O

Figure 6 Chemical structure of acylated homoserine lactone molecules, inducers of the TraR-based system. (A) Agrobacter-ium quorum sensing N-(3-oxo-octanoyl)-homoserine lactone (3-oxo-C8-HSL); (B) Synthetic [N-P-oxo-octanoyl]-homoserine lactam.

an obstacle to future development of this system. In the attempt to design a ligand more suitable for experiments in eukaryotes, Nedderman et al. (79) synthesized [N-p-oxo-octa-noyl]-homoserine lactam (Fig. 6). Notably, the [N-p-oxo-oc-tanoyl]-homoserine lactam was stable under experimental conditions and nontoxic up to a concentration of 500 ^M. The affinity of p65NtraR for the lactam was about 10-fold lower than that for 3-oxo-C8-HSL (EC50 70 ^M vs. 6 ^M); nonetheless, because of the nontoxic nature of the compound, strong induction was achieved by using higher concentration (hundreds ^M) of the compounds (79). Thus, in spite of the striking conservation of the lactone ring in natural quorum sensing signal molecules, it is possible to develop compounds that combine increased stability with biological activity.

In conclusion, although the characterization of this new system is still preliminary, its low basal transcriptional activity and the robust dose-dependent induction offer significant advantages. Its general properties suggest that it is perhaps possible to engineer other LuxR proteins that respond to different AHLs and thus increase the repertoire of genes whose expression could be simultaneously regulated, provided that the cognate AHL for one LuxR protein does not activate the others.

B. Streptogramin- and Macrolide-responsive Systems

In recent years, M. Fussenegger and colleagues have developed two regulatory systems which, similar to the Tet system, are based on naturally evolved mechanisms of antibiotic resistance.

limited by there always being a substantial time lag between cell exposure to AHL and gene activation.

It was observed that the affinity of 3-oxo-C8-HSL for the ligand-binding domain of TraR does not significantly change in the context of the chimeric protein. However, the apparent affinity of 3-oxo-C8-HSL for TraR in eukaryotic systems is lower than in bacterial strains (^M vs. nM, respectively) (79). The reason for this is not presently understood, and future work will be needed to address this issue. P65NtraR and F3-TraR induced transcription of gene clones downstream of TraR-responsive promoters in eukaryotic cells (Fig. 7). The highest inducibility was obtained with p65NtraR acting on a target promoter consisting of seven tra boxes. The leakiness was low, and adding 3-oxo-C8-HSL to the cell culture medium led to strong induction of gene expression, comparable to the activity of the constitutive CMV enhancer/promoter. Robust induction of SEAP expression in the presence of p65NtraR and 3-oxo-C8-HSL was shown in different human cell lines, and TraR induction was dose-dependent for 3-oxo-C8-HSL, reaching up to 1000-fold induction (79).

A lactone moiety, which undergoes spontaneous lactono-lysis upon prolonged exposure to aqueous conditions (82), is invariably present in all the quorum sensing signal molecules of the LuxR family of transcriptional regulators (70). The limited chemical or enzymatic stability of lactones thus poses

1. Streptogramin-responsive System

A gene regulatory system was developed based on a Strepto-myces antibiotic resistance operon (83). To tolerate their own antibiotic products, Streptomyces have developed various resistance mechanisms. A pristinamycin resistance operon was identified and cloned from S. pristinaespiralis. S. pristi-naespiralis produces pristinamycin, a composite strepto-gramin antibiotic consisting of a pair of structurally unrelated molecules: pristinamycin I (PI, also called Streptogramin B), a cyclic hexadepsipeptide, and pristinamycin II (PII; also called Streptogramin A), a polyunsaturated macrolactone (84). S. pristinaespiralis genome codes for an efflux-type pristina-mycin-resistance determinant (ptr), whose expression is induced by pristinamycin itself. A repressor protein, called Pip (pristinamycin induced protein), binds to the ptr promoter (Ptr) and represses transcription of the resistance gene. In the presence of pristinamycin, the antibiotic binds to Pip, and this results in the release of Pip from Ptr and induction of gene expression (85). Similar to the Tet system, the pristinamycin repressible Pip-Ptr interaction was adapted for use in mammalian cells. Two configurations were developed: PipOFF and PipON (Fig. 8).

