Antibodies do not only need to be produced from fungi. Researchers at the Biomedical Sciences Institute (ICB) of the University of São Paulo (USP) have extracted from a spider (Acanthoscurria gomesiana) a substance – named gomesin – which functions as an anti-microbe, more efficient and quicker than conventional anti-microbes. It even has a more comprehensive effect: in the laboratory it demonstrated strong action against twenty four species of bacteria, nine fungi and five yeasts. Likely to be able to be tested on human beings in three years, gomesin represents a new line of antibiotics – formed by molecules used by invertebrate animals to combat micro organisms – and is a promising weapon against the advances of super-resistant bacteria, which demand more powerful and efficient medicines, at a speed which the pharmaceutical industry cannot keep up with. Today, it is no longer so simple to treat an infection as it was just after the discovery of penicillin in 1928. At that time, it was enough to have a five to seven days treatment based on antibiotics and the recovery was complete.
“The use of this new generation of antibiotics on infections is very promising”, says Antonio Gildo Bianchi, a professor at the Department of Parasitology at the ICB and the coordinator of the thematic project Genes, Peptides and Proteins of Arthropods of Medical and Veterinary Interest, which has the financial support of R$ 380,600 and plus US$ 358,000 from FAPESP. “Also it is very interesting to discover this application starting from a basic study of the immunological system of invertebrates”, comments Bianchi. In the cattle tick (Boophilus microplus), another invertebrate studied, the ICB researchers have identified three peptides (molecules of low molecular mass formed by a chain of amino acids) which, though they are not as well described as the ones in the spider, do the same job: they put up a first barrier against invading microorganisms. One of them is found in the intestine of the tick and two, that possess anti-bacteria action, in the hemolymph, the blood of the arthropods is called, the group of animals that also includes insects and spiders.
Sirlei Daffre, a researcher with the ICB who is coordinating the sub-project directed towards the identification and description of these new substances, was surprised to discover that one of the peptides of the tick, which acts against bacteria and fungi, is actually a fragment of the hemoglobin (the molecule that transports oxygen to the cells) of cattle. For her, it is an indication that the tick must have one or more enzymes capable of cutting up the hemoglobin of cattle, extracting from it a fragment that works as protection against infections. “It makes sense”, she says. The ticks inhabit regions close to the bull’s genitals, highly susceptible to contamination, and extract from the digested food itself, a peptide that guarantees immunity to it. “It is the perfect parasite.” she comments.
A serious situation
The problem of resistance to antibiotics, which is now beginning to be mollified, is serious mainly in hospitals where infections find a fertile field for their dissemination, due to the low resistance of patients undergoing treatment. According to the Ministry of Health, hospital infection in Brazil is around 13.1% and is still out of control, even with the preventative measures demanded by the Control Program of Hospital Infection of the National Agency of Sanitary Vigilance (ANVS). The overcrowding in hospitals, the indiscriminate use of antibiotics and the lack of training of health professionals, are some of the reasons pointed by the ANVS for the increase in the rates of hospital infection, today the fourth highest cause of death in Brazil.
Of the antibiotics in the market today, the majority work selectively on a group of bacteria, inhibiting their growth and in this way destroying them. However, their action is slow when compared with the bacterium duplication speed, which is only twenty minutes. In Sirlei’s opinion, this difference of performance leads to the formation of generations of more and more resistant bacteria and check the efficiency of the conventional antibiotics. A specialist in the insects’ immunology and molecular biology, the researcher has found an opposing and much more comfortable situation through the advance of her work with gomesin, extracted from spiders provided by the Butantan Institute and looked after at the ICB by the post graduate student Pedro Ismael da Silva Jr. in the laboratory. In the first step towards the purification and description of the anti-microbe peptides, it is generally he who extracts the hemolymph from the dorsal vein of the spiders. In order to do this, the spiders are shaved and maintained at -20°C for 15 minutes.
“Gomesin has a very fast action, quicker than conventional antibiotics as it acts directly on the bacterium membrane”, explains Sirlei. Experiments indicate that the anti-microbe peptides make holes in the membrane of the bacterium and thus the bacterium death is swift. Conventional antibiotics have another mechanism: they act upon the interior of the cell, in a process of formation of proteins and in the synthesis of nucleic acids such as DNA and RNA. That is the reason for their being much slower. These differences became very evident in an experiment that compared conventional antibiotics with a peptide similar to gomesin, named protegrin, extracted from the leucocytes (blood cells) of pigs, animals that have not lost their oldest defense mechanisms even with a more sophisticated immunological system.
The result: the protegrin took some 10 minutes to provoke the reduction of the number of bacteria from 1,000,000 down to 1,000, whilst conventional antibiotics took some 4 hours (in the case of vancomycin) and 24 hours (in the case of norfloxacin). As well as the reaction time, a further point in favor of the peptides are the indications that they provoke fewer side effects and are only slightly active towards muscular cells and other tissue. In principle, they are also only slightly hemolytic or that is to say, they destroy fewer red blood cells than conventional antibiotics, something which could be a saving grace in extremely serious cases, when one is dealing with a highly debilitated organism.
