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1.  New Players in the Toxin Field: Polymorphic Toxin Systems in Bacteria 
mBio  2015;6(3):e00285-15.
Bacteria have evolved numerous strategies to increase their competitiveness and fight against each other. Indeed, a large arsenal of antibacterial weapons is available in order to inhibit the proliferation of competitor cells. Polymorphic toxin systems (PTS), recently identified by bioinformatics in all major bacterial lineages, correspond to such a system primarily involved in conflict between related bacterial strains. They are typically composed of a secreted multidomain toxin, a protective immunity protein, and multiple cassettes encoding alternative toxic domains. The C-terminal domains of polymorphic toxins carry the toxic activity, whereas the N-terminal domains are related to the trafficking mode. In silico analysis of PTS identified over 150 distinct toxin domains, including putative nuclease, deaminase, or peptidase domains. Immunity genes found immediately downstream of the toxin genes encode small proteins that protect bacteria against their own toxins or against toxins secreted by neighboring cells. PTS encompass well-known colicins and pyocins, contact-dependent growth inhibition systems which include CdiA and Rhs toxins and some effectors of type VI secretion systems. We have recently characterized the MafB toxins, a new family of PTS deployed by pathogenic Neisseria spp. Many other putative PTS have been identified by in silico predictions but have yet to be characterized experimentally. However, the high number of these systems suggests that PTS have a fundamental role in bacterial biology that is likely to extend beyond interbacterial competition.
PMCID: PMC4436062  PMID: 25944858
2.  Evolution of the deaminase fold and multiple origins of eukaryotic editing and mutagenic nucleic acid deaminases from bacterial toxin systems 
Nucleic Acids Research  2011;39(22):9473-9497.
The deaminase-like fold includes, in addition to nucleic acid/nucleotide deaminases, several catalytic domains such as the JAB domain, and others involved in nucleotide and ADP-ribose metabolism. Using sensitive sequence and structural comparison methods, we develop a comprehensive natural classification of the deaminase-like fold and show that its ancestral version was likely to operate on nucleotides or nucleic acids. Consequently, we present evidence that a specific group of JAB domains are likely to possess a DNA repair function, distinct from the previously known deubiquitinating peptidase activity. We also identified numerous previously unknown clades of nucleic acid deaminases. Using inference based on contextual information, we suggest that most of these clades are toxin domains of two distinct classes of bacterial toxin systems, namely polymorphic toxins implicated in bacterial interstrain competition and those that target distantly related cells. Genome context information suggests that these toxins might be delivered via diverse secretory systems, such as Type V, Type VI, PVC and a novel PrsW-like intramembrane peptidase-dependent mechanism. We propose that certain deaminase toxins might be deployed by diverse extracellular and intracellular pathogens as also endosymbionts as effectors targeting nucleic acids of host cells. Our analysis suggests that these toxin deaminases have been acquired by eukaryotes on several independent occasions and recruited as organellar or nucleo-cytoplasmic RNA modifiers, operating on tRNAs, mRNAs and short non-coding RNAs, and also as mutators of hyper-variable genes, viruses and selfish elements. This scenario potentially explains the origin of mutagenic AID/APOBEC-like deaminases, including novel versions from Caenorhabditis, Nematostella and diverse algae and a large class of fast-evolving fungal deaminases. These observations greatly expand the distribution of possible unidentified mutagenic processes catalyzed by nucleic acid deaminases.
PMCID: PMC3239186  PMID: 21890906
3.  A novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and DNA viral systems 
Nucleic Acids Research  2011;39(11):4532-4552.
The use of nucleases as toxins for defense, offense or addiction of selfish elements is widely encountered across all life forms. Using sensitive sequence profile analysis methods, we characterize a novel superfamily (the SUKH superfamily) that unites a diverse group of proteins including Smi1/Knr4, PGs2, FBXO3, SKIP16, Syd, herpesviral US22, IRS1 and TRS1, and their bacterial homologs. Using contextual analysis we present evidence that the bacterial members of this superfamily are potential immunity proteins for a variety of toxin systems that also include the recently characterized contact-dependent inhibition (CDI) systems of proteobacteria. By analyzing the toxin proteins encoded in the neighborhood of the SUKH superfamily we predict that they possess domains belonging to diverse nuclease and nucleic acid deaminase families. These include at least eight distinct types of DNases belonging to HNH/EndoVII- and restriction endonuclease-fold, and RNases of the EndoU-like and colicin E3-like cytotoxic RNases-folds. The N-terminal domains of these toxins indicate that they are extruded by several distinct secretory mechanisms such as the two-partner system (shared with the CDI systems) in proteobacteria, ESAT-6/WXG-like ATP-dependent secretory systems in Gram-positive bacteria and the conventional Sec-dependent system in several bacterial lineages. The hedgehog-intein domain might also release a subset of toxic nuclease domains through auto-proteolytic action. Unlike classical colicin-like nuclease toxins, the overwhelming majority of toxin systems with the SUKH superfamily is chromosomally encoded and appears to have diversified through a recombination process combining different C-terminal nuclease domains to N-terminal secretion-related domains. Across the bacterial superkingdom these systems might participate in discriminating `self’ or kin from `non-self’ or non-kin strains. Using structural analysis we demonstrate that the SUKH domain possesses a versatile scaffold that can be used to bind a wide range of protein partners. In eukaryotes it appears to have been recruited as an adaptor to regulate modification of proteins by ubiquitination or polyglutamylation. Similarly, another widespread immunity protein from these toxin systems, namely the suppressor of fused (SuFu) superfamily has been recruited for comparable roles in eukaryotes. In animal DNA viruses, such as herpesviruses, poxviruses, iridoviruses and adenoviruses, the ability of the SUKH domain to bind diverse targets has been deployed to counter diverse anti-viral responses by interacting with specific host proteins.
PMCID: PMC3113570  PMID: 21306995
4.  Annexin A2 Mediates Mycoplasma pneumoniae Community-Acquired Respiratory Distress Syndrome Toxin Binding to Eukaryotic Cells 
mBio  2014;5(4):e01497-14.
