| Prodiginines are a family of tripyrrole red pigments with antibacterial, antifungal, antiprotozoal, antimalarial, anticancer and immunosuppressive activities, and it has even been proposed as natural dyes applied on food and textile industries. However, the significant difference in biological activity of prodiginines with different chemical structure has been frequently reported. In the present study, we have isolated a red pigmented entomopathogenic bacterium Serratia marcescens strain SCQ1from the hemolymph of diseased silkworm (Bombyx mori) larva with septicemia symptom. By taking advantages of the pathogenicity in silkworms, we chose silkworm pupae as the main carbon and nitrogen sources, optimized the fermentation conditions to enhance the red pigment production of strain SCQ1. Then the red pigment was purified and the chemical structure was determined. Furthermore, we analyzed and hypothesized the physiological functions of prodigiosin. At last, we investigated the antibacterial, antifungal, anticancer bioactivities and especially antiviral effect against Bombyx mori nucleopolyhedroviruses (BmNPV).1. Isolation, Identification and fermentation of S. marcescens strain SCQ1Because of insufficient production, the international market price of prodigiosin was up to500USD/mg that seriously restricted the application of this bioactive compound. We have isolated a red pigmented bacterium strain SCQ1from the hemolymph of diseased silkworm larva, then it was identified as Serratia marcescens which fulfilled Koch’s postulates. The fermentation conditions for the red pigment production, including temperature, pH value, fermentation time, metal ions, carbon and nitrogen sources, were preliminarily optimized as28℃,6.5-8.0,56h, Ca2+, glycerol and yeast extract, respectively, by using one-factor-at-a-time method. Then3key components, Ca2+, glycerol and yeast extract were subsequently optimized by using steepest ascent method. Finally, response surface methodology, involving Box-Behnken design matrix with3variables was applied to optimize the medium components for prodigiosin production. The optimal parameters were obtained as:glycerol14.25g/L, yeast extract14.02g/L and CaCl225.75μmol/L, with the maximum theoretical yields of986.78mg/L. Under this condition, the actual production of prodigiosin was confirmed as974.76±15.33mg/L, which was2.04-fold higher than the initial yields.Considering the high cost of commercial growth medium, we took advantage of the pathogenicity of strain SCQ1, chose silkworm pupae as a low cost substrate to produce prodigiosin. SCQ1showed delayed growth in the medium which only contained silkworm pupae without any other supplements, and the fermentation time was extended to112h, but with a relatively high prodigiosin yield of667.23±15.33mg/L. Then we screened nutrient supplements and found that yeast extract and sucrose could stimulate bacterial growth to promote fermentation process, with the time of72h. We also noticed that CaCl2did not show any positive effect on prodigiosin yields under this condition. As mentioned above, by using response surface methodology, the optimal parameters were obtained as:yeast extract6.36g/L, sucrose4.22g/L and silkworm pupae homogenate (SPH)14.24g/L, with the maximum theoretical yields of2,020.66mg/L. Under this condition, the actual production of prodigiosin was confirmed as1,916.08±13.33mg/L which was2.87-fold higher than the initial yields, and1.97-fold higher than optimized commercial growth medium. These results highlighted the potential application of S. marcescens SCQ1as an industrial strain for prodigiosin production, which was also provided a new way for comprehensive utilization of silkworm pupae resources.2. Purification and identification of prodigiosinThere are various prodigiosin-like pigments with different chemical structures and therefore different bioactivities. We have purified and identified the prodigiosin produced by S. marcescens SCQ1. The hydrophobic prodigiosin predominantly distributed in the bacterial precipitation, and a methanol chloroform mixture (95：5, v/v) was applied to extract the pigment. The prodigiosin was preliminarily determined by thin-layer chromatography (TLC), with the Rf value around0.6(petroleum ether:acetic ether=1:1, v/v) which consistent with previous reports. The crude extraction of prodigiosin was further purified by column chromatograph, then vacuum concentrated and freeze-dried to dark red solid form with a green reflex. Color variation of prodigiosin solution in different pH conditions was observed, and it generally showed purple at pH2-3, orange at pH7.2-7.4, yellow at pH10-12, corresponded with the shifting of maximum absorption wavelength. High performance liquid chromatography (HPLC) analysis indicated that the purity of prodigiosin was around95.8%. The prodigiosin sample was then analyzed by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), and the molecular mass was determined as323, consistent with prodigiosin (C20H25N3O, CAS No.82-89-3). Atomic absorption spectroscopy analysis indicated that the calcium content was38.4μg per gram of dry prodigiosin sample, and some other trace elements were also analyzed. In brief, we have purified and determined the chemical structure of prodigiosin produced by strain SCQ1.3. Physiological function of S. marcescens prodigiosinVarious microorganisms produce pyrrole-derived secondary metabolites, e.g. prodigiosin and tambjamine. However, the physiological role of these metabolites still remained unknown and lack of reasonable explanation. We qualitatively investigated the prodigiosin-metal ion interaction based on the color reaction. We found prodigiosin could interact with various metal ions under neutral condition and might form prodigiosin-metal complexes. We have also noticed that prodigiosin-zinc complex exhibited strong fluorescence effect, from yellow to orange. By using disk diffusion method, we found the biosynthesis of prodigiosin could enhance the tolerance of S. marcescens to excess Cu2+by compared with pigmented SCQ1and non-pigmented SCQ1-W mutant. Furthermore, we have observed a non-homogeneous distribution phenomenon of prodigiosin in S. marcescens colony that this pigment precipitated on the surface of colony center and formed a tree-ring structure. Although prodigiosin produced by S. marcescens was reported as non-diffusible pigment, it could be observed diffusing outside but limited near the edge