Effective coverage
As well as the low concentration necessary for obtaining the desired effect, the gomesin is a strong candidate to be used as an antibiotic peptide since is possesses a wide range of activity. Tests carried out on cell cultures demonstrate that this peptide presents strong action against 14 bacteria of type gram-positive, 10 bacteria of type gram-negative 9 types of fungi and 5 types of yeast. At the top of the list are three bacteria species that cause hospital infections, Staphylococcus aureus, Staphylococcus saprophyticus, Streptococcus pyogenes as well as Pseudomonas aeruginosa, which also causes infections in the urinary tract and on burns. We can add, equally decimated, Staphylococcus saprophyticus, which brings on a urinary infection, Staphylococcus aureus, which causes meningitis and boils, and Streptococcus pyogenes, responsible for rheumatic fever. The list goes on with Klebsiella pneumoniae, which causes pneumonia; Listeria monocytogenes, associated with meningitis and pneumonia; Candida albicans, the origin of candidiasis ; Cryptococcus neoformans, of meningitis; Salmonella thyphimurium, for salmonella; and Tricophyton mentagrophytes, for skin mycosis. Gomesin also acts against the parasite that causes leishmaniosis, the Leishmania amazonensis.
Peptides exert an important role in the immunological system of animals and plants. They constitute the first barriers against invading bacteria and fungi, before the organism works out more efficient responses through the production of antibodies and of defense cells. For the invertebrates, which can count only upon an inherent immunological system, the peptides are vital. “Differently from the vertebrates”, explains Sirlei, “the invertebrates do not produce antibodies and the response to a microorganism invader is always the same.” The vertebrates can count upon peptides such as defensins, protegrins and lysozymes from their inherent system that act in secretions of the mucous membranes of the respiratory tract, in the digestive and urinary tracts, and in the genitals, preventing microorganisms from entering and installing themselves in the organism.
According to Sirlei, the study on the applications of the peptides, in order to advance, will have to have to count upon a more sophisticated research structure as well as the interest of the pharmaceutical industry. Besides the consistent results, attested by publications in scientific magazines of international circulation – the most recent is in the Journal of Biological Chemistry, which has accepted for publication a study to determine the structure of gomesin –, the ICB team has got another stump card for negotiating the next phase: gomesin has been patented for more than two months. Abroad, research is advancing and there has even been testing on human beings, still distant for Brazil. Sirlei herself is working in collaboration with Philippe Bulet, of the National Council of Scientific Research (CNRS) of Strasbourg (France), who has got together with other researchers to open a company and to produce peptides from arthropods on an industrial scale, with therapeutic ends. In the United States, a research line is at the business phase with protegrin, in search of a compound for the treatment of ulcers in mouth mucous. In Canada, a product for cleaning catheters based on these substances has already been released for use.
Prospects
In Sirlei’s opinion, the application of the peptides has been focused on topical use, but tests carried out on mice indicate that they are also efficient in an injected form. “Oral administration would also be viable, but it depends on the development of a composition that doesn’t alter itself with the action of digestive enzymes”, she emphasizes. When compared to some of the almost 500 peptides already identified, gomesin presents an advantage: it has a relatively small molecular chain with only 18 amino acids. For this reason its production through chemical synthesis becomes reasonably simple.
There is a possibility that gomesin could also be obtained by genetic engineering. Sirlei says that, though this study has not yet been completed, the gene of the spider responsible for the production of this peptide has already been cloned. This also opens up the prospect of, through genetic cloning, to genetically modify mosquitoes that transmit diseases in such a way that they become less welcome to parasites – against which that the anti-microbe peptides also react. Thus, the parasite would be attacked by the peptides in the interior of the insect, breaking with the parasitic cycle.
It is in this direction that another sub-project is heading, also coordinated by Bianchi. For more than a year and a half, under the orientation of Osvaldo Marinotti, who implanted in the ICB the research line with genetically transformed mosquitoes, a team has been working on the identification of the sub species of the Anopheles, the genre of mosquito that transmits malaria, along with a study on its relationship with the Plasmodium parasite, the protozoan that causes the illness. What they want is to thwart the survival of the parasite in the insect. ‘The action of the Plasmodium on humans has been widely studied, but little is known about the relationship between the parasite and the mosquito”, says Bianchi. “It is clear that to create a transgenic mosquito implies a series of ethical questions and demands research about the impact it would have on the environment.” The results, although already very pleasing, should not see any possible application as immediately as the control of infections through the extracted peptides of the invertebrates.
Profile:
• Antonio Gildo de Bianchi, 58 years of age, graduated in Biology in 1967, concluded his doctorate in 1972 and was a substitute professor in 1977 at the Chemical Institute of the University of São Paulo (USP). Currently he is an invited professor at the Parasitology Department of the Biomedical Sciences Institute (ICB) of USP.
• Dr. Sirlei Daffre, 42 years of age, graduated in Biology in 1980, finished her master’s in 1983 and her doctorate in 1988 at the Chemical Institute of USP. In 1992 she concluded her post doctorate on the immunological system of the fruit fly, Drosophila melanogaster, at the University of Stockholm in Sweden. She has been a professor at the Parasitology Department of the ICB of USP since 1988.
Project: Genes, Peptides and Proteins of Arthropods of Medical and Veterinary Interest
Investment: R$ 380,670.00 and a further US$ 358,000.00