Mycoplasma pneumoniae synthesizes a novel human surfactant protein A (SP-A)-binding cytotoxin, designated community-acquired respiratory distress syndrome (CARDS) toxin, that exhibits ADP-ribosylating and vacuolating activities in mammalian cells and is directly linked to a range of acute and chronic airway diseases, including asthma. In our attempt to detect additional CARDS toxin-binding proteins, we subjected the membrane fraction of human A549 airway cells to affinity chromatography using recombinant CARDS toxin as bait. A 36-kDa A549 cell membrane protein bound to CARDS toxin and was identified by time of flight (TOF) mass spectroscopy as annexin A2 (AnxA2) and verified by immunoblotting with anti-AnxA2 monoclonal antibody. Dose-dependent binding of CARDS toxin to recombinant AnxA2 reinforced the specificity of the interaction, and further studies revealed that the carboxy terminus of CARDS toxin mediated binding to AnxA2. In addition, pretreatment of viable A549 cells with anti-AnxA2 monoclonal antibody or AnxA2 small interfering RNA (siRNA) reduced toxin binding and internalization. Immunofluorescence analysis of CARDS toxin-treated A549 cells demonstrated the colocalization of CARDS toxin with cell surface-associated AnxA2 upon initial binding and with intracellular AnxA2 following toxin internalization. HepG2 cells, which express low levels of AnxA2, were transfected with a plasmid expressing AnxA2 protein, resulting in enhanced binding of CARDS toxin and increased vacuolization. In addition, NCI-H441 cells, which express both AnxA2 and SP-A, upon AnxA2 siRNA transfection, showed decreased binding and subsequent vacuolization. These results indicate that CARDS toxin recognizes AnxA2 as a functional receptor, leading to CARDS toxin-induced changes in mammalian cells.
Host cell susceptibility to bacterial toxins is usually determined by the presence and abundance of appropriate receptors, which provides a molecular basis for toxin target cell specificities. To perform its ADP-ribosylating and vacuolating activities, community-acquired respiratory distress syndrome (CARDS) toxin must bind to host cell surfaces via receptor-mediated events in order to be internalized and trafficked effectively. Earlier, we reported the binding of CARDS toxin to surfactant protein A (SP-A), and here we show how CARDS toxin uses an alternative receptor to execute its pathogenic properties. CARDS toxin binds selectively to annexin A2 (AnxA2), which exists both on the cell surface and intracellularly. Since AnxA2 regulates membrane dynamics at early stages of endocytosis and trafficking, it serves as a distinct receptor for CARDS toxin binding and internalization and enhances CARDS toxin-induced vacuolization in mammalian cells.
PMCID: PMC4147866  PMID: 25139904
5.  A New Family of Secreted Toxins in Pathogenic Neisseria Species 
PLoS Pathogens  2015;11(1):e1004592.
The genus Neisseria includes both commensal and pathogenic species which are genetically closely related. However, only meningococcus and gonococcus are important human pathogens. Very few toxins are known to be secreted by pathogenic Neisseria species. Recently, toxins secreted via type V secretion system and belonging to the widespread family of contact-dependent inhibition (CDI) toxins have been described in numerous species including meningococcus. In this study, we analyzed loci containing the maf genes in N. meningitidis and N. gonorrhoeae and proposed a novel uniform nomenclature for maf genomic islands (MGIs). We demonstrated that mafB genes encode secreted polymorphic toxins and that genes immediately downstream of mafB encode a specific immunity protein (MafI). We focused on a MafB toxin found in meningococcal strain NEM8013 and characterized its EndoU ribonuclease activity. maf genes represent 2% of the genome of pathogenic Neisseria, and are virtually absent from non-pathogenic species, thus arguing for an important biological role. Indeed, we showed that overexpression of one of the four MafB toxins of strain NEM8013 provides an advantage in competition assays, suggesting a role of maf loci in niche adaptation.
Author Summary
Many bacteria are able to secrete toxins targeted against neighboring cells. In order to protect themselves against their own toxin, they also express an “immunity” protein. In silico analysis of bacterial genomes predicts that numerous genes could encode potential new toxin-immunity systems. The recently described CDI system is involved in contact-dependent inhibition of growth and confers to its host strain a significant advantage in competitive ecosystems such as the gastro-intestinal tract. Indeed, an Escherichia coli CDI+ strain is able to outcompete CDI- strains and to become predominant. Here, we show that a large family of genes called “maf”, found in pathogenic Neisseria species, encodes a toxin-immunity system. We demonstrate that a toxin named MafBMGI-1NEM8013 inhibits the growth of E. coli by degrading RNA and show that the immunity protein MafIMGI-1NEM8013 is able to abolish the toxicity. MafB toxins exhibit highly variable toxic domains. This variability of secreted toxins could be important to compete against bacteria of different species sharing the same reservoir. Since a strain may contain numerous toxin-immunity systems that can all play a role in interbacterial competition, deciphering interactions between these systems will allow a better understanding of complex bacterial communities.
PMCID: PMC4287609  PMID: 25569427
6.  Comprehensive comparative-genomic analysis of Type 2 toxin-antitoxin systems and related mobile stress response systems in prokaryotes 
Biology Direct  2009;4:19.
The prokaryotic toxin-antitoxin systems (TAS, also referred to as TA loci) are widespread, mobile two-gene modules that can be viewed as selfish genetic elements because they evolved mechanisms to become addictive for replicons and cells in which they reside, but also possess "normal" cellular functions in various forms of stress response and management of prokaryotic population. Several distinct TAS of type 1, where the toxin is a protein and the antitoxin is an antisense RNA, and numerous, unrelated TAS of type 2, in which both the toxin and the antitoxin are proteins, have been experimentally characterized, and it is suspected that many more remain to be identified.
We report a comprehensive comparative-genomic analysis of Type 2 toxin-antitoxin systems in prokaryotes. Using sensitive methods for distant sequence similarity search, genome context analysis and a new approach for the identification of mobile two-component systems, we identified numerous, previously unnoticed protein families that are homologous to toxins and antitoxins of known type 2 TAS. In addition, we predict 12 new families of toxins and 13 families of antitoxins, and also, predict a TAS or TAS-like activity for several gene modules that were not previously suspected to function in that capacity. In particular, we present indications that the two-gene module that encodes a minimal nucleotidyl transferase and the accompanying HEPN protein, and is extremely abundant in many archaea and bacteria, especially, thermophiles might comprise a novel TAS. We present a survey of previously known and newly predicted TAS in 750 complete genomes of archaea and bacteria, quantitatively demonstrate the exceptional mobility of the TAS, and explore the network of toxin-antitoxin pairings that combines plasticity with selectivity.
The defining properties of the TAS, namely, the typically small size of the toxin and antitoxin genes, fast evolution, and extensive horizontal mobility, make the task of comprehensive identification of these systems particularly challenging. However, these same properties can be exploited to develop context-based computational approaches which, combined with exhaustive analysis of subtle sequence similarities were employed in this work to substantially expand the current collection of TAS by predicting both previously unnoticed, derived versions of known toxins and antitoxins, and putative novel TAS-like systems. In a broader context, the TAS belong to the resistome domain of the prokaryotic mobilome which includes partially selfish, addictive gene cassettes involved in various aspects of stress response and organized under the same general principles as the TAS. The "selfish altruism", or "responsible selfishness", of TAS-like systems appears to be a defining feature of the resistome and an important characteristic of the entire prokaryotic pan-genome given that in the prokaryotic world the mobilome and the "stable" chromosomes form a dynamic continuum.
