Putative Toxin Research Articles

RNA-seq and high-definition mass spectrometry reveal the complex and divergent venoms of two rear-fanged colubrid snakes.

BackgroundLargely because of their direct, negative impacts on human health, the venoms of front-fanged snakes of the families Viperidae and Elapidae have been extensively characterized proteomically, transcriptomically, and pharmacologically. However, relatively little is known about the molecular complexity and evolution of the venoms of rear-fanged colubrid snakes, which are, with a few notable exceptions, regarded as harmless to humans. Many of these snakes have venoms with major effects on their preferred prey, and their venoms are probably as critical to their survival as those of front-fanged elapids and viperids.ResultsWe sequenced the venom-gland transcriptomes from a specimen of Hypsiglena (Desert Night Snake; family Colubridae, subfamily Dipsadinae) and of Boiga irregularis (Brown Treesnake; family Colubridae, subfamily Colubrinae) and verified the transcriptomic results proteomically by means of high-definition mass spectrometry. We identified nearly 3,000 nontoxin genes for each species. For B. irregularis, we found 108 putative toxin transcripts in 46 clusters with <1% nucleotide divergence, and for Hypsiglena we identified 79 toxin sequences that were grouped into 33 clusters. Comparisons of the venoms revealed divergent venom types, with Hypsiglena possessing a viper-like venom dominated by metalloproteinases, and B. irregularis having a more elapid-like venom, consisting primarily of three-finger toxins.ConclusionsDespite the difficulty of procuring venom from rear-fanged species, we were able to complete all analyses from a single specimen of each species without pooling venom samples or glands, demonstrating the power of high-definition transcriptomic and proteomic approaches. We found a high level of divergence in the venom types of two colubrids. These two venoms reflected the hemorrhagic/neurotoxic venom dichotomy that broadly characterizes the difference in venom strategies between elapids and viperids.

Nemertean toxin genes revealed through transcriptome sequencing.

Nemerteans are one of few animal groups that have evolved the ability to utilize toxins for both defense and subduing prey, but little is known about specific nemertean toxins. In particular, no study has identified specific toxin genes even though peptide toxins are known from some nemertean species. Information about toxin genes is needed to better understand evolution of toxins across animals and possibly provide novel targets for pharmaceutical and industrial applications. We sequenced and annotated transcriptomes of two free-living and one commensal nemertean and annotated an additional six publicly available nemertean transcriptomes to identify putative toxin genes. Approximately 63–74% of predicted open reading frames in each transcriptome were annotated with gene names, and all species had similar percentages of transcripts annotated with each higher-level GO term. Every nemertean analyzed possessed genes with high sequence similarities to known animal toxins including those from stonefish, cephalopods, and sea anemones. One toxin-like gene found in all nemerteans analyzed had high sequence similarity to Plancitoxin-1, a DNase II hepatotoxin that may function well at low pH, which suggests that the acidic body walls of some nemerteans could work to enhance the efficacy of protein toxins. The highest number of toxin-like genes found in any one species was seven and the lowest was three. The diversity of toxin-like nemertean genes found here is greater than previously documented, and these animals are likely an ideal system for exploring toxin evolution and industrial applications of toxins.

Diversity of Shiga toxin-producing Escherichia coli in sheep flocks of Paraná State, southern Brazil

