Abstract:

Since the 1920s and 1930s, the discovery and application of antibiotics have saved countless lives. If people have benefited from it before, now the world has to work to solve the consequences of antibiotic abuse - the emergence and spread of resistant bacteria.

In September 2016, 193 member states attending the UN General Assembly signed a declaration promising to strengthen the control of antibiotics. As countries take actions to promote the reduction of antibiotic use, scientists are also actively looking for alternatives to develop antibiotics.

The feed Research Institute of CAAS has such a long-term team dedicated to the research of antimicrobial peptides and antibiotic substitutes. Twenty four European and American scholars jointly reviewed in the academic journal Lancet Infectious Diseases, and listed the antifungal peptides developed by the team as the first ideal substitute of traditional antibiotics. On November 16, 2016, at the 18th China International hi tech Fair held in Shenzhen, the new antimicrobial peptide preparation developed by the team was awarded the excellent product award.

Wang Jianhua, a researcher at the feed Research Institute of the Chinese Academy of Agricultural Sciences and chief scientist of the research team, was interviewed by the reporter of China Science Daily about his research progress.

The crisis of drug-resistant bacteria needs to be resolved

There are "super super bacteria" besides "super bacteria". South China Agricultural University has found a kind of "super super bacteria". According to reports, this kind of drug-resistant bacteria is resistant to carbapenems and myxorubicin because it carries a heterozygous particle. Among them, the latter has been regarded as the "last defense line" of human resistance to drug-resistant bacteria.

With the discovery of "super bacteria", the lineup of drug-resistant bacteria has become more and more complete and powerful. Countries around the world have gradually realized that the abuse of antibiotics and the emergence of drug-resistant bacteria are becoming a major global crisis.

Especially since 2016, this issue has attracted unprecedented attention. The G20 communique in Hangzhou promised to "promote the prudent use of antibiotics"; 193 member states attending the UN General Assembly signed a declaration promising to strengthen the control of antibiotics. In August 2016, China's health and Family Planning Commission jointly issued a letter calling on the whole society to act together to curb the development of drug resistance.

In addition to strengthening supervision in policy, looking for alternatives is also a way to reduce the use of antibiotics. At present, the development of antibiotic substitutes is becoming a priority topic in the field of biomedicine, among which, antimicrobial peptides become the "star of hope" of antibiotic substitutes because of their broad-spectrum and high-efficiency bactericidal activity to bacteria, and they are not easy to produce drug resistance.

According to the data, in 1980, Swedish scientists first induced polypeptides with antibacterial activity in silkworm chrysalis. Since then, scientists have discovered and isolated peptides with antibacterial activity from bacteria, fungi, amphibians, insects, higher plants, mammals and even human body.

"Antimicrobial peptide is a kind of small molecular peptide with biological activity, which is an important part of innate immune system. It has a wide range of sources and broad-spectrum antibacterial effect. It is not easy for bacteria to produce resistance to it." Wang Jianhua told China Science Daily.

Under the continuous cross support of national agricultural science and technology innovation project, 863 program, National Science and technology support program and natural science foundation, Wang Jianhua led the research team to carry out a series of research on the molecular structure, gene expression, bactericidal characteristics and bactericidal mechanism of anti gram positive bacteria antimicrobial peptide, anti gram negative bacteria antimicrobial peptide, Lfcin and its derived peptide.

Ideal substitute for traditional antibiotics

11 years ago, the discovery of hyphomycin was a bright spot. In 2005, scientists isolated a kind of defensin mycelium from saprophytic ascomycetes growing on the surface of Nordic pine forest for the first time. It can prevent the growth of bacteria by blocking the synthesis of bacterial cell wall, and has a strong defensive effect on Gram-positive bacteria (such as Staphylococcus, Streptococcus, pneumococcus, etc.).

Nz2114, a derivative of hyphomycin, is a better derivative peptide obtained by high-throughput mutation than the parent peptide. Its antibacterial activity against Staphylococcus aureus is significantly increased, which is more than 30 times higher than the parent peptide. In order to further enhance the antibacterial activity of nz2114 against Staphylococcus aureus (hereinafter referred to as Staphylococcus aureus), especially against methicillin resistant Staphylococcus aureus (hereinafter referred to as MRSA), Wang Jianhua's team further modified nz2114 and obtained mutant mp1102.