This paper was reviewed by Kenn Gerdes (nominated by Arcady Mushegian), Daniel Haft, Arcady Mushegian, and Andrei Osterman. For full reviews, go to the Reviewers' Reports section.
PMCID: PMC2701414  PMID: 19493340
7.  Comprehensive analysis of the HEPN superfamily: identification of novel roles in intra-genomic conflicts, defense, pathogenesis and RNA processing 
Biology Direct  2013;8:15.
The major role of enzymatic toxins that target nucleic acids in biological conflicts at all levels has become increasingly apparent thanks in large part to the advances of comparative genomics. Typically, toxins evolve rapidly hampering the identification of these proteins by sequence analysis. Here we analyze an unexpectedly widespread superfamily of toxin domains most of which possess RNase activity.
The HEPN superfamily is comprised of all α-helical domains that were first identified as being associated with DNA polymerase β-type nucleotidyltransferases in prokaryotes and animal Sacsin proteins. Using sensitive sequence and structure comparison methods, we vastly extend the HEPN superfamily by identifying numerous novel families and by detecting diverged HEPN domains in several known protein families. The new HEPN families include the RNase LS and LsoA catalytic domains, KEN domains (e.g. RNaseL and Ire1) and the RNase domains of RloC and PrrC. The majority of HEPN domains contain conserved motifs that constitute a metal-independent endoRNase active site. Some HEPN domains lacking this motif probably function as non-catalytic RNA-binding domains, such as in the case of the mannitol repressor MtlR. Our analysis shows that HEPN domains function as toxins that are shared by numerous systems implicated in intra-genomic, inter-genomic and intra-organismal conflicts across the three domains of cellular life. In prokaryotes HEPN domains are essential components of numerous toxin-antitoxin (TA) and abortive infection (Abi) systems and in addition are tightly associated with many restriction-modification (R-M) and CRISPR-Cas systems, and occasionally with other defense systems such as Pgl and Ter. We present evidence of multiple modes of action of HEPN domains in these systems, which include direct attack on viral RNAs (e.g. LsoA and RNase LS) in conjunction with other RNase domains (e.g. a novel RNase H fold domain, NamA), suicidal or dormancy-inducing attack on self RNAs (RM systems and possibly CRISPR-Cas systems), and suicidal attack coupled with direct interaction with phage components (Abi systems). These findings are compatible with the hypothesis on coupling of pathogen-targeting (immunity) and self-directed (programmed cell death and dormancy induction) responses in the evolution of robust antiviral strategies. We propose that altruistic cell suicide mediated by HEPN domains and other functionally similar RNases was essential for the evolution of kin and group selection and cell cooperation. HEPN domains were repeatedly acquired by eukaryotes and incorporated into several core functions such as endonucleolytic processing of the 5.8S-25S/28S rRNA precursor (Las1), a novel ER membrane-associated RNA degradation system (C6orf70), sensing of unprocessed transcripts at the nuclear periphery (Swt1). Multiple lines of evidence suggest that, similar to prokaryotes, HEPN proteins were recruited to antiviral, antitransposon, apoptotic systems or RNA-level response to unfolded proteins (Sacsin and KEN domains) in several groups of eukaryotes.
Extensive sequence and structure comparisons reveal unexpectedly broad presence of the HEPN domain in an enormous variety of defense and stress response systems across the tree of life. In addition, HEPN domains have been recruited to perform essential functions, in particular in eukaryotic rRNA processing. These findings are expected to stimulate experiments that could shed light on diverse cellular processes across the three domains of life.
This article was reviewed by Martijn Huynen, Igor Zhulin and Nick Grishin
PMCID: PMC3710099  PMID: 23768067
8.  The Long-Lived Nature of Clostridium perfringens Iota Toxin in Mammalian Cells Induces Delayed Apoptosis ▿  
Infection and Immunity  2009;77(12):5593-5601.
Mono-ADP ribosylation of actin by bacterial toxins, such as Clostridium perfringens iota or Clostridium botulinum C2 toxins, results in rapid depolymerization of actin filaments and cell rounding. Here we report that treatment of African green monkey kidney (Vero) cells with iota toxin resulted in delayed caspase-dependent death. Unmodified actin did not reappear in toxin-treated cells, and enzyme-active toxin was detectable in the cytosol for at least 24 h. C2 toxin showed comparable, long-lived effects in cells, while a C2 toxin control lacking ADP-ribosyltransferase activity did not induce cell death. To address whether the remarkable stability of the iota and C2 toxins in cytosol was crucial for inducing cell death, we treated cells with C/SpvB, the catalytic domain of Salmonella enterica SpvB. Although C/SpvB also mono-ADP ribosylates actin as do the iota and C2 toxins, cells treated with a cell-permeating C/SpvB fusion toxin became rounded but recovered and remained viable. Moreover, unmodified actin reappeared in these cells, and ADP-ribosyltransferase activity due to C/SpvB was not detectable in the cytosol after 24 h, a result most likely due to degradation of C/SpvB. Repeated application of C/SpvB prevented recovery of cells and reappearance of unmodified actin. In conclusion, a complete but transient ADP ribosylation of actin was not sufficient to trigger apoptosis, implying that long-term stability of actin-ADP-ribosylating toxins, such as iota and C2, in the cytosol is crucial for inducing delayed, caspase-dependent cell death.
PMCID: PMC2786441  PMID: 19805536
9.  Clostridium difficile Toxin CDT Induces Formation of Microtubule-Based Protrusions and Increases Adherence of Bacteria 
PLoS Pathogens  2009;5(10):e1000626.
Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by production of the Rho GTPase-glucosylating toxins A and B. Recently emerging hypervirulent Clostridium difficile strains additionally produce the binary ADP-ribosyltransferase toxin CDT (Clostridium difficile transferase), which ADP-ribosylates actin and inhibits actin polymerization. Thus far, the role of CDT as a virulence factor is not understood. Here we report by using time-lapse- and immunofluorescence microscopy that CDT and other binary actin-ADP-ribosylating toxins, including Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin, induce redistribution of microtubules and formation of long (up to >150 µm) microtubule-based protrusions at the surface of intestinal epithelial cells. The toxins increase the length of decoration of microtubule plus-ends by EB1/3, CLIP-170 and CLIP-115 proteins and cause redistribution of the capture proteins CLASP2 and ACF7 from microtubules at the cell cortex into the cell interior. The CDT-induced microtubule protrusions form a dense meshwork at the cell surface, which wrap and embed bacterial cells, thereby largely increasing the adherence of Clostridia. The study describes a novel type of microtubule structure caused by less efficient microtubule capture and offers a new perspective for the pathogenetic role of CDT and other binary actin-ADP-ribosylating toxins in host–pathogen interactions.
