Léo RamosDuring the last week of February, Professor Walter Colli received a phone call informing him that he had won the 2014 Almirante Álvaro Alberto award for Science and Technology. On the other end of the line, giving him this auspicious news, were the then Minister of Science and Technology, Marco Antonio Raupp, and the president of the National Council for Scientific and Technological Development (CNPq), Glaucius Oliva. “It came as a shock,” he said in an interview April 4 on the Pesquisa Brasil show, on Radio USP. This is understandable, since after all, as he put it, “this may be the most important award in the field of Brazilian science.”
This award is granted by CNPq in partnership with the Conrado Wessel Foundation and the Brazilian Navy, based on an in-depth assessment of the names put forth by the scientific community itself, through its associations and societies, and by institutions within the national science and technology system. This assessment takes into consideration the contributions made by the researcher over the course of his career towards progress in his field of knowledge. This year, the field chosen was life sciences.
There is no doubt that the many contributions Colli made towards furthering knowledge about the interaction between the protozoon that causes Chagas disease, Trypanosoma cruzi, and its host cell in the human body, were the determining factors in CNPq’s choice. But he rightly understands that the influence he had on the direction his field took, his dedication to science and technology policy in the strictest sense, and his experience on the National Biosecurity Commission (CTNBio) and on a number of boards also played a role. For example, since 2003, he has been working as adjunct coordinator of life sciences at FAPESP. “I was always around. I was an easy-going type of guy and maybe they liked me,” he jokingly said in that interview with Pesquisa Brasil.
|University of São Paulo (USP): Bachelor’s degree (1962), Doctorate (1966) and Full professor (1971); The Public Health Research Institute of the City of New York: Post-doctoral work (1969)|
|Chemistry Institute, USP|
|More than 100 works published in the specialized literature and 22 book chapters. Supervised the work of 24 interns, including Master’s degree and Doctoral degree candidates and post-doctoral students|
In October he will receive the certificate, medal and the R$ 200,000 that the award includes at a ceremony to be held in Brasília during National Science and Technology Week. In the meantime, earning the Álvaro Alberto award is an excellent pretext for Pesquisa FAPESP to bring its readers a little information about Walter Colli’s personal and scientific background, as he tells it. This means that it comes packaged in the prose of one adept at telling stories with a special flavor to them. He describes main characters, their adventures and the context in which they occur, always under a critical and combative eye, which is at times both funny and moving.
This sums up the following interview (see full version only in portuguese), which describes, among other things, the successful trajectory of Walter Colli, scenes from an old São Paulo and two notable traits of his way of being a scientist: the funny informality, the veritable intimacy with which he treats the his main object of research, T. cruzi, and a certain fearlessness in facing delicate or controversial questions. Such questions include the influence Brazil’s poverty in terms of scientific environment has on the individual trajectory of scientists, or to put it another way, on the impossibility Brazilian researchers face in sustaining and furthering certain discoveries they pioneer until such time as they receive international recognition. Or the intense struggle he waged at CTNBio against those who are opposed to cultivating and consuming genetically modified organisms. These are his words:
Where were you born?
I’m from the city of São Paulo, the Brás neighborhood. I was born on Joli street, which is near Bresser street.
That’s an area of Italian immigrants. What was your family like?
Quite poor. My father was a clerk. He worked at his successful brother’s factory, which manufactured a type of ribbon known as fitilhos. Nobody even knows what that is any more, but it’s a kind of flat thread, used in fine packaging. He earned minimum wage. We all, my father, mother, sister and I, lived in a house in a vila, which is what they used to call alleys. The house had one bedroom, a living room and a kitchen. There was a wood-burning stove, and we would take a bath in a basin in the kitchen, because we had no shower. You had to go downstairs to use the outhouse…
When did your parents arrive in Brazil?
