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Schistosoma mansoni

Inside the parasite

Identification of genes of the leads to new ways of fighting schistosomiasis and gains international prominence

FMRP - USPLodged in the intestines, the worms lay eggs that will be released with the feces.FMRP - USP

Soon after starting the colonization of Brazil, the Portuguese established a regular traffic in African slaves for the colony. Hidden in the bowels of the men who crossed the Atlantic in the hold of the ships, an unwanted passenger arrived in the country, which was to turn into a problem in these tropical lands: they were worms of the Schistosoma genus that cause schistosomiasis, one of the oldest human diseases, registered even in Egyptian mummies 5,000 years old. It is only today, almost five centuries later, that one can size up better this unperceived disembarkation: the species of human parasite that has most prospered here, Schistosoma mansoni, found also in other parts of South America, the Caribbean and Africa, infects about 10 million Brazilians. After the dissemination, over the last few decades, of the use of medicines that combat this parasite, which grows to a length of half a centimeter when lodged in the human body, the risk of dying from schistosomiasis has been halved. The disease, caught by contact with water contaminated with larvae of the worm, is also known as water belly, because of the swelling it causes in the abdomen. Even so, schistosomiasis is still common – or endemic – in the whole of the Northeast and in stretches of the Southeast, particularly in Minas Gerais, with outbreaks dispersed over almost the whole of the national territory.

Until last month, the chapter reserved for Brazil in the history of schistosomiasis could be summed up like this. Today, not any more. In an effort of little over two years, researchers from the Schistosoma mansoni Genome project, financed by FAPESP in the ambit of the ONSA network (a virtual consortium of genomic laboratories in the state of São Paulo) are beginning to write, in the national colors, what may be a new chapter in the trajectory of schistosomiasis – this time, with glad tidings. They determined, completely or in part, the sequences of 92% of the estimated 14,000 genes of the parasite. By analogy with the genetic material of other organisms sequenced, they discovered the function of 45% of the worm’s genes. The other 55% represent totally new sequences – it is not yet known which proteins derive from their action. “A little over half of the parasite’s genes have never been identified in other organisms”, explains Sergio Verjovski-Almeida, from the Chemistry Institute of the University of São Paulo (USP), the coordinator of the project, which has a budget of US$ 1 million and is also supported by the National Council for Scientific and Technological Development (CNPq). “Before, the complete sequences of only 163 of the worm’s genes were known. We have brought this figure up to 510 genes.”

To attain this level of unprecedented information about the genes of the schistosomiasis parasite, the scientists generated 163,000 partial sequences of genes active in the six main stages in the worm’s life cycle, ranging from the forms that live freely in fresh water to those than inhabit their intermediate host, the snail, and those that infest man (please see the life cycle of the parasite above). Before the publication of the results from the ONSA network, there were only 16,000 stretches of expressed sequence tags (ESTs) of the schistosomiasis on the public databases, 75% of them derived from the adult stage of the parasite. “We have increased this figures more than tenfold”, commemorates Emmanuel Dias Neto, from the Psychiatry Institute of USP, who is taking part in the project. This feat ought to be celebrated in double: it results from the application, by Brazilians, of a national method for finding ESTs, called Orestes. Along with Andrew Simpson, today in the New York office of the Ludwig Cancer Research Institute, Dias Neto is one of the inventors of this method, which identifies central regions of expressed genes, which lead to the formation of proteins that act on the different stages under analysis.

The results of this work were announced on September 15, in a ceremony that took place in the Bandeirantes Palace, the seat of the São Paulo government, with the presence of representatives of the São Paulo universities, of the scientific community and of the authorities of the State. “The conclusion of the work with Schistosoma mansoni is one more feat of science in São Paulo and Brazil”, stated Governor Geraldo Alckmin. “We are an example for research in South America”, said FAPESP’s president, Carlos Vogt, also present at the event. In Brasilia, on the day following the presentation of the results, Verjovski, the other 37 researchers of the Schistosoma mansoni Genome and FAPESP won public recognition, in the form of a Vote of Applause, conferred by the Senate in the session on September 16.