Sheep constitute an important source of zoonotic pathogens as Shiga toxin-producing Escherichia coli (STEC). In this study, the prevalence, serotypes and virulence profiles of STEC were investigated among 130 healthy sheep from small and medium farms in southern Brazil. STEC was isolated from 65 (50%) of the tested animals and detected in all flocks. A total of 70 STEC isolates were characterized, and belonged to 23 different O:H serotypes, many of which associated with human disease, including hemolytic-uremic syndrome (HUS). Among the serotypes identified, O76:H19 and O65:H– were the most common, and O75:H14 and O169:H7 have not been previously reported in STEC strains. Most of the STEC isolates harbored only stx1, whereas the Stx2b subtype was the most common among those carrying stx2. Enterohemolysin (ehxA) and intimin (eae) genes were detected in 61 (87.1%) and four (5.7%) isolates, respectively. Genes encoding putative adhesins (saa, iha, lpfO113) and toxins (subAB and cdtV) were also observed. The majority of the isolates displayed virulence features related to pathogenesis of STEC, such as adherence to epithelial cells, high cytotoxicity and enterohemolytic activity. Ovine STEC isolates belonged mostly to phylogenetic group B1. PFGE revealed particular clones distributed in some farms, as well as variations in the degree of genetic similarity within serotypes examined. In conclusion, STEC are widely distributed in southern Brazilian sheep, and belonged mainly to serotypes that are not commonly reported in other regions, such as O76:H19 and O65:H–. A geographical variation in the distribution of STEC serotypes seems to occur in sheep.

Entericidin is required for a probiotic treatment (Enterobacter sp. strain C6-6) to protect trout from cold-water disease challenge.

Flavobacterium psychrophilum causes bacterial cold-water disease in multiple fish species, including salmonids. An autochthonous Enterobacter strain (C6-6) inhibits the in vitro growth of F. psychrophilum, and when ingested as a putative probiotic, it provides protection against injection challenge with F. psychrophilum in rainbow trout. In this study, low-molecular-mass (≤3 kDa) fractions from both Enterobacter C6-6 and Escherichia coli K-12 culture supernatants inhibited the growth of F. psychrophilum. The ≤3-kDa fraction from Enterobacter C6-6 was analyzed by SDS-PAGE, and subsequent tandem mass spectroscopy identified EcnB, which is a small membrane lipoprotein that is a putative pore-forming toxin. Agar plate diffusion assays demonstrated that ecnAB knockout strains of both Enterobacter C6-6 and E. coli K-12 no longer inhibited F. psychrophilum (P < 0.001), while ecnAB-complemented knockout strains recovered the inhibitory phenotype (P < 0.001). In fish experiments, the engineered strains (C6-6 ΔecnAB and C6-6 ΔecnAB<pET101::ecnAB>) and the wild-type strain (C6-6) were added to the fish diet every day for 38 days. On day 11, the fish were challenged by injection with a virulent strain of F. psychrophilum (CSF 259-93). Fish that were fed C6-6 had significantly longer survival than fish fed the ecnAB knockout strain (P < 0.0001), while fish fed the complemented knockout strain recovered the probiotic phenotype (P = 0.61). This entericidin is responsible for the probiotic activity of Enterobacter C6-6, and it may present new opportunities for therapeutic and prophylactic treatments against similarly susceptible pathogens.

Toxic oligomer species of amyloid-β in Alzheimer's disease, a timing issue.

A decade following the paradigm-shifting concept that endogenous forms of soluble, non-fibrillar amyloid-β (Aβ) might constitute the major bioactive entity causing synaptic loss and cognitive decline in Alzheimer's disease (AD), our understanding of these oligomeric species still remains conspicuously superficial. The current lack of direct evaluation tools for each endogenous Aβ oligomer hampers our ability to readily address crucial question such as: (i) where they form and accumulate?; (ii) when they first appear in human brains and body fluids?; (iii) what is the longitudinal expression of these putative toxins during the course of the disease?; (iv) and how do these soluble Aβ assemblies alter synaptic and neuronal function in the brain? Despite these limitations, indirect ex vivo measurement and isolation from biological specimens has been possible and have allowed parsing out intrinsic differences between putative endogenous Aβ oligomers. In this review, I integrated recent findings and extrapolated emerging hypotheses derived from these studies with the hope to provide a clarified view on the putative role of endogenous Aβ oligomers in AD, with a particular emphasis on the timing at which these soluble species might act in the aging and diseased brain.

Rethinking cycad metabolite research.

