抗微生物胜肽與海洋生物技術實驗室
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陳志毅Jyh-Yih Chen
研究員Research Fellow- 專長：Antimicrobial peptides, Marine biotechnology
- 信箱：zoocjy@gate.sinica.edu.tw
- 電話：03-9880544 ext.15
- 位置：B103/MRS
實驗室簡介展開收合
Summary of research accomplishments
The Marine Research Station (MRS) is located at Jiaushi, Ilan, Taiwan. It is a facility of the Institute of Cellular and Organismic Biology for conducting research and development in the fields of marine molecular biology and biotechnology with specific focus on gene transfer, ecophysiology, immunology and pathology of aquatic organisms. My research performance is listed below.
Project 1: Production of transgenic fish with fish or shrimp antimicrobial peptide-fluorescent protein with increased resistance to bacterial pathogens and a bright skin color
Recently, in my laboratory, antimicrobial functions against several pathogens of several antimicrobial peptides (AMPs) were characterized. AMPs are part of organisms’ innate defense against bacteria, fungi, viruses, and other harmful microbes. These AMPs have been shown to lyse bacterial membranes, indicating their potential as drugs against bacterial infections. In Taiwan, many aquaculturists use antibiotics to treat diseased fish, and this practice can lead to antibiotic-resistant bacterial strains. Consuming contaminated fish may induce production of antibiotic-resistant bacterial strains in human. With goal of helping aquaculture industry to find new drugs for the future, we began to study AMP functions with transgenic fish approach. Our results on AMP functions suggest that AMPs can potentially be applied in the aquaculture industry as alternatives to the overuse of antibiotics.
Secondly, ornamental fish are aquaculture species with high economic value. Together with AMP function using transgenic technology, we also produced a large and brightly colour fluorescent fish. We constructed plasmid with mylz promoter driving the green fluorescence protein gene and an antimicrobial peptide gene. The first convict cichlid with the improved muscle-promoter fluorescence protein gene was obtained in my lab on August 15, 2008. These transgenic fluorescent fish were transferred to the Jy Lin Co. In the meantime, the Jy Lin Co. is awaiting approval under laws governing genetically modified organisms (GMOs).
For the above-described project, our important contributions are:
1. One mission of the MRS is to help aquaculture industry improve their products. In my lab, we are the first to produce fluorescent convict cichlid (Cichlasoma nigrofasciatus) in the world and shown below. The transgenic fluorescent convict cichlid can help a Taiwan biotechnology company made earning of NT$100~$200 million over the next few years, as quoted from a fish breeder's speech. The transgenic fluorescent convict cichlid (Cichlasoma nigrofasciatus) helped us won the 2013 Taipei Biotechnology Award shown below.
Project 2: Recombinant antimicrobial peptide protein as fish fodder protected fish from Vibrio vulnificus infection and enhanced immunomodulatory functions
In my laboratory, the antimicrobial functions of several antimicrobial peptides (AMPs) against certain pathogens were characterized. One interesting peptide of 21 amino acids, named epinecidin-1, was isolated from a marine grouper (Epinephelus coioides); epinecidin-1 was found to exhibit considerable antimicrobial activity against bacteria, fungi, viruses, and other harmful microbes. However, there are no specific therapeutic methods for treating fish disease brought about by Vibrio vulnificus infection. Vibrio vulnificus is the causative agent of vibriosis, a hemorrhagic septicaemia affecting a variety of fish species and other aquatic animals, which brings about large economic losses in the aquaculture industry worldwide. Expression of recombinant epinecidin-1 protein in either an E. coli protein expression system or Artemia, and then using the resulting fodder or Artemia to feed fish, may be of potential benefit for aquaculture, as epinecidin-1 in the diet may act as an immunostimulant and antimicrobial agent against Vibrio vulnificus infection in fish. For the above-described project, our important contributions are as follows:
1) Our study used electroporation technology to improve the efficiency of introduction of plasmid DNA into Artemia, and the bactericidal activity of Artemia-expressed epinecidin-1 in zebrafish against Vibrio vulnificus (204) infection.