Author Summary
Clostridium difficile is responsible for ∼20 percent of antibiotic-related cases of diarrhea and nearly all cases of pseudomembranous colitis. The pathogens produce two protein toxins (toxins A and B), which inactivate Rho-GTPases of host cells by glucosylation. Recently emerging hypervirulent strains of C. difficile release higher amounts of toxins A and B, are resistant towards fluoroquinolones and produce an additional protein toxin called C. difficile transferase (CDT). CDT is a binary toxin, which modifies G-actin by ADP-ribosylation, thereby inhibiting actin polymerization. So far the pathogenetic role of CDT is not clear. Here we studied the effects of CDT on human colon carcinoma cells and show that the toxin causes rearrangement of microtubules and formation of long cellular protrusions. The microtubule-based protrusions form a dense meshwork at the cell surface, which wrap and embed Clostridia, thereby increasing adherence of the pathogens. We observed similar effects with other members of the family of binary actin-ADP-ribosylating toxins like C. botulinum C2 toxin and C. perfringens iota toxin. Our findings show a novel type of microtubule structures induced by actin-ADP-ribosylating toxins and propose an important role of these toxins in host–pathogen interactions by their effects on adherence and colonization of Clostridia.
PMCID: PMC2757728  PMID: 19834554
10.  Live virus-free or die: coupling of antivirus immunity and programmed suicide or dormancy in prokaryotes 
Biology Direct  2012;7:40.
The virus-host arms race is a major theater for evolutionary innovation. Archaea and bacteria have evolved diverse, elaborate antivirus defense systems that function on two general principles: i) immune systems that discriminate self DNA from nonself DNA and specifically destroy the foreign, in particular viral, genomes, whereas the host genome is protected, or ii) programmed cell suicide or dormancy induced by infection.
Presentation of the hypothesis
Almost all genomic loci encoding immunity systems such as CRISPR-Cas, restriction-modification and DNA phosphorothioation also encompass suicide genes, in particular those encoding known and predicted toxin nucleases, which do not appear to be directly involved in immunity. In contrast, the immunity systems do not appear to encode antitoxins found in typical toxin-antitoxin systems. This raises the possibility that components of the immunity system themselves act as reversible inhibitors of the associated toxin proteins or domains as has been demonstrated for the Escherichia coli anticodon nuclease PrrC that interacts with the PrrI restriction-modification system. We hypothesize that coupling of diverse immunity and suicide/dormancy systems in prokaryotes evolved under selective pressure to provide robustness to the antivirus response. We further propose that the involvement of suicide/dormancy systems in the coupled antivirus response could take two distinct forms:
1) induction of a dormancy-like state in the infected cell to ‘buy time’ for activation of adaptive immunity; 2) suicide or dormancy as the final recourse to prevent viral spread triggered by the failure of immunity.
Testing the hypothesis
This hypothesis entails many experimentally testable predictions. Specifically, we predict that Cas2 protein present in all cas operons is a mRNA-cleaving nuclease (interferase) that might be activated at an early stage of virus infection to enable incorporation of virus-specific spacers into the CRISPR locus or to trigger cell suicide when the immune function of CRISPR-Cas systems fails. Similarly, toxin-like activity is predicted for components of numerous other defense loci.
Implications of the hypothesis
The hypothesis implies that antivirus response in prokaryotes involves key decision-making steps at which the cell chooses the path to follow by sensing the course of virus infection.
This article was reviewed by Arcady Mushegian, Etienne Joly and Nick Grishin. For complete reviews, go to the Reviewers’ reports section.
PMCID: PMC3506569  PMID: 23151069
11.  CRISPR-Cas 
RNA Biology  2013;10(5):679-686.
The CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR-associated genes) is an adaptive immunity system in bacteria and archaea that functions via a distinct self-non-self recognition mechanism that is partially analogous to the mechanism of eukaryotic RNA interference (RNAi). The CRISPR-Cas system incorporates fragments of virus or plasmid DNA into the CRISPR repeat cassettes and employs the processed transcripts of these spacers as guide RNAs to cleave the cognate foreign DNA or RNA. The Cas proteins, however, are not homologous to the proteins involved in RNAi and comprise numerous, highly diverged families. The majority of the Cas proteins contain diverse variants of the RNA recognition motif (RRM), a widespread RNA-binding domain. Despite the fast evolution that is typical of the cas genes, the presence of diverse versions of the RRM in most Cas proteins provides for a simple scenario for the evolution of the three distinct types of CRISPR-cas systems. In addition to several proteins that are directly implicated in the immune response, the cas genes encode a variety of proteins that are homologous to prokaryotic toxins that typically possess nuclease activity. The predicted toxins associated with CRISPR-Cas systems include the essential Cas2 protein, proteins of COG1517 that, in addition to a ligand-binding domain and a helix-turn-helix domain, typically contain different nuclease domains and several other predicted nucleases. The tight association of the CRISPR-Cas immunity systems with predicted toxins that, upon activation, would induce dormancy or cell death suggests that adaptive immunity and dormancy/suicide response are functionally coupled. Such coupling could manifest in the persistence state being induced and potentially providing conditions for more effective action of the immune system or in cell death being triggered when immunity fails.
PMCID: PMC3737325  PMID: 23439366
CRISPR-Cas; adaptive immunity; innate immunity; programmed cell death; dormancy; RRM domain
12.  Mycoplasma pneumoniae Community Acquired Respiratory Distress Syndrome toxin expression reveals growth phase and infection-dependent regulation 
Molecular Microbiology  2010;76(5):1127-1141.
Mycoplasma pneumoniae causes acute and chronic respiratory infections, including tracheobronchitis and community acquired pneumonia, and is linked to asthma and an array of extra-pulmonary disorders. Recently, we identified an ADP-ribosylating and vacuolating toxin of M. pneumoniae, designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin. In this study we analysed CARDS toxin gene (annotated mpn372) transcription and identified its promoter. We also compared CARDS toxin mRNA and protein profiles in M. pneumoniae during distinct in vitro growth phases. CARDS toxin mRNA expression was maximal, but at low levels, during early exponential growth and declined sharply during mid-to-late log growth phases, which was in direct contrast to other mycoplasma genes examined. Between 7% and 10% of CARDS toxin was localized to the mycoplasma membrane at mid-exponential growth, which was reinforced by immunogold electron microscopy. No CARDS toxin was released into the medium. Upon M. pneumoniae infection of mammalian cells, increased expression of CARDS toxin mRNA was observed when compared with SP-4 broth-grown cultures. Further, confocal immunofluorescence microscopy revealed that M. pneumoniae readily expressed CARDS toxin during infection of differentiated normal human bronchial epithelial cells. Analysis of M. pneumoniae-infected mouse lung tissue revealed high expression of CARDS toxin per mycoplasma cell when compared with M. pneumoniae cells grown in SP-4 medium alone. Taken together, these studies indicate that CARDS toxin expression is carefully controlled by environmental cues that influence its transcription and translation. Further, the acceleration of CARDS toxin synthesis and accumulation in vivo is consistent with its role as a bona fide virulence determinant.