Cycads are among the most ancient of extant Spermatophytes, and are known for their numerous pharmacologically active compounds. One compound in particular, β-methylamino-L-alanine (BMAA), has been implicated as the cause of amyotrophic lateral sclerosis/Parkinson dementia complex (ALS/PDC) on Guam. Previous studies allege that BMAA is produced exclusively by cyanobacteria, and is transferred to cycads through the symbiotic relationship between these cyanobacteria and the roots of cycads. We recently published data showing that Cycas micronesica seedlings grown without endophytic cyanobacteria do in fact increase in BMAA, invalidating the foundation of the BMAA hypothesis. We use this example to suggest that the frenzy centered on BMAA and other single putative toxins has hindered progress. The long list of cycad-specific compounds may have important roles in signaling or communication, but these possibilities have been neglected during decades of attempts to force single metabolites into a supposed anti-herbivory function. We propose that an unbiased, comprehensive approach may be a more appropriate means of proceeding with cycad biochemistry research.

Analysis of emetic toxin production by Bacillus species using cellular cytotoxicity, molecular, and chromatographic assays

In this study, we analyzed production of the emetic toxin, cereulide, in 30 Bacillus strains by using a cellular cytotoxicity (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; MTT) assay, polymerase chain reaction (PCR), and micellar electrokinetic chromatographycapillary electrophoresis (MEKC-CE) analysis. The Bacillus cereus emetic strains produced 60 ∼ 227 µg/mL of cereulide when analyzed using MEKC-CE. Some other Bacillus species, including B. subtilis, B. pumilus, and B. megaterium, produced putative cereulide at levels similar to those produced by emetic B. cereus, whereas B. mycoides and B. thuringiensis did not produce the putative toxin or produced it at a concentration less than 2 µg/mL. Only B. cereus emetic strains were found to be toxic to bovine fibroblast cells, with the exception of one Bacillus diarrheal strain. The PCR results correlated with the MTT assay results, except in the case of one B. cereus diarrheal strain. This strain may produce either unusually heat-stable enterotoxins or is a co-producer of emetic and diarrheal toxins. Collectively, our results indicate that several Bacillus species produce toxin(s) with structures and properties similar to those of cereulide.

Testing the Toxicofera: Comparative transcriptomics casts doubt on the single, early evolution of the reptile venom system

The identification of apparently conserved gene complements in the venom and salivary glands of a diverse set of reptiles led to the development of the Toxicofera hypothesis – the single, early evolution of the venom system in reptiles. However, this hypothesis is based largely on relatively small scale EST-based studies of only venom or salivary glands and toxic effects have been assigned to only some putative Toxicoferan toxins in some species. We set out to examine the distribution of these proposed venom toxin transcripts in order to investigate to what extent conservation of gene complements may reflect a bias in previous sampling efforts. Our quantitative transcriptomic analyses of venom and salivary glands and other body tissues in five species of reptile, together with the use of available RNA-Seq datasets for additional species, shows that the majority of genes used to support the establishment and expansion of the Toxicofera are in fact expressed in multiple body tissues and most likely represent general maintenance or “housekeeping” genes. The apparent conservation of gene complements across the Toxicofera therefore reflects an artefact of incomplete tissue sampling. We therefore conclude that venom has evolved multiple times in reptiles.

A bioinformatics survey for conotoxin-like sequences in three turrid snail venom duct transcriptomes