of bacteria colony with prolonged incubation. We found the bacterial prodigiosin was concentrated and stored in a bubble-like structure inside the colony which subsequently secreted outside the colony during certain growth stage. The bubbles fulfilled with prodigiosin was moved for a short distance on the medium surface, then exploded and the red pigment was released. We also found that under normal conditions, even high concentration of prodigiosin did not affect the colony growth, no matter whether secreted by S. marcescens or artificially supplemented. By using Cu2+or Mn2+to regulate bacterial growth, we discovered the metal ion transmission function of "prodigiosin bubble". In one hand, the bacterial growth was extremely inhibited when reached the "prodigiosin bubble" rich in Cu2+, and that eventually led to a hollowed bacteria-free area in the colony with prolonged incubation. On the other hand, the bacterial growth was significantly stimulated when reached the "prodigiosin bubble" rich in Mn2+that led to a raised colony edge.Thus, we hypothesize that prodigiosin may be functionally as a metal ion pool of S. marcescens, and involve with metal ion redistribution in the bacterial community. This physiological role of prodigiosin may enhance both metal ion tolerance and competition of S. marcescens, thus beneficial for environmental adaptation. 4. Antibacterial and anticancer activities of prodigiosinThe antibacterial activity of prodigiosin (C20H25N3O) of many reports are quite different. Although it could be ascribed to different tested strains, purification and testing methods, etc., the key problem is the application of crude prodigiosin extract, which may contain other antibacterial compounds as reported before. Our results indicated that purified prodigiosin did not show any significant antibacterial activity against several clinical isolates, including Staphylococcus aureus, Enterococcus faecalis, Salmonella typhosa, Salmonella paratyphi A, Shigella dysenteriae, Bacillus subtilis, Escherichia coli ATCC25922and multiple-drug-resistant E. coli strains. Besides, prodigiosin only showed slight antifungal effect against Candida albicans and Phialophora verrucosa, but inactive to Cryptococcus neoformans, Aspergillus nidulans, Scedosporium apiospermum. Thus, prodigiosin (C20H25N3O) is generally inactive against several bacterial and fungal clinical isolates, therefore worthless for further investigation.According to2012Chinese cancer registry annual report, the top5cancers in China were lung (18.74%), gastric (12.67%), colorectal (10.30%), liver (10.04%) and esophageal (7.74%) cancer. Besides, lung, liver and gastric cancer showed the highest motility. Prodigiosin was reported as anticancer compound against several cancer cell lines, with significantly different inhibitory efficiency. We found that prodigiosin showed significant anticancer activity against human lung cancer cell line A549, liver cancer cell line HepG2, gastric cancer cell line MGC803and SGC7901, with the50%inhibitory concentration (IC50) value of6.81,8.23,15.62and0.37μmol/L, respectively. Human bone marrow stem cell line (MSCs) was applied to evaluate the cytotoxicity of prodigiosin against non-malignant cells, and the IC50value was26.93μmol/L. The IC50value of prodigiosin against MSCs, which was much higher than the average IC50value over60cancer cell lines reported by American national cancer institute (NIC), revealed the selective cytotoxicity of prodigiosin. The cytotoxic effects of prodigiosin against A549cells was analyzed as an example. We have observed an interesting phenomenon that prodigiosin accumulated in small vesicles, along with the cell death. Early stage of apoptosis was detected by flow cytometry, but the rate of apoptotic cells was stayed at low level, whereas the rate of dead cells was dramatically increased along with the concentration of pordigiosin or the treatment time. While treated with5mg/L prodigiosin, the death rate of A549cells could be increased rapidly from11.2%of4hto94.3%of20h.These results indicate that prodigiosin (CAS No.82-89-3) has selective cytotoxicity against human cancer cells by inducing apoptosis and rapidly leading to death. 5. Anti-BmNPV effect of prodigiosinBmNPV is one of the most common pathogen of silkworms that often lead to serious damage in sericulture industry. BmNPV possesses a very special and highly efficient infection strategy that an outbreak of nucleopolyhedrosis might be caused by only a few infected larvae due to intensive breeding. Therefore, the release of viral occlusion bodies from dead larva is the key step of BmNPV horizontal transmission. Prodigiosin showed unique antiviral effects that blocked BmNPV lytic replication, reduced polyhedra production at a low concentration of10nmol/L, but rapidly killed all infected cells at a relatively high concentration of300-600nmol/L, without cytotoxic effects. Despite of different cell viability, prodigiosin treatment significantly reduced polyhedra production. Actually,300nmol/L prodigiosin treatment rapidly killed70%infected cells within12h, and the rate was increased to90%after24h. Furthermore, prodigiosin (10mg per larva) did not cause any acute toxic effects on healthy silkworm, but infected larvae were rapidly killed that only17.59%±7.46%producing polyhedra. Previous studies indicated that, under cell-free conditions, prodigiosin inhibited both Topo Ⅰ and Ⅱ catalytic activity by directly interfering with the enzyme or bounding with DNA. We found that prodigiosin primarily located in the cytoplasm that cannot directly interact with DNA or DNA topoisomerase. The specific caspase3inhibitor Ac-DEVD-CHO didn’t show any inhibitory effect on BmNPV infected cells, but surprisingly, the general caspase inhibitor Z-VAD-FMK significantly inhibited polyhedra production, suggesting that activation of initiator caspase might be involved with BmNPV replication. Because caspase involved with NF-κB activation, which is also the target of prodigiosin, a specific NF-κB inhibitor BAY11-7082was introduced to BmNPV infected cells. The polyhedra production was inhibited by BAY11-7082which suggested NF-κB might be required for efficient replication of BmNPV.Our research, for the first time, clearly indicated the antiviral activity of prodigiosin (CAS No.82-89-3). Thus, the mechanisms and application of prodigiosin against BmNPV worth for further study. |
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