The results showed that the antibacterial activity of MRSA of MP1102 was 15 times higher than that of NZ2114. For example, if mp1102 is used to treat mice infected with Staphylococcus aureus at concentrations of 5mg / kg, 10mg / kg and 20mg / kg, it can kill 90% of Staphylococcus aureus within 12 hours.

At the same time, mp1102 has antibacterial activity against Clostridium perfringens, which can cause gas gangrene, food poisoning and other diseases. "Mechanism study shows that mp1102 has double bactericidal mechanism of destroying cell membrane and interfering with DNA in Clostridium perfringens." Wang Jianhua said that the antibacterial activity of mp1102 against Clostridium perfringens was better than that of mycelium, nz2114 or other antibiotics such as bacitracin zinc, aureomycin and Virginia mycin.

Since then, the team has introduced net positive charge and hydrophobic amino acids into the mycelium mycomycin sequence, and designed the antibacterial peptide mp1106, so that its anti Staphylococcus aureus activity is nearly 40 times higher than the parent peptide. In order to improve the bactericidal targeting of Staphylococcus aureus, the research team connected Staphylococcus aureus pheromone to mycelium to construct Staphylococcus aureus targeted antimicrobial peptide. The results showed that the target peptide had specific bactericidal effect on the target bacteria, but had no bacteriostatic activity on other probiotics such as bacillus, Streptococcus lactis and Enterococcus faecalis.

In addition, Wang Jianhua's team has also successively established efficient production systems of mycelium mycomycin, nz2114, mp1102 and mp1106 to achieve efficient secretion and expression of this series of products, with production of 748 mg / L, 2390 mg / L, 695 mg / L and 2134 mg / L respectively, which is the highest production of antimicrobial peptides of the same type.

The team's work has attracted the attention of international peers. Twenty four European and American scholars jointly reviewed the Lancet Infectious Diseases published this year, and listed the antifungal peptide developed by the team as "the best alternative to traditional antibiotics".

Development prospect
At the 18th China International hi tech Fair held in Shenzhen, Wang Jianhua's team won the excellent product award for the research and development of "new antimicrobial peptide preparation specially for anti gram positive bacteria".

Antimicrobial peptide industry is a new biotechnological industry. For a long time, due to the wide sources and many kinds of antimicrobial peptides, small molecular weight and low content in nature, it is difficult to separate and purify antimicrobial peptides, high cost of chemical synthesis, etc., so it is urgent to establish a high-efficiency and low-cost production path of transgenic. Wang Jianhua's team has made breakthroughs in a series of bottleneck technologies, such as antimicrobial peptide product design and low-cost production.

Tanja Schneider, a representative scholar of German antibacterial microorganism research, and other scholars believe that the high-efficiency production platform of fungal defensins antimicrobial peptides developed by the team is characterized by low cost, easy operation, high yield, and has great development prospects.

Wang Jianhua disclosed to reporters that several MP series varieties have completed the pilot test, have the industrialization level, start some evaluation experiments, and have independent intellectual property rights.

In addition to a series of work on the research of fungal defensins, Wang Jianhua's team also carried out continuous research in the fields of molecular structure, recombinant expression, bactericidal characteristics and bactericidal mechanism of earthworm peptide and its derived peptide, Lfcin and its derived peptide. For example, Wang Jianhua's team designed derivative peptides N2 and N6 on the basis of nz17074, a derivative peptide of earthworm peptide 3. Compared with the parent peptide, N2 and N6 had significantly higher antibacterial activity and stronger environmental stability. At the same time, the team also successfully obtained the active recombinant antibacterial peptide.

Wang Jianhua said that the antibacterial peptide has the function of promoting growth and treating diseases for livestock and poultry. It is a non-toxic, harmless and residue free green product and is expected to become an alternative to antibiotics. "The industrialization of antimicrobial peptide feed additive and its application in pig, chicken, fish and other feeds are of great significance to reduce the use of traditional feed antibiotics and curb the generation of drug resistance of pathogenic bacteria." He said.

Michael zasloff, one of the founders of antimicrobial peptides, highly praised the research of Chinese scientists in his monograph published this year. He said that the fungal defensins developed by Wang Jianhua's team and two other antimicrobial peptides are likely to become the three "first graduates" of antimicrobial peptides successfully entering the clinical treatment field.

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