The repertoire of venom peptides produced by Conoidean snails has shown to be useful for therapeutic and neuropharmacologic applications. Despite their dominance in terms of species number, the Family Turridae is the least studied among their other Conoidean counterparts. They provide a vast resource of pharmacological material only hindered by the inaccessibility of their deep water habitat for sample collection and their small size that allows only a limited amount of material from their venom duct amenable for analysis. Using high-throughput transcriptome sequencing, toxin transcripts can be extracted bioinformatically to fast-track toxin discovery. This approach was utilized on the venom duct transcriptomes of three species of turrids: Unedogemmula bisaya, Crassispira cerithina, and Gemmula speciosa and resulted in the discovery of 41, 22, and 74 putative turrid toxin genes, respectively. Comparisons among these turrid toxin genes to conotoxins show (i) similar superfamily precursors between conotoxins and turrid toxins for the classes D, I2, L, M, O1, O2, and P, (ii) a wider range of peptide lengths of up to 190 amino acids long for mature turritoxin, and (iii) nondisulfide-rich turritoxins with the B2 signal sequence. Novel superfamilies and cysteine frameworks including a novel 14-cysteine residue framework were also obtained.

Next generation sequencing analysis of nine Corynebacterium ulcerans isolates reveals zoonotic transmission and a novel putative diphtheria toxin-encoding pathogenicity island.

BackgroundToxigenic Corynebacterium ulcerans can cause a diphtheria-like illness in humans and have been found in domestic animals, which were suspected to serve as reservoirs for a zoonotic transmission. Additionally, toxigenic C. ulcerans were reported to take over the leading role in causing diphtheria in the last years in many industrialized countries.MethodsTo gain deeper insights into the tox gene locus and to understand the transmission pathway in detail, we analyzed nine isolates derived from human patients and their domestic animals applying next generation sequencing and comparative genomics.ResultsWe provide molecular evidence for zoonotic transmission of C. ulcerans in four cases and demonstrate the superior resolution of next generation sequencing compared to multi-locus sequence typing for epidemiologic research. Additionally, we provide evidence that the virulence of C. ulcerans can change rapidly by acquisition of novel virulence genes. This mechanism is exemplified by an isolate which acquired a prophage not present in the corresponding isolate from the domestic animal. This prophage contains a putative novel virulence factor, which shares high identity with the RhuM virulence factor from Salmonella enterica but which is unknown in Corynebacteria so far. Furthermore, we identified a putative pathogenicity island for C. ulcerans bearing a diphtheria toxin gene.ConclusionThe novel putative diphtheria toxin pathogenicity island could provide a new and alternative pathway for Corynebacteria to acquire a functional diphtheria toxin-encoding gene by horizontal gene transfer, distinct from the previously well characterized phage infection model. The novel transmission pathway might explain the unexpectedly high number of toxigenic C. ulcerans.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-014-0113-3) contains supplementary material, which is available to authorized users.

Host specificity and clade dependent distribution of putative virulence genes in Moritella viscosa

Moritella viscosa is the aetiological agent of winter-ulcer disease in farmed salmonids in the North Atlantic. Previously, two major (typical and variant) genetic clades have been demonstrated within this bacterial species, one of which is almost solely related to disease in Atlantic salmon (Salmo salar). In the present study infection trials demonstrated that ‘typical’ M. viscosa isolated from Norwegian Atlantic salmon was highly virulent in this fish species but resulted in lower levels of mortality in rainbow trout. ‘Variant’ M. viscosa isolated from rainbow trout resulted in modest mortality levels in both Atlantic salmon and rainbow trout. To investigate the possible genetic background for inter-strain virulence differences, 38 M. viscosa isolates of diverse geographical origin and host species and a number of other Moritella spp. were investigated for the presence/absence of putative virulence related homologs. All isolates were positive for DNA sequences coding for; the Type VI secretion ATPase (clpV), hemolysin co-regulated protein (hcp), bacterioferritins (bfrA and bfrB), lectin (hemG), phospholipase D (pld), multifunctional autoprocessing repeats-in-toxin (martxA), aerolysin (aer), invasin (inv), and cytotoxic necrotizing factor (cnf), with the exception of one isolate in which cnf could not be confirmed. The product of an ABC transporter metal-binding lipoprotein (mat) was consistently detected although 11 isolates, all phylogenetically related, appear to produce a truncated version. A putative insecticidal toxin complex (mitABC) was detected almost exclusively in ‘typical’ Atlantic salmon isolates, and our data indicate that this complex of genes is expressed and co-transcribed. Transmission electron microscopy investigation revealed pili and flagella surface structures on nine M. viscosa representing both typical and variant isolates. Our results provide strong support for the existence of host specificity/high virulence in ‘typical’ M. viscosa related to Atlantic salmon. The gene distribution also provides further support for the genetic division within M. viscosa, and constitutes a basis for further study of the importance of the mitABC complex in winter-ulcer pathogenesis.

