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• 1. Introduction: An Overview of Bacterial Toxin-Antitoxin Systems

These cells were then transfected with a plasmid from which the gene of interest and the kis antitoxin gene were transcriptionally coupled using an internal ribosome entry site IRES that enabled a bicistronic arrangement in eukaryotic cells. Expression data from three different transgenes luciferase, eGFP and an IgG antibody, the genes of which were driven by an SV40 promoter , showed that transfectants that expressed the Kid toxin steadily increased their transgene expression over several weeks up to fold increase for the IgG antibody , whereas in the absence of toxin expression, transgene expression in the transfectants dropped over the same period of time [ 73 ].

The increased expression within the pools of kid -transfected cells was possibly due to a selection process for highly expressing clones created by random integration of the transgene- kis cassette. Cells with reduced expression levels are likely eliminated or are over-grown. Thus, to apply this system in other cells, the lethal effect of kid or any other TA toxin expression needs to be validated in the cell line of interest.

Furthermore, coupled expression of the gene of interest with the kis or other antitoxin genes must be achieved [ 73 ]. The development of TA systems for application in antiviral gene therapy is seemingly feasible, as has been demonstrated in several elegant studies, and strategies to employ them as possible drugs were thoroughly described previously [ 31 ]. As most of the toxins of TA systems are ribonucleases, this would have potential in particular for the control of RNA viruses.

To exploit TA systems as antivirals, specificity has become a major concern, as while bacterial endoribonuclease toxins usually cleave at specific RNA sequences or sites, they are not cell-specific. Activation of toxin effect through a viral promoter in response to its specific protein, and activation through cleavage by a specific viral protease, are two rather clever approaches to explicitly tackle the viral-infected cells. The first approach is exemplified by the Tat transactivator of transcription protein, which is a viral regulator protein produced during the early stage of HIV-1 infection by HIV-1 viruses.

The Tat regulator protein will bind to the specific long terminal repeat LTR , termed the transactivation response TAR sequence, that consequently induces the production of various HIV-1 proteins for infection purposes [ 74 ]. Thus, therapy using a construct with a toxin gene placed downstream of a TAR sequence in a retroviral vector, during the early stage of HIV-1 infection, should result in activation of expression of the toxin gene upon the binding of the Tat regulator protein to the TAR promoter sequence.

MazF is one of the most well-studied TA system toxins, and the E. In addition, the CD4 level was also not affected.

Fortunately, although MazF was also able to cleave cellular mRNA, the levels of induced MazF did not seem to cause serious cell damage and thus normal cellular growth was maintained. Therefore, the inhibition of viral growth by the MazF-based therapy to control and protect the cells from HIV viruses and to maintain the immune system is somewhat important. As a result, even though the levels of MazF-Tmac cells in the peripheral blood gradually decreased, their level was still significantly detected throughout the entire experimental period and MazF-Tmac cells were also detected in the lymphoid tissues and the spleen.

Strikingly, no lesions were observed and antibodies against MazF were also not detected.

Moreover, the gene-modified cells harvested from the monkeys more than half a year post-infusion were still able to inhibit the replication of SHIV These combined results reflect the persistency and safety of this promising MazF-based anti-HIV virus approach. Besides Tat-dependent activation of the MazF toxin, another interesting approach made use of viral proteases, which cleave at specific cleavage sites, to activate the toxin to explicitly target the infected cells. MazF can be activated by incubating the inert proteins with NS3 protease that cleaves the linker in between the MazE and MazF proteins, thereby releasing the MazF toxin [ 77 ].

The same principle applies to other viral proteases such as HIV-1 protease and factor Xa [ 77 ]. However, unlike the MazF-Tmac cells, cytotoxic effects were observed although this was with low levels of NS3 [ 78 ]. Thus, the dosage of MazF needs be fine-tuned to eliminate its harmfulness to human cells to make this antiviral approach more feasible. Although TAs are absent in eukaryotic cells, TA toxins have been found to inhibit growth in Saccharomyces cerevisiae and Arabidopsis thaliana see above. More interestingly, these toxins were able to trigger apoptosis in human cells [ 34 , 35 ].

This finding has opened new avenues to explore the feasibility of using TAs as tools to develop anti-tumor drugs [ 79 , 80 ]. The hypothesis was based on the finding that the toxins Kid from plasmid R1 [ 81 ], and MazF from the chromosome of E. Even though not many studies on the activity of prokaryotic toxins on oncogenic cells have been reported, it has been recently shown that toxins VapC 22 from the chromosome of Mycobacterium tuberculosis H37Rv , and PasB from plasmid pTF-FC2 from Thiobacillus ferroxidans exhibit pro-apoptotic activity in diverse human cancer cell lines [ 83 ].

However, most of these studies have been conducted using cell-cultures i. Then, there are a number of remaining questions to be tackled, such as how to target cancer cells with TAs while avoiding potential harmfulness to the normal cells? Are there activator-regulatory proteins present in oncogenic cells that could be used to mimic the antiviral therapy mentioned above? And last, but not least, what ways can be envisaged to deliver the desired anti-cancer toxin into tumor cells and not into healthy cells? Stable expression of a foreign gene integrated within the chromosome of a mammalian cell would depend greatly on a number of factors, but especially on the region where integration occurred, on the surrounding DNA context adjacent genes, DNA structure , and on the promoter used to express the transgene.

In many instances, integration occurs randomly if no targeted site has been chosen. This makes it very tedious and difficult to detect the cells that have integrated the transgene. Consequently, a targeted integration site would be generally favored. An excellent and imaginative approach to tackle the above questions and to achieve stable expression of heterologous genes in eukaryotic cells is proposed using the Agrobacterium tumefaciens Ti plasmid machinery, or any other T4 secretion system T4SS protein complex, to specifically integrate the desired genetic information within any eukaryotic chromosome [ 85 ].