The date for making the work public was not chosen by chance. Precisely on this date, the electronic version of the Nature Genetics magazine, one of the scientific periodicals of the greatest prestige, anticipated on its page on the Internet the content of two articles about parasites that cause schistosomiasis: one ten-page text written by the Brazilian scientists from the Schistosoma mansoni Genome project, and another, of nine pages, written by a Chinese group that analyzed genes and proteins of Schistosoma japonicum , the species of the worm that causes the disease in Asia. Done by rival groups, both the articles earned the main headline on the cover of the printed version of the October issue of Nature Genetics.

In the project that rivaled the ONSA initiative, researchers from the Chinese National Human Genome Center, in Shanghai, used the traditional way of looking for ESTs and generated 43,707 fragments of expressed sequences (four times less than the Brazilians), using material collected from only two stages in the life of S. japonicum – the ONSA project worked with six stages of the cycled of S. mansoni. Even so, the Chinese say that their sequences are related to 13,131 of the 15,000 genes that, according to them, constitute the genome of S. japonicum. Only 35% of the genes identified in the Chinese project had never been found before in other organisms, while in the Brazilian work, as was verified, this level reached 55%.

This amount of unprecedented genes may be a result of S. mansoni being a very old worm. According to calculations by the Brazilian team, the parasite differentiated itself from other species of multicellular animals, from a common ancestor, 1 billion years ago. And so originated an independent lineage of beings that have symmetrical left and right sides of the body, but do not have a digestive tube, nor a cavity containing the internal organs. “The S. mansoni is the oldest animal with sequenced genes to show sexual dimorphism”, explains Verjovski. That is: the male and the female of the worm show distinct forms: the specimens of the male sex are larger than those of the female sex. The importance attributed to the article by the São Paulo researchers in Nature Genetics recalls another recent feat of members of the ONSA network. In July 2002, they made the cover of Nature magazine, from which Nature Genetics is derived, when they became the first group in the world to finish the complete sequencing of the genome of a bacterium that attacks plants: Xylella fastidiosa, which causes Citrus Variegated Chlorosis (CVC), or the yellowing disease. The difference is that, now, the feat was divided with the Chinese, perhaps the emerging country that most competes with Brazil in the area of genomics.

In two years, the Schistosoma mansoni Genome involved 37 researchers and nine laboratories from the ONSA network and generated outstanding results, such as the discovery of 46 genes that in principle may be useful in the search for new medicines, besides 28 genes that are candidates for being a target as a new vaccine against the disease. To guarantee the intellectual protection of possible products derived from their findings, the scientists from ONSA have asked for a patent in the United States covering the right of use of 1,000 active fragments of the parasite’s DNA. These bits of genes may be of great value too for creating more effective ways of diagnosing or preventing schistosomiasis. “We want to avoid the biopiracy of our data and to seek partnerships with companies, in order to meet the costs of the studies for developing drugs and vaccines”, FAPESP’s scientific director José Fernando Perez explains. “The genome of the Schistosoma mansoni is an enterprise with a great social impact.”

Today, there are indications that Praziquantel, the medicine most used against schistosomiasis, is losing efficiency, particularly in Africa, the continent most affected. There is still no vaccine against the disease, which affects 200 million persons in 75 countries – a figure surpassed only by malaria, with 300 million cases. The data about the number of deaths as a result of schistosomiasis vary enormously: they range from 11,000 to 200,000 deaths a year, according to the World Health Organization (WHO). There are also permanent damages and a loss in the quality of life associated with the more serious cases of the disease, some 10% of the total. Schistosomiasis is a typical ailment of poor countries or regions, where there is a lack of running water and a sewage network – of basic sanitation, in short -, as well as of educational campaigns capable of preventing contamination. Man eliminates in his feces the worm’s eggs, which, in places where there is no proper sewage system, finds their intermediate host: the freshwater snails of the Biomphalaria genus. The Schistosoma reproduces itself in this mollusk for about 30 days and returns to the water – and this is the stage when it becomes capable of infecting human beings.