Identification of a mosquitocidal toxin from Bacillus thuringiensis using mass spectrometry.

The Bacillus thuringiensis strain S2160-1 has previously been identified as being highly toxic to mosquito larvae and a viable alternative to strains currently used commercially to control these insects. A PCR approach had identified the presence of four putative insecticidal toxin genes (cry30Ea, cry30 Ga, cry50Ba and cry54Ba) in this strain, but did not identify the genes that encoding three of the main crystal toxin proteins of size 140 and 130 and 30 kDa. In this study we used mass spectrometry to identify the 130 kDa toxin as a rare Cry4 toxin (Cry4Cb3). The gene encoding this toxin was cloned and expressed and the toxin shown to have mosquitocidal activity against Culex quinquefasciatus.

A Polychaete's powerful punch: venom gland transcriptomics of Glycera reveals a complex cocktail of toxin homologs.

Glycerids are marine annelids commonly known as bloodworms. Bloodworms have an eversible proboscis adorned with jaws connected to venom glands. Bloodworms prey on invertebrates, and it is known that the venom glands produce compounds that can induce toxic effects in animals. Yet, none of these putative toxins has been characterized on a molecular basis. Here we present the transcriptomic profiles of the venom glands of three species of bloodworm, Glycera dibranchiata, Glycera fallax and Glycera tridactyla, as well as the body tissue of G. tridactyla. The venom glands express a complex mixture of transcripts coding for putative toxin precursors. These transcripts represent 20 known toxin classes that have been convergently recruited into animal venoms, as well as transcripts potentially coding for Glycera-specific toxins. The toxins represent five functional categories: Pore-forming and membrane-disrupting toxins, neurotoxins, protease inhibitors, other enzymes, and CAP domain toxins. Many of the transcripts coding for putative Glycera toxins belong to classes that have been widely recruited into venoms, but some are homologs of toxins previously only known from the venoms of scorpaeniform fish and monotremes (stonustoxin-like toxin), turrid gastropods (turripeptide-like peptides), and sea anemones (gigantoxin I-like neurotoxin). This complex mixture of toxin homologs suggests that bloodworms employ venom while predating on macroscopic prey, casting doubt on the previously widespread opinion that G. dibranchiata is a detritivore. Our results further show that researchers should be aware that different assembly methods, as well as different methods of homology prediction, can influence the transcriptomic profiling of venom glands.

Meclofenamate elicits a nephropreventing effect in a rat model of ischemic acute kidney injury by suppressing indoxyl sulfate production and restoring renal organic anion transporters.

