Although analysis of toxin-antitoxin (TA) systems can be instructive, to date, there is no information on the prevalence and identity of TA systems based on a large panel of Acinetobacter baumannii clinical isolates. The aim of the current study was to screen for functional TA systems among clinical isolates of A. baumannii and to identify the systems' locations. For this purpose, we screened 85 A. baumannii isolates collected from different clinical sources for the presence of the mazEF, relBE and higBA TA genes. The results revealed that the genes coding for the mazEF TA system were commonly present in all clinical isolates of A. baumannii. Reverse transcriptase-polymerase chain reaction analysis showed that transcripts were produced in the clinical isolates. Our findings showed that TA genes are prevalent, harboured by chromosomes and transcribed within A. baumannii. Hence, activation of the toxin proteins in the mazEF TA system should be investigated further as an effective antibacterial strategy against this bacterium.
Type II (proteic) toxin-antitoxin systems (TAs) are widely distributed among bacteria and archaea. They are generally organized as operons integrated by two genes, the first encoding the antitoxin that binds to its cognate toxin to generate a harmless protein⁻protein complex. Under stress conditions, the unstable antitoxin is degraded by host proteases, releasing the toxin to achieve its toxic effect. In the Gram-positive pathogen Streptococcus pneumoniae we have characterized four TAs: pezAT, relBE, yefM-yoeB, and phD-doc, although the latter is missing in strain R6. We have assessed the role of the two yefM-yoeB and relBE systems encoded by S. pneumoniae R6 by construction of isogenic strains lacking one or two of the operons, and by complementation assays. We have analyzed the phenotypes of the wild type and mutants in terms of cell growth, response to environmental stress, and ability to generate biofilms. Compared to the wild-type, the mutants exhibited lower resistance to oxidative stress. Further, strains deleted in yefM-yoeB and the double mutant lacking yefM-yoeB and relBE exhibited a significant reduction in their ability for biofilm formation. Complementation assays showed that defective phenotypes were restored to wild type levels. We conclude that these two loci may play a relevant role in these aspects of the S. pneumoniae lifestyle and contribute to the bacterial colonization of new niches.
Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.
GCN5-related N-acetyltransferase (GNAT) is a huge superfamily of proteins spanning the prokaryotic and eukaryotic domains of life. GNAT proteins usually transfer an acetyl group from acetyl-CoA to a wide variety of substrates ranging from aminoglycoside antibiotics to large macromolecules. Type II toxin-antitoxin (TA) modules are typically bicistronic and widespread in bacterial and archael genomes with diverse cellular functions. Recently, a novel family of type II TA toxins was described, which presents a GNAT-fold and functions by acetylating charged tRNA thereby precluding translation. These GNAT toxins are usually associated with a corresponding ribbon-helix-helix-fold (RHH) antitoxin. In this issue, Qian et al. describes a unique GNAT-RHH TA system, designated KacAT, from a multidrug resistant strain of the pathogen, Klebsiella pneumoniae. As most type II TA loci, kacAT is transcriptionally autoregulated with the KacAT complex binding to the operator site via the N-terminus region of KacA to repress kacAT transcription. The crystal structure of the KacT toxin is also presented giving a structural basis for KacT toxicity. These findings expand our knowledge on this newly discovered family of TA toxins and the potential role that they may play in antibiotic tolerance and persistence of bacterial pathogens.
Bacterial toxin-antitoxin (TAs) loci usually consist of two genes organized as an operon, where their products are bound together and inert under normal conditions. However, under stressful circumstances the antitoxin, which is more labile, will be degraded more rapidly, thereby unleashing its cognate toxin to act on the cell. This, in turn, causes cell stasis or cell death, depending on the type of TAs and/or time of toxin exposure. Previously based on in silico analyses, we proposed that Streptococcus pneumoniae, a pathogenic Gram-positive bacterium, may harbor between 4 and 10 putative TA loci depending on the strains. Here we have chosen the pneumococcal strain Hungary(19A)-6 which contains all possible 10 TA loci. In addition to the three well-characterized operons, namely relBE2, yefM-yoeB, and pezAT, we show here the functionality of a fourth operon that encodes the pneumococcal equivalent of the phd-doc TA. Transcriptional fusions with gene encoding Green Fluorescent Protein showed that the promoter was slightly repressed by the Phd antitoxin, and exhibited almost background values when both Phd-Doc were expressed together. These findings demonstrate that phd-doc shows the negative self-regulatory features typical for an authentic TA. Further, we also show that the previously proposed TAs XreA-Ant and Bro-XreB, although they exhibit a genetic organization resembling those of typical TAs, did not appear to confer a functional behavior corresponding to bona fide TAs. In addition, we have also discovered new interesting bioinformatics results for the known pneumococcal TAs RelBE2 and PezAT. A global analysis of the four identified toxins-antitoxins in the pneumococcal genomes (PezAT, RelBE2, YefM-YoeB, and Phd-Doc) showed that RelBE2 and Phd-Doc are the most conserved ones. Further, there was good correlation among TA types, clonal complexes and sequence types in the 48 pneumococcal strains analyzed.