PMCID: PMC2883071  PMID: 20199607
13.  A Group A Streptococcus ADP-Ribosyltransferase Toxin Stimulates a Protective Interleukin 1β-Dependent Macrophage Immune Response 
mBio  2015;6(2):e00133-15.
The M1T1 clone of group A Streptococcus (GAS) is associated with severe invasive infections, including necrotizing fasciitis and septicemia. During invasive M1T1 GAS disease, mutations in the covRS regulatory system led to upregulation of an ADP-ribosyltransferase, SpyA. Surprisingly, a GAS ΔspyA mutant was resistant to killing by macrophages and caused higher mortality with impaired bacterial clearance in a mouse intravenous challenge model. GAS expression of SpyA triggered macrophage cell death in association with caspase-1-dependent interleukin 1β (IL-1β) production, and differences between wild-type (WT) and ΔspyA GAS macrophage survival levels were lost in cells lacking caspase-1, NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), or pro-IL-1β. Similar in vitro findings were identified in macrophage studies performed with pseudomonal exotoxin A, another ADP-ribosylating toxin. Thus, SpyA triggers caspase-1-dependent inflammatory cell death in macrophages, revealing a toxin-triggered IL-1β-dependent innate immune response pathway critical in defense against invasive bacterial infection.
Group A Streptococcus (GAS) is a leading human pathogen capable of producing invasive infections even in healthy individuals. GAS bacteria produce a toxin called SpyA that modifies host proteins through a process called ADP ribosylation. We describe how macrophages, frontline defenders of the host innate immune system, respond to SpyA by undergoing a specialized form of cell death in which they are activated to release the proinflammatory cytokine molecule interleukin 1β (IL-1β). Release of IL-1β activates host immune cell clearance of GAS, as we demonstrated in tissue culture models of macrophage bacterial killing and in vivo mouse infectious-challenge experiments. Similar macrophage responses to a related toxin of Pseudomonas bacteria were also shown. Thus, macrophages recognize certain bacterial toxins to activate a protective immune response in the host.
PMCID: PMC4453525  PMID: 25759502
14.  Identification of Functional Toxin/Immunity Genes Linked to Contact-Dependent Growth Inhibition (CDI) and Rearrangement Hotspot (Rhs) Systems 
PLoS Genetics  2011;7(8):e1002217.
Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiA/CdiB family of two-partner secretion proteins. Each CdiA protein exhibits a distinct growth inhibition activity, which resides in the polymorphic C-terminal region (CdiA-CT). CDI+ cells also express unique CdiI immunity proteins that specifically block the activity of cognate CdiA-CT, thereby protecting the cell from autoinhibition. Here we show that many CDI systems contain multiple cdiA gene fragments that encode CdiA-CT sequences. These “orphan” cdiA-CT genes are almost always associated with downstream cdiI genes to form cdiA-CT/cdiI modules. Comparative genome analyses suggest that cdiA-CT/cdiI modules are mobile and exchanged between the CDI systems of different bacteria. In many instances, orphan cdiA-CT/cdiI modules are fused to full-length cdiA genes in other bacterial species. Examination of cdiA-CT/cdiI modules from Escherichia coli EC93, E. coli EC869, and Dickeya dadantii 3937 confirmed that these genes encode functional toxin/immunity pairs. Moreover, the orphan module from EC93 was functional in cell-mediated CDI when fused to the N-terminal portion of the EC93 CdiA protein. Bioinformatic analyses revealed that the genetic organization of CDI systems shares features with rhs (rearrangement hotspot) loci. Rhs proteins also contain polymorphic C-terminal regions (Rhs-CTs), some of which share significant sequence identity with CdiA-CTs. All rhs genes are followed by small ORFs representing possible rhsI immunity genes, and several Rhs systems encode orphan rhs-CT/rhsI modules. Analysis of rhs-CT/rhsI modules from D. dadantii 3937 demonstrated that Rhs-CTs have growth inhibitory activity, which is specifically blocked by cognate RhsI immunity proteins. Together, these results suggest that Rhs plays a role in intercellular competition and that orphan gene modules expand the diversity of toxic activities deployed by both CDI and Rhs systems.
Author Summary
Recent work from our laboratories has shown that many bacteria express contact-dependent growth inhibition (CDI) systems in which stick-like proteins on the cell surface deliver toxic tips into target cells. Over 60 distinct toxic tips have been identified in bacteria, and our data indicate that each CDI+ cell expresses a specific immunity protein that binds to its cognate toxin and inactivates it to prevent cell suicide. Here we identify genes for toxic tips that are not attached to the stick protein. Each of these “orphan” tips has toxic activity, which is blocked by its associated immunity protein. Remarkably, the orphan tips of some bacterial species are often found on the stick proteins in other species, suggesting that cells load and deliver different tips. We also report on a system called Rhs, which encodes another predicted stick-like protein that also carries variable tips. We found that the tips of Rhs proteins are toxic and that Rhs systems encode immunity proteins that specifically block toxin activity. CDI and Rhs toxin tip diversity may represent a microbial arms race, driven by the competition for environmental resources.
PMCID: PMC3150448  PMID: 21829394
15.  Dissecting the Urokinase Activation Pathway Using Urokinase-Activated Anthrax Toxin 
Anthrax toxin is a three-part toxin secreted by Bacillus anthracis, consisting of protective antigen (PrAg), edema factor (EF), and lethal factor (LF). To intoxicate host mammalian cells, PrAg, the cell-binding moiety of the toxin, binds to cells and is then proteolytically activated by furin on the cell surface, resulting in the active heptameric form of PrAg. This heptamer serves as a protein-conducting channel that translocates EF and LF, the two enzymatic moieties of the toxin, into the cytosol of the cells where they exert cytotoxic effects. The anthrax toxin delivery system has been well characterized. The amino-terminal PrAg-binding domain of LF (residues 1–254, LFn) is sufficient to allow translocation of fused “passenger” polypeptides, such as the ADP-ribosylation domain of Pseudomonas exotoxin A, to the cytosol of the cells in a PrAg-dependent process. The protease specificity of the anthrax toxin delivery system can also be reengineered by replacing the furin cleavage target sequence of PrAg with other protease substrate sequences. PrAg-U2 is such a PrAg variant, one that is selectively activated by urokinase plasminogen activator (uPA). The uPA-dependent proteolytic activation of PrAg-U2 on the cell surface is readily detected by Western blotting analysis of cell lysates in vitro, or cell or animal death in vivo. Here we describe the use of PrAg-U2 as a molecular reporter tool to test the controversial question of what components are required for uPAR-mediated cell surface pro-uPA activation. The results demonstrate that both uPAR and plasminogen play critical roles in pro-uPA activation both in vitro and in vivo.