In the PipOFF system, a pristinamycin inducible transacti-vator (PIT) was obtained by fusing Pip to the VP16 activation domain, and a PIT responsive promoter, called Ppir (pristina-mycin repressible promoter) was constructed by combining

Figure 7 TraR-based regulatory system. The activator consists of TraR fused to an activation domain. In the absence of the ligand, the protein is unfolded: drug administration promotes appropriate folding of the chimera, which acquires the capability of binding and activating a target promoter containing multimeric TraR binding sites. See text for additional details.

Figure 7 TraR-based regulatory system. The activator consists of TraR fused to an activation domain. In the absence of the ligand, the protein is unfolded: drug administration promotes appropriate folding of the chimera, which acquires the capability of binding and activating a target promoter containing multimeric TraR binding sites. See text for additional details.

three Pip operator sequences with a minimal promoter from Drosophila heat-shock protein 70 gene (Phsp70min). In the absence of the ligand, PIT binds and activates Ppir, whereas the system is silenced in the presence of the antibiotic, which disrupts PIT-Pir interaction (83).

The PipON system is based on the use of Pip coupled with a pristinamycin-inducible promoter (PpirON, also called PSV40-PIR3), which consists of multiple Pip operator sequences cloned downstream of the SV40 strong constitutive promoter. In the absence of the antibiotic, Pip binds to PpirON and blocks the SV40 driven expression: in the presence of the antibiotic, Pip is released and gene expression induced. In the PipON configuration, fusing Pip with the KRAB repressing domain further enhanced its repressing capability: in this configuration, the system displays a 10-fold lower basal activity but also a reduced maximal activity (83).

Both systems have so far been tested only ex vivo in mammalian cells, by transient transfection or retroviral infection.

They enabled hundreds-fold induction of reporter gene expression and displayed a relatively low basal activity. Interestingly, the PipON and PipOFF systems are only responsive to PI (Fig. 9), while PII had no effect. This finding is of interest when translated into a human therapeutic context: in fact, the use of PI alone as a stimulatory molecule is unlikely to elicit broad antibiotic resistance against composite streptogramin (PI + PII) (85).

2. Macrolide-responsive System

More recently, Fussenegger and colleagues developed a system regulated by macrolide antibiotics, such as erythromycin (EM), whose structure is shown in Fig. 10. Also in this case, the key elements derive from an antibiotic resistance operon. The Mph(A) gene codes for a 2'-phosphotransferase that inactivates 14-membered macrolides (85). Its expression in basal conditions is repressed by a prokaryotic repressor, MphR(A), which binds to a 35-bp operator sequence (ETR) overlapping

Figure 8 Streptogramin-responsive system. PipOFF: the chimeric activator (called PIT) consists of Pip fused to an activation domain. In the absence of pristinamycin, it binds and activates the target promoter consisting of three Pip binding sites (operator sequences) upstream of a minimal promoter. In the presence of the ligand, PIT is released from DNA and transcription is terminated. PipON: the chimeric repressor consists of Pip fused to the KRAB repressor domain and the target promoter consists of Pip operator sequences cloned downstream of the SV40 promoter. In the absence of the drug, the Pip-KRAB fusion binds to its target sequences, thus silencing the SV40 promoter. Drug administration results in release of the Pip-KRAB fusion from DNA and consequent derepression of the SV40 promoter. The macrolide-responsive system operates according to the same principles. See text for additional details.

Figure 8 Streptogramin-responsive system. PipOFF: the chimeric activator (called PIT) consists of Pip fused to an activation domain. In the absence of pristinamycin, it binds and activates the target promoter consisting of three Pip binding sites (operator sequences) upstream of a minimal promoter. In the presence of the ligand, PIT is released from DNA and transcription is terminated. PipON: the chimeric repressor consists of Pip fused to the KRAB repressor domain and the target promoter consists of Pip operator sequences cloned downstream of the SV40 promoter. In the absence of the drug, the Pip-KRAB fusion binds to its target sequences, thus silencing the SV40 promoter. Drug administration results in release of the Pip-KRAB fusion from DNA and consequent derepression of the SV40 promoter. The macrolide-responsive system operates according to the same principles. See text for additional details.

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