Toxin-antitoxin (TA) genes were first reported in plasmids and were considered expendable genetic cassettes involved in the stable maintenance of the plasmid replicon by interfering with growth and/or viability of bacteria in which the plasmid was lost. TAs were later found in bacterial chromosomes and also in integrated mobile genetic elements; they were proposed to be involved in the bacterial response to stressful situations. At present, 100s of TAs have been identified and classified in up to six families (I to VI), with those belonging to the type II (constituted by two protein components) being the most studied. Based on well-characterized examples of several type II TAs, we discuss in this review that irrespective of their locations in plasmids or chromosomes, TAs functionally overlap as indicated by: (i) in both locations they can mediate the maintenance of genetic elements to which they are physical linked, and (ii) they can induce persistence or virulence in response to stress situations. Examples of functional confluences in homologous TA systems with different locations are also given. We also consider whether the physiological role of TAs is due to their genetic organization as operons or to their inherent properties, like the short lifespan of the antitoxin component.
The toxin-antitoxin (TA) system is a regulatory system where two sets of genes encode the toxin and its corresponding antitoxin. In this study, the prevalence of TA systems in independently isolated clinical isolates of Enterococcus faecium and Enterococcus faecalis was determined, the dominant TA system was identified, different virulence genes in E. faecium and E. faecalis were surveyed, the level of expression of the virulence and TA genes in normal and stress conditions was determined, and finally their associations with the TA genes were defined. Remarkably, the analysis demonstrated higBA and mazEF in all clinical isolates, and their locations were on chromosomes and plasmids, respectively. On the other hand, a quantitative analysis of TA and virulence genes revealed that the expression level in both genes is different under normal and stress conditions. The results obtained by anti-mazF peptide nucleic acids demonstrated that the expression level of virulence genes had decreased. These findings demonstrate an association between TA systems and virulence factors. The mazEF on the plasmids and the higBA TA genes on the chromosomes of all E. faecium and E. faecalis strains were dominant. Additionally, there was a decrease in the expression of virulence genes in the presence of anti-mazF peptide nucleic acids. Therefore, it is suggested that mazEF TA systems are potent and sensitive targets in all E. faecium and E. faecalis strains.
Specific antibody to Pseudomonas pseudomallei exotoxin was detected in sheep sera exposed to natural infection. An enzyme-linked immunosorbent assay (ELISA) was used. Serum antitoxin was present in 49.3% of sera obtained from a flock of sheep naturally exposed to P. pseudomallei infection. Among these sera, 17.0% gave titers of 10,000. In contrast, serum antitoxin was present in only 6.0% of sera collected from sheep kept on a melioidosis-free farm. The ELISA reactivity of all positive sera could be completely absorbed with purified P. pseudomallei exotoxin. Similarly, preincubation of the exotoxin-coated wells with specific antiserum inhibited the ELISA reactivity of sheep sera. The results indicate that exotoxin is produced in vivo during infection by P. pseudomallei.
The protective effects of Mucuna pruriens seed extract (MPE) against the cardio-respiratory depressant and neuromuscular paralytic effects induced by injection of Calloselasma rhodostoma (Malayan pit viper) venom in anaesthetized rats were investigated. While MPE pretreatment did not reverse the inhibitory effect of the venom on the gastrocnemius muscle excitability, it significantly attenuated the venom-induced cardio-respiratory depressant effects (p < 0.05). The protection effects may have an immunological mechanism, as indicated by the presence of several proteins in the venom that are immunoreactive against anti-MPE. However, we cannot rule out the possibility that the pretreatment may exert a direct, non-immunological protective action against the venom.
The toxin-antitoxin (TA) systems are systems in which an unstable antitoxin inhibits a stable toxin. This review aims to introduce the TA system and its biological application in bacteria. For this purpose, first we introduce a new classification for the TA systems based on how the antitoxin can neutralize the toxin, we then describe the functions of TA systems and finally review the application of these systems in biotechnology.
In their initial stages of discovery, prokaryotic toxin-antitoxin (TA) systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I-VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA antitoxins. We shall also look into some interesting deviations from the canonical type II TA systems such as tripartite TA systems where the regulatory role is played by a third party protein and not the antitoxin, and a unique TA system encoding a single protein with both toxin as well as antitoxin domains.
Cholera caused by the O139 serogroup still remains a public health concern in certain regions of the world and the existing O1 vaccines do not cross-protect cholera caused by this serogroup. An aminolevulinic acid (ALA) auxotroph vaccine candidate against the O139 serogroup, designated as VCUSM2, was recently developed. It was found to be immunogenic in animal model studies but showed mild reactogenic effects due to the presence of two intact copies of Vibrio cholerae toxin (CTX) genetic element. In the present study we have modified the ctx operon by systematic allelic replacement methodology to produce a mutant strain, designated as VCUSM14. This strain has two copies of chromosomally integrated and mutated ctxA gene, encoding immunogenic but not toxic cholera toxin A subunit (CT-A). The amino acids arginine and glutamic acid at position 7th and 112th, respectively, in CT-A of VCUSM14 were substituted with lysine (R7K) and glutamine (E112Q), respectively. Two copies of the ace and zot genes present in the ctx operon were also deleted. Cholera toxin-ELISA using GM1 ganglioside showed that the both wild type CT and mutated CT were recognized by anti-CT polyclonal antibodies. VCUSM14 produced comparatively less amount of antigenic cholera toxin when compared to the VCUSM2 and Bengal wild type strain. VCUSM14 did not elicit fluid accumulation when inoculated into rabbit ileal loops at doses of 10(6) and 10(8) CFU. The colonization efficiency of VCUSM14 was one log lower than the parent strain, VCUSM2, which can be attributed to the ALA auxotrophy and less invasive properties of VCUSM14. VCUSM14, thus a non-reactogenic auxotrophic vaccine candidate against infection by O139 V. cholerae.