Indoxyl sulfate (IS), a putative low-molecular weight uremic toxin, is excreted in the urine under normal kidney function, but is retained in the circulation and tissues during renal dysfunction in acute kidney injury and chronic kidney disease. IS, which is one of the most potent inducers of oxidative stress in the kidney and cardiovascular system, is enzymatically produced in the liver from indole by cytochrome P450-mediated hydroxylation to indoxyl, followed by sulfotransferase-mediated sulfate conjugation. We used rat liver S9 fraction to identify inhibitors of IS production. After testing several compounds, including phytochemical polyphenols, we identified meclofenamate as a potent inhibitor of IS production with an apparent IC50 value of 1.34 μM. Ischemia/reperfusion (I/R) of rat kidney caused a marked elevation in the serum IS concentration 48 hours after surgery. However, intravenous administration of meclofenamate (10 mg/kg) significantly suppressed this increase in the serum level of IS. Moreover, IS concentrations in both kidney and liver were dramatically elevated by renal I/R treatment, but this increase was blocked by meclofenamate. Serum creatinine and blood urea nitrogen were markedly elevated in rats after renal I/R treatment, but these increases were significantly restored by administration of meclofenamate. Renal expression of both basolateral membrane-localized organic anion transporters rOAT1 and rOAT3 was downregulated by I/R treatment. However, expression of rOAT1 and rOAT3 recovered after administration of meclofenamate, which is associated with the inhibition of I/R-evoked elevation of prostaglandin E2. Our results suggest that meclofenamate inhibits hepatic sulfotransferase-mediated production of IS, thereby suppressing serum and renal accumulation of IS. Meclofenamate also prevents the prostaglandin E2-dependent downregulation of rOAT1 and rOAT3 expression. In conclusion, meclofenamate was found to elicit a nephropreventive effect in ischemic acute kidney injury.

Characterization of SEMA3A-encoded semaphorin as a naturally occurring Kv4.3 protein inhibitor and its contribution to Brugada syndrome.

Semaphorin 3A (SEMA3A)-encoded semaphorin is a chemorepellent that disrupts neural patterning in the nervous and cardiac systems. In addition, SEMA3A has an amino acid motif that is analogous to hanatoxin, an inhibitor of voltage-gated K(+) channels. SEMA3A-knockout mice exhibit an abnormal ECG pattern and are prone to ventricular arrhythmias and sudden cardiac death. Our aim was to determine whether SEMA3A is a naturally occurring protein inhibitor of Kv4.3 (Ito) channels and its potential contribution to Brugada syndrome. Kv4.3, Nav1.5, Cav1.2, or Kv4.2 were coexpressed or perfused with SEMA3A in HEK293 cells, and electrophysiological properties were examined via whole-cell patch clamp technique. SEMA3A selectively altered Kv4.3 by significantly reducing peak current density without perturbing Kv4.3 cell surface protein expression. SEMA3A also reduced Ito current density in cardiomyocytes derived from human-induced pluripotent stem cells. Disruption of a putative toxin binding domain on Kv4.3 was used to assess physical interactions between SEMA3A and Kv4.3. These findings in combination with coimmunoprecipitations of SEMA3A and Kv4.3 revealed a potential direct binding interaction between these proteins. Comprehensive mutational analysis of SEMA3A was performed on 198 unrelated SCN5A genotype-negative patients with Brugada syndrome, and 2 rare SEMA3A missense mutations were identified. The SEMA3A mutations disrupted SEMA3A's ability to inhibit Kv4.3 channels, resulting in a significant gain of Kv4.3 current compared with wild-type SEMA3A. This study is the first to demonstrate SEMA3A as a naturally occurring protein that selectively inhibits Kv4.3 and SEMA3A as a possible Brugada syndrome susceptibility gene through a Kv4.3 gain-of-function mechanism.

Emerging types of Shiga toxin-producing E. coli (STEC) O178 present in cattle, deer, and humans from Argentina and Germany.