PMCID: PMC3393601  PMID: 19377974
Anthrax toxin; plasminogen; protective antigen; urokinase plasminogen activator; urokinase plasminogen activator receptor
16.  Toxins from Bacteria 
EXS  2010;100:1-29.
Bacterial toxins damage the host at the site of bacterial infection or distanced from the site of infections. Bacterial toxins can be single proteins or organized as oligomeric protein complexes and are organized with distinct AB structure-function properties. The A domain encodes a catalytic activity; ADP-ribosylation of host proteins is the earliest post-translational modification determine to be performed by bacterial toxin, and now include glucosylation and proteolysis among other s. Bacterial toxins also catalyze the non-covalent modification of host protein function or can modify host cell properties through direct protein-protein interactions. The B domain includes two functional domains: a receptor-binding domain, which defines the tropism of a toxin for a cell and a translocation domain that delivers A domain across a lipid bilayer, either on the plasma membrane or the endosome. Bacterial toxins are often characterized based upon the section mechanism that delivers the toxin out of the bacterium, termed type I–VII. This review will overview the major families of bacterial toxins and will also describe the specific structure-function properties of the botulinum neurotoxins.
PMCID: PMC3564551  PMID: 20358680
Toxins; ADP-ribosylation; post-translational modification; secretion systems; diphtheria toxin; cholera toxin; superantigens; botulinum neurotoxins
17.  Clostridium difficile Binary Toxin CDT Induces Clustering of the Lipolysis-Stimulated Lipoprotein Receptor into Lipid Rafts 
mBio  2013;4(3):e00244-13.
Clostridium difficile is the leading cause of antibiotics-associated diarrhea and pseudomembranous colitis. Hypervirulent C. difficile strains produce the binary actin-ADP-ribosylating toxin CDT (C. difficile transferase), in addition to the Rho-glucosylating toxins A and B. We recently identified the lipolysis-stimulated lipoprotein receptor (LSR) as the host receptor that mediates uptake of CDT into target cells. Here we investigated in H1-HeLa cells, which ectopically express LSR, the influence of CDT on the plasma membrane distribution of the receptor. We found by fluorescence microscopy that the binding component of CDT (CDTb) induces clustering of LSR into subcompartments of the plasma membrane. Detergent extraction of cells treated with CDTb, followed by sucrose gradient fractionation, uncovered accumulation of LSR in detergent-resistant membranes (DRMs) that contained typical marker proteins of lipid rafts. Membrane cholesterol depletion with methyl-β-cyclodextrin inhibited the association of LSR with DRMs upon addition of CDTb. The receptor-binding domain of CDTb also triggered LSR clustering into DRMs. CDTb-triggered clustering of LSR into DRMs could be confirmed in Caco-2 cells. Our data suggest that CDT forces its receptor to cluster into lipid rafts and that oligomerization of the B component might enhance but is not essential for this process.
C. difficile binary toxin CDT is a member of the iota-like, actin ADP-ribosylating toxin family. The mechanism that mediates endocytic uptake of these toxins still remains elusive. Previous studies highlighted the importance of lipid rafts for oligomerization of the binding component of these toxins and for cell entry. Recently, the host cell receptor for this toxin family, namely, the lipolysis-stimulated lipoprotein receptor (LSR), has been identified. Our study now demonstrates that the binding component of CDT (CDTb) induces clustering of LSR into lipid rafts. Importantly, LSR clustering is efficiently induced also by the receptor-binding domain of CDTb, suggesting that oligomerization of the B component of CDT is not the main trigger of this process. The current work extends our knowledge on the cooperative play between iota-like toxins and their receptor.
PMCID: PMC3648903  PMID: 23631918
18.  Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25+ cells 
Regulatory T cells (Treg) constitute a major inhibitory cell population which suppresses immune responses. Thus, Treg have proven to be key players in the induction of transplantation tolerance, protection from autoimmune disease and prevention of the development of effective anti-tumor immune reactions. Treg express high levels of the high affinity interleukin-2 receptor (IL-2R) consisting of IL-2Rα (CD25) together with IL-2Rβ (CD122) and the common γ-chain (CD132). An effective reagent capable of depleting Treg in vivo would facilitate better cancer treatment and allow mechanistic studies of the role of Treg in transplantation tolerance and the development of autoimmune disease. In this study, we have developed a novel bivalent human IL-2 fusion toxin along with an Ontak®-like monovalent human IL-2 fusion toxin and compared the functional ability of these reagents in vitro. Here we show that genetically linking two human IL-2 domains in tandem, thereby generating a bivalent fusion toxin, results in significantly improved capacity in targeting human CD25+ cells in vitro. Binding analysis by flow cytometry showed that the bivalent human IL-2 fusion toxin has notably increased affinity for human CD25+ cells. In vitro functional analysis demonstrated that the bivalent isoform has an increased potency of approximately 2 logs in inhibiting cellular proliferation and protein synthesis in human CD25+ cells compared to the monovalent human IL-2 fusion toxin. Additionally, we performed two inhibition assays in order to verify that the fusion toxins target the cells specifically through binding of the human IL-2 domain of the fusion toxin to the human IL-2 receptor on the cell surface. These results demonstrated that 1) both monovalent and bivalent human IL-2 fusion toxins are capable of blocking the binding of biotinylated human IL-2 to human CD25 by flow cytometry; and 2) human IL-2 blocked the fusion toxins from inhibiting protein synthesis and cellular proliferation in vitro, thus confirming that the human IL-2 fusion toxins target the cells specifically through binding to the human IL-2 receptor. We believe that the bivalent human IL-2 fusion toxin will be a more potent, and therefore, more optimal agent than the current clinically-used monovalent fusion toxin (denileukin diftitox, Ontak®) for in vivo depletion of Treg.
PMCID: PMC4120078  PMID: 24462799
Human IL-2; Diphtheria toxin; Fusion toxin; Regulatory T cell; Pichia pastoris expression
19.  New connections in the prokaryotic toxin-antitoxin network: relationship with the eukaryotic nonsense-mediated RNA decay system 
Genome Biology  2003;4(12):R81.
Sequence profile analysis of the RelE- and ParE-type post-segregational cell killing (PSK) toxins from diverse bacteria and archaea has unified these proteins into a single superfamily. Further comparative analysis suggests that the core of the eukaryotic nonsense-mediated RNA decay system has probably evolved from a PSK-related system.