Creating a vaccine against schistosomiasis is not easy. It is a task for five or ten years, should some research line prove really promising. The Butantan Institute – one of the centers that participated in the study of the worm’s genome, along with the State University of Campinas (Unicamp) and the Adolfo Lutz Institute – has now tested on mice sequences of DNA that could act as a vaccine against schistosomiasis. In this approach, the researchers alter genes of the parasite that are possibly linked to the development of the disease. Afterwards, they inoculate the rodents with a preparation containing the modified genes and infect them with a large number of worms. Up until now, the team from Butantan has now tested vaccines with eight altered genes. The initial results with two genes proved to be reasonable and are acting as a stimulus for the work to be carried on. “We managed about 35% immunization”, says Luciana Cezar de Cerqueira Leite, from Butantan. “The WHO says that a vaccine with 45% of effectiveness is acceptable.” It should be made clear what these figures mean. In this case, a 35% protection means that, if they come into contact with a population of one hundred parasites, the animals immunized with the most efficient candidates to being DNA vaccines will be infected by 65 parasites. Gene immunization manages to reduce the population of worms in the animal, but does not prevent schistosomiasis from developing.

Intelligent arsenal
Some recently identified genes of S. mansoni may be useful for creating drugs, to the extent that they provide important clues about the mechanisms that make it possible for the parasite to trick the human immune system and remain safe in the bloodstream. One of the most interesting discoveries concerns a group of four genes with similar functions. The cells of the worm use them to make four kinds of chemical compounds that set off an allergic reaction in the human organism – the allergenic toxins. Oddly enough, each one of these toxins is similar to the poison of distinct species of wasp. Synthesizing this chemical arsenal triggers off a specific allergic response in the immune system, which produces antibodies against these toxins, but does not direct its action against the parasite itself. Another discovery of weight: the S. mansoni has a gene that is responsible for producing an anticoagulant protein similar to a toxin found in the poison of snakes. As it does not let the blood of a contaminated individual coagulate around the parasite, this toxin makes it difficult for the Schistosoma mansoni to be identified by the defense system of the organism in which it is installed. As such, it works as one more way for the worm to mislead the immune system.

The research is helping to elucidate the mechanism associated with resistance to Praziquantel. The suspicion was that this medicine acted on a sort of pores in the cells, the calcium channels, produced from an already known gene of the parasite. The drug would thus succeed in neutralizing the transmission of chemical messages between the worm’s cells, making it difficult for them to grow. But, according to this research, there are two other genes capable of leading to the production of other calcium channels, which could replace the blocked ones, besides keeping up communication between the cells of the Schistosoma. The loss of the medicine’s effectiveness may be related to alterations in some of these genes, which could lead to the production of slightly different calcium channels.

Despite being little mentioned in the news in recent years, overshadowed by other tropical diseases, such as the outbreaks of dengue in big cities, schistosomiasis continues to be a serious public health problem in Brazil. Data from the National Health Foundation (Funasa) show that the use of medicines, along with the traditional measures of controlling the population of snails of the Biomphalaria genus and of expanding the sewage systems, has reduced by half the number of deaths resulting from the disease in Brazilian territory. In 1979, seven deaths occurred for each 1 million inhabitants. In 1999, this rate fell to three per million. In the Northeast, the region most affected by the disease, a decrease in deaths was also to be seen, in the order of 50%, over these 20 years. The problem is that the number of deaths per 1 million inhabitants has fallen from 12 to 6. That is, the rate of mortality from schistosomiasis in the Northeast is still double the Brazilian average.

One should not, however, think that this ailment is a scourge that is exclusive to the people in the Northeast. “There is no doubt that the use of medicines has brought down the mortality rate associated with the disease all over the country”, observes parasitologist Paulo Marcos Zech Coelho, the overall coordinator of the Integrated Schistosomiasis Program at the Oswaldo Cruz Foundation (Fiocruz). “But schistosomiasis is still expanding geographically in Brazil, far from being controlled.” According to Coelho, outbreaks of the disease have been discovered in the last few years even in the states of the South, areas previously free from this problem. “Creating an effective vaccine against the disease is difficult, but the data from the genome of the Schistosoma mansoni can help in developing new drugs to treat schistosomiasis.” The hope is that Praziquantel will thus cease to be the only weapon against the disease.

The Project
Schistosoma mansoni Genome; Modality Genome Program – FAPESP;
Coordinator Sergio Verjovski-Almeida – Chemical Institute at USP; Investiment US$ 500,000.00 (FAPESP) and US$ 500,000.00 (CNPq)