Loessner, H, Endmann, A, Leschner, S, Westphal, K, Rohde, M, Miloud, T et al.. Remote control of tumour-targeted Salmonella enterica serovar Typhimurium by the use of L-arabinose as inducer of bacterial gene expression in vivo. Cell Microbiol 9: 1529-1537

Molecular Immunology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany.
Cellular Microbiology (Impact Factor: 4.92). 07/2007; 9(6):1529-37. DOI: 10.1111/j.1462-5822.2007.00890.x
Source: PubMed


We have used Salmonella enterica serovar Typhimurium (S. typhimurium) which are able to colonize tumours besides spleen and liver. Bacteria were equipped with constructs encoding green fluorescent protein or luciferase as reporters under control of the promoter PBAD that is inducible with L-arabinose. Reporter genes could be induced in culture but also when the bacteria resided within the mouse macrophages J774A.1. More important, strong expression of reporters by the bacteria could be detected in mice after administration of L-arabinose. This was especially pronounced in bacteria colonizing tumours. Histology demonstrated that the bacteria had accumulated in and close to necrotic areas of tumours. Bacterial gene induction was observed in both regions. PBAD is tightly controlled also in vivo because gene E of bacteriophage PhiX174 could be introduced as inducible suicide gene. The possibility to deliberately induce genes in bacterial carriers within the host should render them extremely powerful tools for tumour therapy.

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    • "This plasmid was cut with BspLUII, blunt ended and again digested with BsrGI. The 1307 bp partial lux fragment was subsequently inserted into the mini-Tn7 transposon plasmid pHL289 (Loessner et al., 2007). For this, plasmid pHL289 was opened with NheI, blunt ended and thereafter cut with BsrGI. "
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    ABSTRACT: Chromosomal integration of expression modules for transgenes is an important aspect for the development of novel Salmonella vectors. Mini-Tn7 transposons have been used for the insertion of one such module into the chromosomal site attTn7, present only once in most Gram-negative bacteria. However, integration of multiple mini-Tn7 copies might be suitable for expression of appropriate amounts of antigen or combination of different modules. Here we demonstrate that integration of a 9.6 kb mini-Tn7 harbouring the luciferase luxCDABE (lux) occurs at the natural attTn7 site and simultaneously other locations of the Salmonella chromosome, which were engineered using λ-Red recombinase to contain one or two additional artificial attTn7 sites (a-attTn7). Multicopy integration even at closely spaced attTn7 sites was unexpected in light of the previously reported distance-dependent Tn7 target immunity. Integration of multiple copies of a mini-Tn7 containing a gfp cassette resulted in increasing green fluorescence of bacteria. Stable consecutive integration of two mini-Tn7 encoding lacZ and lux was achieved by initial transposition of lacZ-mini-Tn7, subsequent chromosomal insertion of a-attTn7 and a second round of transposition with lux-mini-Tn7. Mini-Tn7 thus constitutes a versatile method for multicopy integration of expression cassettes into the chromosome of Salmonella and possibly other bacteria. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.
    Microbial Biotechnology 12/2014; 8(1). DOI:10.1111/1751-7915.12187 · 3.02 Impact Factor
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    • "Direct triggering with chemical inducers, however, is hampered by (1) rapid clearance from the blood (Seri et al., 1996), (2) poor penetration into tissue, and (3) cellular consumption (Foley et al., 1978; Loessner et al., 2007). Brief exposure does not activate enough bacteria and reduces the amount of protein drug produced. "
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    ABSTRACT: Bacterial therapies have the potential to overcome resistances that cause chemotherapies to fail. When using bacteria to produce anticancer agents in tumors, triggering gene expression is necessary to prevent systemic toxicity.The use of chemical triggers, however, is hampered by poor delivery of inducing molecules, which reduces the number of activated bacteria. To solve this problem, we created a cell-communication system that enables activated bacteria to induce inactive neighbors. We hypothesized that introducing cellcommunication into Salmonella would improve direct triggering strategies by increasing protein production, increasing sensitivity to inducer molecules, and enabling expression in tumor tissue. To test these hypotheses we integrated the P(BAD) promoter into the quorum-sensing machinery from Vibrio fischeri. The expression of a fluorescent reporter gene was compared to expression from non-communicating controls. Function in three-dimensional tissue was tested in a tumor-on-a-chip device. Bacterial communication increased fluorescence 40-fold and increased sensitivity to inducer molecules more than 10,000-fold. The system enabled bacteria to activate neighbors and increased the time-scale of protein production. Gene expression was controllable and tightly regulated. At the optimal inducing signal, communicating bacteria produced 350 times more protein than non-communicating bacteria. The cell-communication system created in this study has uses beyond cancer therapy, including protein manufacturing, bioremediation and biosensing. It would enable amplified induction of gene expression in any environment that limits availability of inducer molecules. Ultimately, because inducible cellular communication enables gene expression in tissue, it will be a critical component of bacterial anticancer therapies. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 06/2013; 110(6). DOI:10.1002/bit.24816 · 4.13 Impact Factor
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    • "Several attenuated Salmonella strains have been developed for tumor targeting studies. SL7207, which has a defect in the aroA gene and is a derivative of similar attenuated strains28, has been used by several groups2930313233, although it can affect the health of immuno-compromised mice2933. Deletions in purI and msbB gave rise to VNP200092134 which has been used for gene-targeted pro-drug therapy35 and tested for oral delivery36 and in clinical trials3738. "
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    ABSTRACT: Using bacteria as therapeutic agents against solid tumors is emerging as an area of great potential in the treatment of cancer. Obligate and facultative anaerobic bacteria have been shown to infiltrate the hypoxic regions of solid tumors, thereby reducing their growth rate or causing regression. However, a major challenge for bacterial therapy of cancer with facultative anaerobes is avoiding damage to normal tissues. Consequently the virulence of bacteria must be adequately attenuated for therapeutic use. By placing an essential gene under a hypoxia conditioned promoter, Salmonella Typhimurium strain SL7207 was engineered to survive only in anaerobic conditions (strain YB1) without otherwise affecting its functions. In breast tumor bearing nude mice, YB1 grew within the tumor, retarding its growth, while being rapidly eliminated from normal tissues. YB1 provides a safe bacterial vector for anti-tumor therapies without compromising the other functions or tumor fitness of the bacterium as attenuation methods normally do.
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