Optimized Doxycycline-Inducible Gene Expression System for Genetic Programming of Tumor-Targeting Bacteria

Purpose In the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (PtetA) was 100-fold higher in expression strength than tetR promoter (PtetR). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter. Procedures In this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by PtetA and PtetR, and Doxy releases TetR from tetO to de-repress PtetA and PtetR. Results In Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (PtetA:PtetR = 4~6:1) compared with that of pJL87 (PtetA:PtetR = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division. Conclusions Overall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.

Long-term exposure to octenidine in a simulated sink-trap environment results in selection of Pseudomonas aeruginosa, Citrobacter and Enterobacter isolates with mutations in efflux pump regulators.

Octenidine based disinfection products are becoming increasingly popular for infection control of multidrug resistant (MDR) Gram-negative isolates. When a waste trap was removed from a hospital and allowed to acclimatise in a standard tap rig in our laboratory, it was shown that Klebsiella pneumoniae, Pseudomonas aeruginosa, Citrobacter and Enterobacter spp. were readily isolated. This study aimed to understand the potential impact of prolonged exposure to low doses of a commercial product containing octenidine on these bacteria. Phenotypic and genotypic analysis showed that P. aeruginosa strains had increased tolerance to octenidine which was characterised by mutations in the Tet-repressor SmvR. Enterobacter species demonstrated increased tolerance to many other cationic biocides, though not octenidine, as well as the antibiotics ciprofloxacin, chloramphenicol and ceftazidime through mutations in another Tet-repressor RamR. Citrobacter species with mutations in RamR and MarR were identified following octenidine exposure and this is linked to development of resistance to ampicillin, piperacillin and chloramphenicol as well as an increased MIC level for ciprofloxacin. Isolates were able to retain fitness as characterised by growth, biofilm formation and virulence in Galleria mellonella, after prolonged contact with octenidine, although there were strain to strain differences. These results demonstrate that continued low-level octenidine exposure in a simulated sink-trap environment selects for mutations which affect smvR. It may also promote microbial adaptation to other cationic biocides and cross-resistance to antibiotics, whilst not incurring a fitness cost. This suggests that hospital sink traps may act as a reservoir for more biocide tolerant organisms. Importance Multi-drug resistant (MDR) strains of bacteria are a major clinical problem and several reports have linked outbreaks of MDR bacteria with bacterial populations in hospital sinks. Biocides such as octenidine are used clinically in body washes and other products such as wound dressings for infection control. Therefore, increased tolerance to these biocides would be detrimental to infection control processes. Here, we exposed bacterial populations originally from hospital sink traps to repeated dosing with an octenidine containing product over several weeks and observed how particular species adapted. We found mutations in genes related to biocide and antibiotic susceptibility which resulted in increased tolerance, although this was species dependent. Bacteria which became more tolerant to octenidine also showed no loss of fitness. This shows that prolonged octenidine exposure has the potential to promote microbial adaptation in the environment and that hospital sink traps may act as a reservoir for increased biocide and antibiotic tolerant organisms.

TetR-Based Gene Regulation Systems for Francisella tularensis

ABSTRACTThere are a number of genetic tools available for studyingFrancisella tularensis, the etiological agent of tularemia; however, there is no effective inducible or repressible gene expression system. Here, we describe inducible and repressible gene expression systems forF. tularensisbased on the Tet repressor, TetR. For the inducible system, atetoperator sequence was cloned into a modifiedF. tularensis groESLpromoter sequence and carried in a plasmid that constitutively expressed TetR. To monitor regulation the luminescence operon,luxCDABE, was cloned under the hybridFrancisellatetracycline-regulated promoter (FTRp), and transcription was initiated with addition of anhydrotetracycline (ATc), which binds TetR and alleviates TetR association withtetO.Expression levels measured by luminescence correlated with ATc inducer concentrations ranging from 20 to 250 ng ml−1. In the absence of ATc, luminescence was below the level of detection. The inducible system was also functional during the infection of J774A.1 macrophages, as determined by both luminescence and rescue of a mutant strain with an intracellular growth defect. The repressible system consists ofFTRpregulated by a reverse TetR mutant (revTetR), TetR r1.7. Using this system with theluxreporter, the addition of ATc resulted in decreased luminescence, while in the absence of ATc the level of luminescence was not significantly different from that of a construct lacking TetR r1.7. Utilizing both systems, the essentiality of SecA, the protein translocase ATPase, was confirmed, establishing that they can effectively regulate gene expression. These two systems will be invaluable in exploringF. tularensisprotein function.

Vectors for improved Tet repressor-dependent gradual gene induction or silencing in Staphylococcus aureus

A set of vectors for improved tetracycline-dependent gene regulation in Staphylococcus aureus is presented. Plasmid pRAB11 was generated from pRMC2 by adding a second tet operator within the TetR-regulated promoter Pxyl/tet. Pronounced repression was observed in the absence of anhydrotetracycline (ATc) combined with high induction in the presence of the drug, as demonstrated for pRAB11 bearing staphylococcal nuclease nuc1, lacZ or gfp. Also, in plasmid pCG261, the pRAB11 tetR–Pxyl/tet regulatory architecture permitted tight repression and a stepwise increase in transcript amounts of the target gene rny (putative RNase) correlated with rising ATc concentrations. Additionally, pRAB11-derived vectors harbouring semi-rationally designed Pxyl/tet-like fragments, mutated at up to six defined positions, were constructed. Sixteen mutant sequences with single to quadruple exchanges were analysed for transcriptional strength and ATc-dependent inducibility. A set of promoters with gradually decreased activities and improved repression is presented. Finally, the implementation of reverse TetR revtetR-r2, which exhibits three amino acid exchanges and binds to tetO in the presence of ATc, yielded an efficiently co-repressible vector within the pRAB11 system. Intriguingly, revtetR was found to contain a fourth mutation only after propagation in S. aureus. We predict that the described vectors constitute valuable tools for staphylococcal genetics.