Several prokaryotic plasmids maintain themselves in their hosts by means of diverse post-segregational cell killing systems. Recent findings suggest that chromosomally encoded copies of toxins and antitoxins of post-segregational cell killing systems - such as the RelE system - might function as regulatory switches under stress conditions. The RelE toxin cleaves ribosome-associated transcripts, whereas another post-segregational cell killing toxin, ParE, functions as a gyrase inhibitor.
Using sequence profile analysis we were able unify the RelE- and ParE-type toxins with several families of small, uncharacterized proteins from diverse bacteria and archaea into a single superfamily. Gene neighborhood analysis showed that the majority of these proteins were encoded by genes in characteristic neighborhoods, in which genes encoding toxins always co-occurred with genes encoding transcription factors that are also antitoxins. The transcription factors accompanying the RelE/ParE superfamily may belong to unrelated or distantly related superfamilies, however. We used this conserved neighborhood template to transitively search genomes and identify novel post-segregational cell killing-related systems. One of these novel systems, observed in several prokaryotes, contained a predicted toxin with a PilT-N terminal (PIN) domain, which is also found in proteins of the eukaryotic nonsense-mediated RNA decay system. These searches also identified novel transcription factors (antitoxins) in post-segregational cell killing systems. Furthermore, the toxin Doc defines a potential metalloenzyme superfamily, with novel representatives in bacteria, archaea and eukaryotes, that probably acts on nucleic acids.
The tightly maintained gene neighborhoods of post-segregational cell killing-related systems appear to have evolved by in situ displacement of genes for toxins or antitoxins by functionally equivalent but evolutionarily unrelated genes. We predict that the novel post-segregational cell killing-related systems containing a PilT-N terminal domain toxin and the eukaryotic nonsense-mediated RNA decay system are likely to function via a common mechanism, in which the PilT-N terminal domain cleaves ribosome-associated transcripts. The core of the eukaryotic nonsense-mediated RNA decay system has probably evolved from a post-segregational cell killing-related system.
PMCID: PMC329420  PMID: 14659018
20.  Effect of site-directed mutagenic alterations on ADP-ribosyltransferase activity of the A subunit of Escherichia coli heat-labile enterotoxin. 
Infection and Immunity  1991;59(9):2870-2879.
Previous studies of the S1 subunit of pertussis toxin, an NAD(+)-dependent ADP-ribosyltransferase, suggested that a small amino-terminal region of amino acid sequence similarity to the active fragments of both cholera toxin and Escherichia coli heat-labile enterotoxin represents a region containing critical active-site residues that might be involved in the binding of the substrate NAD+. Other studies of two other bacterial toxins possessing ADP-ribosyltransferase activity, diphtheria toxin and Pseudomonas exotoxin A, have revealed the presence of essential glutamic acid residues vicinal to the active site. To help determine the relevance of these observations to activities of the enterotoxins, the A-subunit gene of the E. coli heat-labile enterotoxin was subjected to site-specific mutagenesis in the region encoding the amino-terminal region of similarity to the S1 subunit of pertussis toxin delineated by residues 6 through 17 and at two glutamic acid residues, 110 and 112, that are conserved in the active domains of all of the heat-labile enterotoxin variants and in cholera toxin. Mutant proteins in which arginine 7 was either deleted or replaced with lysine exhibited undetectable levels of ADP-ribosyltransferase activity. However, limited trypsinolysis of the arginine 7 mutants yielded fragmentation kinetics that were different from that yielded by the wild-type recombinant subunit or the authentic A subunit. In contrast, mutant proteins in which glutamic acid residues at either position 110 or 112 were replaced with aspartic acid responded like the wild-type subunit upon limited trypsinolysis, while exhibiting severely depressed, but detectable, ADP-ribosyltransferase activity. The latter results may indicate that either glutamic acid 110 or glutamic acid 112 of the A subunit of heat-labile enterotoxin is analogous to those active-site glutamic acids identified in several other ADP-ribosylating toxins.
PMCID: PMC258107  PMID: 1908825
21.  Delivery of CdiA Nuclease Toxins into Target Cells during Contact-Dependent Growth Inhibition 
PLoS ONE  2013;8(2):e57609.
Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CDI systems deploy a variety of distinct toxins, which are contained within the polymorphic C-terminal region (CdiA-CT) of CdiA proteins. Several CdiA-CTs are nucleases, suggesting that the toxins are transported into the target cell cytoplasm to interact with their substrates. To analyze CdiA transfer to target bacteria, we used the CDI system of uropathogenic Escherichia coli 536 (UPEC536) as a model. Antibodies recognizing the amino- and carboxyl-termini of CdiAUPEC536 were used to visualize transfer of CdiA from CDIUPEC536+ inhibitor cells to target cells using fluorescence microscopy. The results indicate that the entire CdiAUPEC536 protein is deposited onto the surface of target bacteria. CdiAUPEC536 transfer to bamA101 mutants is reduced, consistent with low expression of the CDI receptor BamA on these cells. Notably, our results indicate that the C-terminal CdiA-CT toxin region of CdiAUPEC536 is translocated into target cells, but the N-terminal region remains at the cell surface based on protease sensitivity. These results suggest that the CdiA-CT toxin domain is cleaved from CdiAUPEC536 prior to translocation. Delivery of a heterologous Dickeya dadantii CdiA-CT toxin, which has DNase activity, was also visualized. Following incubation with CDI+ inhibitor cells targets became anucleate, showing that the D.dadantii CdiA-CT was delivered intracellularly. Together, these results demonstrate that diverse CDI toxins are efficiently translocated across target cell envelopes.
PMCID: PMC3585180  PMID: 23469034
22.  The Type III Toxins of Pseudomonas aeruginosa Disrupt Epithelial Barrier Function▿  
Journal of Bacteriology  2007;190(8):2814-2821.
The type III secreted toxins of Pseudomonas aeruginosa are important virulence factors associated with clinically important infection. However, their effects on bacterial invasion across mucosal surfaces have not been well characterized. One of the most commonly expressed toxins, ExoS, has two domains that are predicted to affect cytoskeletal integrity, including a GTPase-activating protein (GAP) domain, which targets Rho, a major regulator of actin polymerization; and an ADP-ribosylating domain that affects the ERM proteins, which link the plasma membrane to the actin cytoskeleton. The activities of these toxins, and ExoS specifically, on the permeability properties of polarized airway epithelial cells with intact tight junctions were examined. Strains expressing type III toxins altered the distribution of the tight junction proteins ZO-1 and occludin and were able to transmigrate across polarized airway epithelial monolayers, in contrast to ΔSTY mutants. These effects on epithelial permeability were associated with the ADP-ribosylating domain of ExoS, as bacteria expressing plasmids lacking expression of the ExoS GAP activity nonetheless increased the permeation of fluorescent dextrans, as well as bacteria, across polarized airway epithelial cells. Treatment of epithelial cells with cytochalasin D depolymerized actin filaments and increased permeation across the monolayers but did not eliminate the differential effects of wild-type and toxin-negative mutants on the epithelial cells, suggesting that additional epithelial targets are involved. Confocal imaging studies demonstrated that ZO-1, occludin, and ezrin undergo substantial redistribution in human airway cells intoxicated by ExoS, -T, and -Y. These studies support the hypothesis that type III toxins enhance P. aeruginosa's invasive capabilities by interacting with multiple eukaryotic cytoskeletal components.