2) Zebrafish were fed on Artemia expressing recombinant epinecidin-1 peptide-GFP fusion for 7, 14, or 21 days, and our data suggest that the fusion protein may play a role as an immunostimulant after intake into the intestines by enhancing a specific defense mechanism in this organ, thereby increasing disease resistance.
3) We described an E. coli expression system for the large-scale production of the recombinant epinecidin-1/DsRed fusion protein, which showed strong antibacterial activity at microgram concentrations against several bacterial species.
4) V. vulnificus (204) numbers were reduced and survival rates were enhanced by the use of recombinant epinecidin-1/DsRed fusion protein mixed with eel powder as fodder for 30 days.
5) One goal of the MRS is to help the aquaculture industry improve their products. My lab was the first in the world to produce recombinant epinecidin-1 protein in an E. coli protein expression system and Artemia. The transgenic Artemia (containing an epinecidin-1 transgene) can help in Taiwan aquaculture to culture fish larvae that are resistant to pathogen infection. Artemia expressing CMV-gfp-epi plasmid conferred efficient antimicrobial activity in larval fish without introducing drug residues or inducing bacterial drug resistance. Older larvae to younger small fish can be fed on fodder mixed with recombinant epinecidin-1/DsRed fusion protein obtained using the E. coli protein expression system. Both technologies may play important roles as a culture chain from larval to fry (or young fish) for use in aquaculture.
Project 3: Demonstration of antitumor function of fish antimicrobial peptides
We have studied the mechanisms and economic value of naturally-occurring AMPs with activity against various tumor types. In our laboratory, we studied pardaxin (GE33) using molecular approaches employed in cancer research. We found pardaxin (GE33) exerts antitumor function and modulates immune responses in mammalian immune systems
For the above-described project, our important contributions are as follows:
1) Our results indicate that pardaxin selectively targets human cervical carcinoma HeLa cells in an electrostatic manner, resulting in production of ROS to induce oxidative stress and UPR to trigger signal transduction of JNK/c-Jun and PERK/eIF2α/CHOP; in turn, this leads to caspase activation and AIF-dependent apoptotic events, such as loss of mitochondrial membrane potential, decrease of RhoGDI (which is postulated to induce the initial morphology of apoptosis by regulating actin polymerization), and chromatin condensation. Proteomic analysis revealed that pardaxin triggers apoptotic signaling pathways in human cervical carcinoma HeLa cells (cross talk among the UPR, c-Jun, and ROS).
2) Our research results demonstrate that pardaxin selectively triggers the death of cancer cells through a molecular mechanism that involves ER targeting and c-FOS induction. Transcriptome analysis of pardaxin-treated HT-1080 (fibrosarcoma cell line) cells revealed induction of the gene encoding c-FOS, an AP-1 transcription factor. Pardaxin mediates cell death by activating c-FOS, but not other AP-1 transcription factors. Overexpression of c-FOS caused a dramatic increase in cell death, while knockdown of c-FOS induced pardaxin resistance; such effects were observed in both an in vitro cell model and an in vivo xenograft tumor model.
3) An antitumor effect was observed when pardaxin (25 mg/kg; 0.5 mg/day) was used to treat mice for 14 days, which caused significant inhibition of murine MN-11 tumor cell growth in mice. To obtain a greater understanding of the antitumor effects of pardaxin, we examined the antitumor activity, toxicity profile, and maximally-tolerated dose of pardaxin treatment in dogs with different types of refractory tumor. Local injection of pardaxin resulted in a significant reduction of perianal gland adenoma growth between 28 and 38 days post-treatment. Surgical resections of canine histiocytomas appear as large areas of ulceration, suggesting that pardaxin acts like a lytic peptide. However, pardaxin treatment was not associated with significant variations in blood biochemical parameters or secretion of immune-related proteins. Our research results indicate that pardaxin has strong therapeutic potential for treating perianal gland adenomas in dogs. These data suggest that pardaxin may be suitable for veterinary application, and also provide valuable information for veterinary medicine and future human clinical trials. We have applied for Taiwanese patents for the use of pardaxin in dog cancer therapy, and are now conferring with a biotechnological company for technology licensing and technology transfer.
