a protein able to counteract the toxicity, are widespread among archaea and bacteria. These loci are implicated in persistence, and as addiction modules to ensure stable inheritance of plasmids and phages. In type I toxin‐antitoxin systems, a small RNA acts as an antitoxin, which prevents the synthesis of the toxin. Most type I toxins are small hydrophobic membrane proteins generally assumed to induce pores, or otherwise permeabilise the cytoplasmic membrane and, as a result, induce cell death by energy starvation. Here we show that this mode of action is not a conserved property of type I toxins. The analysis of the cellular toxicity caused by Bacillus subtilis prophage SPβ‐encoded toxin BsrG revealed that, surprisingly, it neither dissipates membrane potential nor affects cellular ATP‐levels. In contrast, BsrG strongly interferes with the cell envelope biosynthesis, causes membrane invaginations together with delocalisation of the cell wall synthesis machinery and triggers autolysis. Furthermore, efficient inhibition of protein biosynthesis is observed. These findings question the simplistic assumption that small membrane targeting toxins generally act by permeabilising the membrane.     

Regulation of growth and death in Escherichia coli by toxin-antitoxin systems

Escherichia coli K-12 contains at least 36 toxin genes, the expression of which causes growth inhibition and eventual death. These toxins are usually co-expressed with their cognate antitoxins in operons called toxin-antitoxin (TA) modules. Under normal growth conditions, toxins and antitoxins form stable complexes. However, stress-induced proteases preferentially eliminate unstable antitoxins, releasing free toxins to inhibit various cellular functions. TA systems have important roles in the physiology of cells in their natural habitats, including functions in biofilm formation and multidrug resistance. In this Review, we describe these TA systems in light of their functions and roles in the regulation of cell growth and death.     

Toxins-antitoxins: diversity,evolution and function

Genes for toxin-antitoxin (TA) complexes are widespread in prokaryote genomes, and species frequently possess tens of plasmid and chromosomal TA loci. The complexes are categorized into three types based on genetic organization and mode of action. The toxins universally are proteins directed against specific intracellular targets, whereas the antitoxins are either proteins or small RNAs that neutralize the toxin or inhibit toxin synthesis. Within the three types of complex, there has been extensive evolutionary shuffling of toxin and antitoxin genes leading to considerable diversity in TA combinations. The intracellular targets of the protein toxins similarly are varied. Numerous toxins, many of which are sequence-specific endoribonucleases, dampen protein synthesis levels in response to a range of stress and nutritional stimuli. Key resources are conserved as a result ensuring the survival of individual cells and therefore the bacterial population. The toxin effects generally are transient and reversible permitting a set of dynamic, tunable responses that reflect environmental conditions. Moreover, by harboring multiple toxins that intercede in protein synthesis in response to different physiological cues, bacteria potentially sense an assortment of metabolic perturbations that are channeled through different TA complexes. Other toxins interfere with the action of topoisomersases, cell wall assembly, or cytoskeletal structures. TAs also play important roles in bacterial persistence, biofilm formation and multidrug tolerance, and have considerable potential both as new components of the genetic toolbox and as targets for novel antibacterial drugs.     

Toxins-antitoxins: diversity, evolution and function

Genes for toxin-antitoxin (TA) complexes are widespread in prokaryote genomes, and species frequently possess tens of plasmid and chromosomal TA loci. The complexes are categorized into three types based on genetic organization and mode of action. The toxins universally are proteins directed against specific intracellular targets, whereas the antitoxins are either proteins or small RNAs that neutralize the toxin or inhibit toxin synthesis. Within the three types of complex, there has been extensive evolutionary shuffling of toxin and antitoxin genes leading to considerable diversity in TA combinations. The intracellular targets of the protein toxins similarly are varied. Numerous toxins, many of which are sequence-specific endoribonucleases, dampen protein synthesis levels in response to a range of stress and nutritional stimuli. Key resources are conserved as a result ensuring the survival of individual cells and therefore the bacterial population. The toxin effects generally are transient and reversible permitting a set of dynamic, tunable responses that reflect environmental conditions. Moreover, by harboring multiple toxins that intercede in protein synthesis in response to different physiological cues, bacteria potentially sense an assortment of metabolic perturbations that are channeled through different TA complexes. Other toxins interfere with the action of topoisomersases, cell wall assembly, or cytoskeletal structures. TAs also play important roles in bacterial persistence, biofilm formation and multidrug tolerance, and have considerable potential both as new components of the genetic toolbox and as targets for novel antibacterial drugs.     

Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans

The protein toxins produced by Bacillus thuringiensis (Bt) are the most widely used natural insecticides in agriculture. Despite successful and extensive use of these toxins in transgenic crops, little is known about toxicity and resistance pathways in target insects since these organisms are not ideal for molecular genetic studies. To address this limitation and to investigate the potential use of these toxins to control parasitic nematodes, we are studying Bt toxin action and resistance in Caenorhabditis elegans. We demonstrate for the first time that a single Bt toxin can target a nematode. When fed Bt toxin, C. elegans hermaphrodites undergo extensive damage to the gut, a decrease in fertility, and death, consistent with toxin effects in insects. We have screened for and isolated 10 recessive mutants that resist the toxin's effects on the intestine, on fertility, and on viability. These mutants define five genes, indicating that more components are required for Bt toxicity than previously known. We find that a second, unrelated nematicidal Bt toxin may utilize a different toxicity pathway. Our data indicate that C. elegans can be used to undertake detailed molecular genetic analysis of Bt toxin pathways and that Bt toxins hold promise as nematicides.     

Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives

A large number of plant and bacterial toxins with enzymatic activity on intracellular targets are now known. These toxins enter cells by first binding to cell surface receptors, then they are endocytosed and finally they become translocated into the cytosol from an intracellular compartment. In the case of the plant toxin ricin and the bacterial toxin Shiga toxin, this happens after retrograde transport through the Golgi apparatus and to the endoplasmic reticulum. The toxins are powerful tools to reveal new pathways in intracellular transport. Furthermore, knowledge about their action on cells can be used to combat infectious diseases where such toxins are involved, and a whole new field of research takes advantage of their ability to enter the cytosol for therapeutic purposes in connection with a variety of diseases. This review deals with the mechanisms of entry of ricin and Shiga toxin, and the attempts to use such toxins in medicine are discussed.     

Secondary metabolite toxins and nutrition of plant pathogenic fungi

Fungal pathogens derive nutrition from the plants they invade. Some fungi can subvert plant defence responses such as programmed cell death to provide nutrition for their growth and colonisation. Secondary metabolite toxins produced by fungi often play a role in triggering these responses. Knowledge of the biosynthesis of these toxins, and the availability of fungal genome sequences and gene disruption techniques, allows the development of tools for experiments aimed at discovering the role of such toxins in triggering plant cell death and plant disease.     

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

ABSTRACT: 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 conflict. 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 in intra-specific and inter-specific interactions between bacteria. These systems provide insights regarding the emergence of key systems at different points in eukaryotic evolution, such as ADP ribosylation, interaction of myosin VI with cargo proteins, mediation of apoptosis, hyphal heteroincompatibility, hedgehog signaling, arthropod toxins, cell-cell interaction molecules like teneurins and different signaling messengers.     

Modification of plant Rac/Rop GTPase signalling using bacterial toxin transgenes

Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co‐expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive‐like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co‐expressed constitutively active Rop4, blocking the hypersensitive response caused by over‐expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop‐dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop‐dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.     

球形芽孢杆菌杀蚊毒素蛋白及其 遗传操作研究进展

蚊虫是多种人类传染疾病的主要传播媒介,如疟疾、丝虫病、乙型脑炎、黄热病和登革热等,对人类的健康造成了极大的危害［１］。控制蚊虫被认为是消除这些蚊媒疾病的有效途径。在过去的４５年里,尽管化学杀虫剂和各种抗病药物的使用对降低疟疾和蚊媒疾病的发病率和死亡率...     

Small Toxic Proteins and the Antisense RNAs That Repress Them

Summary: There has been a great expansion in the number of small regulatory RNAs identified in bacteria. Some of these small RNAs repress the synthesis of potentially toxic proteins. Generally the toxin proteins are hydrophobic and less than 60 amino acids in length, and the corresponding antitoxin small RNA genes are antisense to the toxin genes or share long stretches of complementarity with the target mRNAs. Given their short length, only a limited number of these type I toxin-antitoxin loci have been identified, but it is predicted that many remain to be found. Already their characterization has given insights into regulation by small RNAs, has suggested functions for the small toxic proteins at the cell membrane, and has led to practical applications for some of the type I toxin-antitoxin loci.     

Conjugation of folate via gelonin carbohydrate residues retains ribosomal-inactivating properties of the toxin and permits targeting to folate receptor positive cells

Conjugation of folate to proteins permits receptor-mediated endocytosis via the folate receptor (FR) and delivery of the conjugate into the cytoplasm of cells. Since many cancers up-regulate the FR it has enabled the targeting of toxins to tumor cells resulting in specific cell death. However, current conjugation methods rely on chemistries that can affect certain catalytic subunits, such as the A-chain of the plant toxin gelonin. As a result many folate-targeted toxins are a compromise between receptor/ligand interaction and toxin activity. We describe the first example of folate conjugated to a protein via carbohydrate residues, using a novel SH-folate intermediate. The folate-gelonin conjugate retains over 99% of toxin activity in a cell-free translational assay compared with unmodified gelonin and is able to bind the FR at the same affinity as free folic acid (10(-10) m). Additionally, the conjugate exhibits prolonged inhibition of protein synthesis in FR positive cell lines in vitro. Folate linked to gelonin via amino conjugation exhibits the same affinity for FR as free folic acid but the toxin is 225-fold less active in a cell-free translational assay. The effect of different conjugation methods on toxin activity and the implications for folate targeting of other glycoproteins are discussed.     

Retrograde transport of protein toxins through the Golgi apparatus

A number of protein toxins from plants and bacteria take advantage of transport through the Golgi apparatus to gain entry into the cytosol where they exert their action. These toxins include the plant toxin ricin, the bacterial Shiga toxins, and cholera toxin. Such toxins bind to lipids or proteins at the cell surface, and they are endocytosed both by clathrin-dependent and clathrin-independent mechanisms. Sorting to the Golgi and retrograde transport to the endoplasmic reticulum (ER) are common to these toxins, but the exact mechanisms turn out to be toxin and cell-type dependent. In the ER, the enzymatically active part is released and then transported into the cytosol, exploiting components of the ER-associated degradation system. In this review, we will discuss transport of different protein toxins, but we will focus on factors involved in entry and sorting of ricin and Shiga toxin into and through the Golgi apparatus.