PMCID: PMC2293221  PMID: 18165298
23.  The VgrG Proteins Are “à la Carte” Delivery Systems for Bacterial Type VI Effectors* 
The Journal of Biological Chemistry  2014;289(25):17872-17884.
Background: The type VI secretion system (T6SS) is involved in bacterial warfare.
Results: Novel H1-T6SS-dependent toxins from Pseudomonas aeruginosa are identified using an in vitro bacterial killing assay.
Conclusion: The newly identified toxins showed a requirement for specific VgrG proteins.
Significance: VgrG proteins may represent an independent form of T6SS-dependent secretion, resembling the contact-dependent growth inhibition (CDI) system.
The bacterial type VI secretion system (T6SS) is a supra-molecular complex akin to bacteriophage tails, with VgrG proteins acting as a puncturing device. The Pseudomonas aeruginosa H1-T6SS has been extensively characterized. It is involved in bacterial killing and in the delivery of three toxins, Tse1–3. Here, we demonstrate the independent contribution of the three H1-T6SS co-regulated vgrG genes, vgrG1abc, to bacterial killing. A putative toxin is encoded in the vicinity of each vgrG gene, supporting the concept of specific VgrG/toxin couples. In this respect, VgrG1c is involved in the delivery of an Rhs protein, RhsP1. The RhsP1 C terminus carries a toxic activity, from which the producing bacterium is protected by a cognate immunity. Similarly, VgrG1a-dependent toxicity is associated with the PA0093 gene encoding a two-domain protein with a putative toxin domain (Toxin_61) at the C terminus. Finally, VgrG1b-dependent killing is detectable upon complementation of a triple vgrG1abc mutant. The VgrG1b-dependent killing is mediated by PA0099, which presents the characteristics of the superfamily nuclease 2 toxin members. Overall, these data develop the concept that VgrGs are indispensable components for the specific delivery of effectors. Several additional vgrG genes are encoded on the P. aeruginosa genome and are not linked genetically to other T6SS genes. A closer inspection of these clusters reveals that they also encode putative toxins. Overall, these associations further support the notion of an original form of secretion system, in which VgrG acts as the carrier.
PMCID: PMC4067218  PMID: 24794869
Bacterial Genetics; Bacterial Toxin; Microbiology; Protein Translocation; Pseudomonas aeruginosa (P. aeruginosa); Bacterial Cell Envelope; Type VI Secretion System; VgrG
24.  Guinea-pig treatment with pertussis toxin suppresses macrophage-dependent bronchoconstriction by fMLP and fails to inhibit the effects of PAF. 
British Journal of Pharmacology  1992;107(4):1029-1036.
1. Bronchoconstriction and thromboxane B2 (TxB2) release following the intra-tracheal administration of the secretagogue N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) to lungs from pertussis toxin-treated guinea-pigs in vivo and in vitro were inhibited as compared to saline-treated animals, under conditions where the responses to PAF were modified less effectively. 2. The cell target accounting for bronchoconstriction by fMLP and for inhibition by pertussis toxin is located in the airways and is probably the alveolar macrophage. Indeed (a) fMLP-induced superoxide anions and TxB2 formation by alveolar macrophages were inhibited by pertussis toxin given in vivo; (b) Gi proteins of membranes from alveolar macrophages were ADP-ribosylated in vivo by pertussis toxin and (c) bronchoconstriction and TxB2 release in response to the intra-tracheal administration of fMLP to lungs from pertussis toxin-treated animals were restored when alveolar macrophages from control guinea-pigs were transferred into the airways of pertussis toxin-treated animals before lung isolation. 3. Pertussis toxin administered to guinea-pigs in vivo, reduced the subsequent TxB2 formation and superoxide anion release by alveolar macrophages stimulated with PAF, but failed to inhibit PAF-induced bronchoconstriction. 4. Formation of TxB2 by alveolar macrophages following the intra-tracheal administration of fMLP accounts for bronchoconstriction and requires pertussis toxin-sensitive Gi proteins. PAF operates via a different mechanism, which is independent of Gi-like protein and involves mediators other than TxB2 and superoxide anions.
PMCID: PMC1907936  PMID: 1334747
25.  Importance of ADP-ribosylation in the morphological changes of PC12 cells induced by cholera toxin. 
Infection and Immunity  1994;62(10):4176-4185.
Cholera toxin (CTX) is composed of two subunits, subunit A, which possesses ADP-ribosyltransferase activity, and subunit B, which is responsible for receptor binding. It has previously been shown that agents that increase cyclic AMP (cAMP) levels in cells induce differentiation of PC12 cells into neurite-like cells. In this report, we show that as little as 100 pg of CTX per ml induces such changes. CTX was found to ADP-ribosylate at least four membrane proteins of PC12 cells in vitro and in vivo and to increase intracellular cAMP levels. We have developed an inducible ctx gene expression system in Vibrio cholerae by using the tac promoter. The culture medium of the CTX-producing bacteria was able to induce the morphological changes and the ADP-ribosylation of the PC12 cell membrane proteins. We have constructed two CTX-cross-reactive mutant proteins (CTX-CRM) by site-directed mutagenesis. The choice of glutamic acid 29 as the target amino acid was based on sequence similarities with other bacterial toxins. CTX-CRM-E29 delta, in which the Glu-29 of the A subunit was deleted, showed strongly reduced ADP-ribosyltransferase activity and did not induce significant morphological changes of PC12 cells. In contrast, CTX-CRM-E29D, in which the Glu-29 was replaced by an aspartic acid, was as active as the wild-type protein. We conclude that the ADP-ribosylation activity of CTX is important for the toxin-induced differentiation of PC12 cells. Pertussis toxin, which had no visible effect on PC12 cell morphology, was also able to ADP-ribosylate a membrane-bound protein(s) in vitro and in vivo. Pertussis toxin alone did not significantly increase cAMP levels in PC12 cells, but it acted synergistically with CTX.
PMCID: PMC303093  PMID: 7927673

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