More than 400 serotypes of Shiga toxin-producing Escherichia coli (STEC) have been implicated in outbreaks and sporadic human diseases. In recent years STEC strains belonging to serogroup O178 have been commonly isolated from cattle and food of bovine origin in South America and Europe. In order to explore the significance of these STEC strains as potential human pathogens, 74 German and Argentinean E. coli O178 strains from animals, food and humans were characterized phenotypically and investigated for their serotypes, stx-genotypes and 43 virulence-associated markers by a real-time PCR-microarray. The majority (n = 66) of the O178 strains belonged to serotype O178:H19. The remaining strains divided into O178:H7 (n = 6), O178:H10 (n = 1), and O178:H16 (n = 1). STEC O178:H19 strains were mainly isolated from cattle and food of bovine origin, but one strain was from a patient with hemolytic uremic syndrome (HUS). Genotyping of the STEC O178:H19 strains by pulsed-field gel electrophoresis revealed two major clusters of genetically highly related strains which differ in their stx-genotypes and non-Stx putative virulence traits, including adhesins, toxins, and serine-proteases. Cluster A-strains including the HUS-strain (n = 35) carried genes associated with severe disease in humans (stx2a, stx2d, ehxA, saa, subAB1, lpfAO113, terE combined with stx1a, espP, iha). Cluster B-strains (n = 26) showed a limited repertoire of virulence genes (stx2c, pagC, lpfAO113, espP, iha). Among O178:H7 strains isolated from deer meat and patients with uncomplicated disease a new STEC variant was detected that is associated with the genotype stx1c/stx2b/ehxA/subAB2/espI/[terE]/espP/iha. None of the STEC O178 strains was positive for locus of enterocyte effacement (LEE)- and nle-genes. Results indicate that STEC O178:H19 strains belong to the growing group of LEE-negative STEC that should be considered with respect to their potential to cause diseases in humans.

A ray of venom: Combined proteomic and transcriptomic investigation of fish venom composition using barb tissue from the blue-spotted stingray (Neotrygon kuhlii).

These results show an efficient manner for removing mucus from fish venoms. These results are the first insights into the evolution of proteins present on stingrayvenom barbs.

The VgrG Proteins Are “à la Carte” Delivery Systems for Bacterial Type VI Effectors

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.

Involvement of toxin-antitoxin modules inBurkholderia cenocepaciabiofilm persistence

Biofilms are involved in the recalcitrance of infections due to the presence of persister cells. Although the molecular basis of persistence is still largely unknown, toxin-antitoxin modules (TA) are thought to play a role in this process. In this study, we investigated whether TA modules contribute to persistence toward antibiotics in Burkholderia cenocepacia J2315. Sixteen pairs of genes were identified based on their apparent similarity to TA modules. Overexpression of the putative toxins had various effects on growth, persistence, and biofilm formation. Toxins, whose overexpression resulted in growth inhibition, often increased the number of surviving persisters; in contrast, overexpression of putative toxins showing no effects on growth had no positive influence on the number of surviving persisters. Furthermore, the expression of the TA modules was compared between treated and untreated sessile and planktonic wild-type cultures. For 10 toxin-encoding genes, the expression was higher in untreated sessile cells than in untreated planktonic cells. Nine toxin-encoding genes were upregulated after treatment with tobramycin, but none after treatment with ciprofloxacin. These results indicate that most, but not all TA modules contribute to persistence in B. cenocepacia J2315 and that this contribution depends on the mode of growth and the antibiotic used.

Characterization of putative toxin/antitoxin systems in Vibrio parahaemolyticus

To obtain more information about the toxin/antitoxin (TA) systems in the Vibrio genus and also to examine their involvement in the induction of a viable but nonculturable (VBNC) state, we searched homologues of the Escherichia coli TA systems in the Vibrio parahaemolyticus genome. We found that a gene cluster, vp1842/vp1843, in the V. parahaemolyticus genome database has homology to that encoding the E. coli TA proteins, DinJ/YafQ. Expression of the putative toxin gene vp1843 in E. coli cells strongly inhibited the cell growth, while coexpression with the putative antitoxin gene vp1842 neutralized this effect. Mutational analysis identified Lys37 and Pro45 in the gene product VP1843 of vp1843 as crucial residues for the growth retardation of E. coli cells. VP1843, unlike the E. coli toxin YafQ, has no protein synthesis inhibitory activity, and that instead the expression of vp1843 in E. coli caused morphological change of the cells. The gene cluster vp1842/vp1843 encodes the V. parahaemolyticus TA system; VP1843 inhibits cell growth, whereas VP1842 serves as an antitoxin by forming a stable complex with VP1843. The putative toxin, VP1843, may be involved in the induction of the VBNC state in V. parahaemolyticus by inhibiting cell division.