Project 4: Demonstration of antibacterial function and application of fish antimicrobial peptides
Almost all antimicrobial peptides (AMPs) studied in my lab show direct antibacterial or bacteriostatic function against Gram-negative and -positive strains. We studied both the application and basic mechanisms of AMP function.
For the above-described project, our important contributions are as follows:
1) Our research results demonstrated the antimicrobial functions of epinecidin-1, tilapia piscidin 4 (TP4), and tilapia piscidin 3 (TP3) against skin trauma injury-mediated MRSA infection in mice. An excision of one square centimeter was made in the outer skin of mice, and a lethal dose of MRSA was applied in the presence or absence of methicillin, vancomycin, epinecidin-1, TP3, or TP4. Mice that received MRSA or MRSA together with methicillin died in four days, whereas the presence of epinecidin-1, TP3, or TP4 protected the mice against MRSA infection. Epinecidin-1, TP3, or TP4 decreased MRSA bacterial counts in the wounded region, improved wound closure, and increased angiogenesis in the wounded region. In conclusion, these three AMPs may act as an effective antimicrobial agent against clinical antibiotic-resistant MRSA, and may be considered for clinical studies against MRSA infection. These research results were obtained in collaboration with Dr. Chang-Jer Wu (Department of Food Science, National Taiwan Ocean University), as the Marine Research Station is lacking facilities for animal experiments. We have applied for US and Taiwanese patents for these innovations, and are negotiating with biotechnology companies for the technological transfer of epinecidin-1 and TP4 as components of burn treatment medical products.
2) Burns are incredibly painful, and many patients die due to bacterial infections of wounds or burns every year. Pain control and therapeutic treatment for burns and wounds remain challenging due to the complex natural history, unclear aetiology, and poor response towards drugs. Neuropathic pain is a widespread health problem associated with nerve injury, prolonged tissue damage, or injury to the peripheral or central nervous system (CNS), which arise through a number of complex changes occurring at various levels in nociceptive pathways. We cooperated with Dr. Zhi-Hong Wen (Department of Marine Biotechnology and Resources, National Sun Yat-Sen University) and applied his animal system to study the use of the antimicrobial peptide piscidin (PCD)-1 as a novel anti-nociceptive agent. For our in vivo studies, mononeuropathy in rat was induced by chronic constriction injury (CCI), and antinociceptive behaviors were compared between naïve rats and CCI models in the presence or absence of PCD-1 (delivered by intrathecal injection). Similar to gabapentin, PCD-1 exerted antinociceptive activity with an approximately 50% effective dose of 9.5 μg against thermal hyperalgesia in CCI rat models. In CCI rats, PCD-1 had antinociceptive effects against mechanical and cold allodynia, thermal hyperalgesia, and weight-bearing threshold. CCI-mediated activation of microglia, astrocytes, and neuron cells were regulated by PCD-1 in the dorsal horn of rat lumbar spinal cord sections. These research results demonstrated that the marine AMP PCD-1 has anti-nociceptive effects, and thus may have potential for development as an alternative pain-alleviating agent; PCD-1 may be combined with epinecidin-1 or TP4 to exert anti-nociceptive and antimicrobial activity for treatment of wounds or burns. We have applied for US and Taiwanese patents for these inventions.
3)We demonstrated the antimicrobial properties of epinecidin-1 against multi-drug resistant clinical isolates of P. aeruginosa (P. aeruginosa (R)) and P. aeruginosa from ATCC (P. aeruginosa (19660)), both in vitro and in vivo. The minimum inhibitory concentrations (MICs) of the peptide against P. aeruginosa (R) and P. aeruginosa (19660) were studied in comparison with those of imipenem. Our results suggested that P. aeruginosa (R) was more susceptible than P. aeruginosa (19660) to epinecidin-1 in vitro. Epinecidin-1 was highly effective at combating peritonitis infection caused by P. aeruginosa (R) or P. aeruginosa (19660) in mouse models, and did not induce adverse effects on the liver or kidney, or on behavior. These findings indicate that epinecidin-1 enhances the survival rate of mice against the bacterial pathogen P. aeruginosa through both antimicrobial and immunomodulatory roles.