Killed Bacillus subtilis spores expressing streptavidin: a novel carrier of drugs to target cancer cells.

Carriers of drugs in cancer therapy are required to reduce side-effects of the drugs to normal cells. Here we constructed killed recombinant Bacillus subtilis spores (SA1) that expressed streptavidin as a chimeric fusion to the spore coat protein CotB and used the spores as bioparticle carrier. When bound with biotinylated cetuximab these spores could specifically target to the epidermal growth factor receptor on HT 29 colon cancer cells, thereby delivered paclitaxel to the cells with 4-fold higher efficiency, as indicated by fluorescent intensity of paclitaxel Oregon Green 488 bound to HT29 cells. Based on real-time monitoring of cell index, the IC50 of growth of HT29 cells by paclitaxel-SA1-cetuximab was estimated to be 2.9 nM approximately 5-fold lower than water-soluble paclitaxel (14.5 nM). Instability of DNA content was observed when cells were treated with 16 nM paclitaxel-SA1-cetuximab, resulting in a 2-fold enhancement in polyploidy cells. Thus, by targeting the release of paclitaxel to HT29 cells, spore-associated cetuximab augmented the inhibitory effect of paclitaxel on cell division and proliferation. The SA1 could be used as a "universal" drug carrier to target specific biomarkers on cancer cells by conjugating with suitable biotinylated antibodies.

Nucleotide sequences and organization of the genes for carotovoricin (Ctv) from Erwinia carotovora indicate that Ctv evolved from the same ancestor as Salmonella typhi prophage.

Carotovoricin Er (CtvEr), which is produced by a plant soft rot disease causative agent, Erwinia carotovora subsp. carotovora Er, is a high-molecular-weight bacteriocin showing Myoviridae phage-tail-like morphology with contractile sheath and plural tail fibers. We determined the complete nucleotide sequences of CtvEr genes on the E. carotovora Er chromosome and report that CtvEr genes consist of lysis cassette, major and minor structural protein gene clusters. Four promoters were identified. The lysis gene cassette, which is composed of the genes for lysis enzyme and holin, was also identified and characterized. The nucleotide sequences and organization of the genes for CtvCGE, which is produced by E. carotovora strain CGE234-M403 with the morphology similar to CtvEr, were also determined and compared to that of CtvEr, and it was found that CtvCGE is almost identical to CtvEr except for tail fibers which are involved in the killing spectra of both bacteriocins. We also explain that the gene organization and the deduced amino acid sequences of both carotovoricins are very close to those of prophage, which is lysogenized in the chromosome on Salmonella enterica serovar Typhi CT18. These findings strongly suggest that Ctv evolved as a phage tail-like bacteriocin from a common ancestor with Salmonella typhi prophage.

Temperature-dependent production of carotovoricin Er and pectin lyase in phytopathogenic Erwinia carotovora subsp. carotovora Er.

The production of pectin lyase (Pnl) and carotovoricin (Ctv), as well as cell lysis in the plant-pathogenic bacterium Erwinia carotovora subsp. carotovora Er are induced by mitomycin C. Here, Pnl and Ctv production and cell lysis were found to be temperature-dependent. The optimal temperature for Pnl production was 30 degrees C. However, the optimal temperature for both Ctv production and cell lysis was 23 degrees C, at which Pnl production was reduced to 47% of the maximum. These data suggest the possible existence of novel regulation system(s) for the production of Pnl and Ctv, and cell lysis, in addition to the well-documented regulation system of recA, rdgA, and rdgB genes.