T4SS is used by bacteria to translocate and transport DNA-protein complexes from a donor to a recipient cell Figure 3. This strategy has been used to develop gene cassettes that make use of the site-specific recombinase and integrase ability of some bacterial plasmid-encoded relaxases proteins devoted to conjugative transfer between bacteria or between bacteria and eukaryotes. In some cases, it has been shown that the target sequence for the relaxase to perform a strand transfer reaction could be as short as 17 nucleotides, and furthermore, the relaxase can integrate the transferred DNA into the nucleus of eukaryotic cells if it finds sequences homologous to its target in the recipient cells [ 86 ].

We speculate that the relaxase activity could be also used to efficiently integrate the desired genes into eukaryotic chromosomes, provided some stretches of homologous DNA are cloned within the incoming plasmid DNA [ 86 , 87 ]. Employment of these cassettes is predicted to function in an efficient manner for gene therapy with specific targeting into the recipient chromosome, rather than random integration [ 88 ].

If the incoming plasmid harbors a toxin-encoding gene and some specific tumor-related DNA region, we could envisage that, at least as a preliminary experimental approach, integration of the toxin gene into the desired chromosome region of tumor cells would be feasible. If, in addition, the toxin gene is cloned under the control of any oncogenic-specific promoter, or any inducible promoter that can be used in eukaryotic cells, then we could also consider that some targeted-vectors could be used to employ toxins as specific anti-cancer tools.

Possible way to deliver an RNase toxin into the chromosome of a tumor cell. The plasmid-encoded relaxase depicted in pink harbors a toxin gene denoted as a red arrow , and a DNA stretch homologous to the chromosome of the recipient cell shown as green lines. The relaxase recognizes and initiates transfer from the donor bacterial cell by means of the protein-DNA complex formed by the relaxase, auxiliary proteins green , and oriT shadowed.

This complex is pumped into the recipient tumor cell by the coupling protein CP, shown in orange and the T4SS export system in blue. The relaxase-DNA enters into the nucleus and integrates its cargo transgene into the chromosome of the recipient cell. Induction of the toxin leads to RNA cleavage and tumor cell stasis. Our knowledge of bacterial TA systems has improved tremendously over the past two decades since their initial discovery in bacterial plasmids and then, bacterial chromosomes, where they were postulated to mediate programmed bacterial cell death [ 8 ].

These small genetic modules are now known to be almost ubiquitous in bacterial and archeael genomes and to play essential roles in diverse cellular processes [ 2 , 3 , 89 ]. As we have shown in the above review, the finding that TA systems are functional in eukaryotic cells has opened the door to various innovative biotechnological and biomedical applications. With the inevitable growth in our fundamental knowledge of TA systems and with more novel TA systems being discovered, further refinements to existing applications will be seen and even more interesting and novel applications will be presented.

TA systems have indeed came a long way since the time when they were viewed as small, curious genetic entities that helped to maintain the stability of bacterial plasmids to their present position as one of the important tools in the toolbox used in the current on-going biotechnology revolution. National Center for Biotechnology Information , U.

Journal List Toxins Basel v. Published online Feb Find articles by Wai Ting Chan. Find articles by Manuel Espinosa. Anton Meinhart, Academic Editor. Author information Article notes Copyright and License information Disclaimer. Received Jan 17; Accepted Feb This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution CC-BY license http: This article has been cited by other articles in PMC.

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Abstract 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. Expression of TA Systems in Yeasts: TA Systems as Tools for Containment in Yeasts The increasing use of genetically modified organisms GMOs in various bioprocesses necessitates appropriate containment strategies to address concerns over the accidental release of these GMOs into the environment, or in cases where the deliberate release of the GMOs into the environment are required for biotechnological applications such as bioremediation, bioleaching and biopesticides [ 36 , 37 ].

Open in a separate window. TA Systems as Tools for the Genetic Manipulation of Yeasts Expression of another endoribonuclease toxin, MazF, from the tightly controlled, methanol-inducible AOX1 promoter in the methylotrophic yeast Pichia pastoris , was found to be lethal.

1. Introduction: An Overview of Bacterial Toxin-Antitoxin Systems

Cell Ablation for the Containment of Genetically Modified Plants Transgenic crops have become integrated into modern agriculture and are increasingly adopted worldwide. TA Systems as Tools for Overproduction of Heterologous Proteins in Eukaryotic Cells The strength and stability of transgene expression in transfected mammalian cell lines depends mainly on the chromosomal integration site, which occurs mostly at random. TA Systems in Gene Therapy 5. Antiviral Gene Therapy The development of TA systems for application in antiviral gene therapy is seemingly feasible, as has been demonstrated in several elegant studies, and strategies to employ them as possible drugs were thoroughly described previously [ 31 ].

Anticancer Gene Therapy Although TAs are absent in eukaryotic cells, TA toxins have been found to inhibit growth in Saccharomyces cerevisiae and Arabidopsis thaliana see above. Conclusions Our knowledge of bacterial TA systems has improved tremendously over the past two decades since their initial discovery in bacterial plasmids and then, bacterial chromosomes, where they were postulated to mediate programmed bacterial cell death [ 8 ].

Abbreviations The following abbreviations are used in this manuscript: Conflicts of Interest The authors declare no conflict of interest. Prokaryotic toxin-antitoxin stress response loci. Diversity, evolution and function.

Toxin-antitoxin systems in bacteria and archaea.