They were Brazilian; it was my grandparents who came from Italy. My father had some talents; he played the violin and was a good house painter. At that time, it was the style to paint a frieze with flowers at the top of the walls, and he did this very well, this was his second profession, but he was barely able to support us. When I was nine and my sister was four, he developed Hodgkin’s lymphoma, which was a fatal disease back then, and died at the age of 45. We were in a hopeless situation. My mother had two brothers who lived with my grandmother in a bigger house. One was the epitome of the bachelor who never married, and the other had a general store on one of the cross streets of Santa Rosa, close to the Municipal Market. That uncle moved away and we went to live with my grandmother and the bachelor uncle. The uncle who moved said he would pay for a typing course for me (nowadays it would be called keyboarding), so today I have a complete course in typing under my belt, with a diploma that I still keep. Then he asked if I could do the invoicing and banking deposits at the factory. In exchange, I would continue to go to school with his help.
Where did you go to elementary school?
To a private school, I think it was a very cheap one, called Externato São João, in Brás. Two sisters, Emygdia and Aquilina de Souza were the owners. They were old-style teachers who were strict, but not too much. They were great and they taught Portuguese well. I will never forget one day when Emygdia came to class – both the Getúlio Vargas dictatorship and the war were winding down, and she said: “Now I can show you something.” She opened the box and took out the flag of São Paulo State.
Until then, the flag had been prohibited.
Absolutely prohibited, since 1932. Afterwards, I started my Junior High studies at a school that would give me access to the Colégio Estadual Antônio Firmino de Proença, in Mooca. I began to go there, and from then on, went only to public institutions. After Junior High school, I went to Roosevelt, which had a separate admissions process.
What were your favorite subjects?
I always liked Portuguese very much, and I liked Latin a lot, which I studied for four years, from my first year on. I was the best student in French class, and later, I ended up going to the United States, and had to speak English, sink or swim! Later, I worked with an Argentine professor for 30 years, so I can speak Spanish, not “Portunhol” (a mixture of Portuguese and Spanish). I also speak a little Italian, because I would always hear my grandmother speaking it.
Speaking of which, during the time you lived with that grandmother, did your mother have to work to support your family?
No, she helped my grandmother at home. She would go buy fresh things at the market every day, and she would walk four blocks there and then come back with a full shopping basket.
At that time, neighborhoods had the smell of fresh greens.
Well, that neighborhood had more than just that, because there was one general store after the other, with rice and beans on the street, sometimes when it would rain, it would smell bad. The street I lived on, Benjamin de Oliveira, as well as Assunção street and Santa Rosa street, would all flood. In general, the houses were raised up and there was a storage area on the bottom. The building my uncle rented had been his and his father’s workshop. Both of them had been artisans and had gone to the School of Fine Arts, which today is at the Pinacoteca Art Gallery.
When you started secondary school, how did you like physics, chemistry and biology at first?
The best part of Roosevelt was the human sciences classes. But I had an exceptional math teacher, Antônio Alves Cruz, who today has a school named after him in Sumaré. He would arrive in the classroom and say: “So-and-so, up to the blackboard! In our last class, we showed that X is equal to this and that, so…” And the student had to know what the correct answer was. The more prepared students would study from another pupil’s notes from the year before, since they knew he always taught the same thing. During the first year, I was held back into the second phase [remedial work]. That scared me, and in the second year, I was the best student in math. To achieve this, I, who was from section 2 B [the classes were divided alphabetically], would go to class in section A, memorize everything and then go to class in my own section. When Professor Cruz would call on me, I would already know the answers. I was bad in physics. I was good in chemistry at the beginning, and so-so in biology. I had one very old teacher, who was also a sister of [Crodowaldo] Pavan, Aída Pavan, but she was a teacher’s aide, she didn’t give theory classes. It was only in the 3rd year that students who were going on to study medicine, biology, veterinary studies, pharmacy etc., would have classes with her, and get to use a microscope. In some of my 3rd year classes, we would go down São Joaquim street to a bar on the corner of Glória street and play pool. One day, the director, a young man who used pomade on his hair, realized what was going on and went there after us. He got up on the pool table and said that when we hit the pinnacles of glory, we would remember him, and he ended by saying: “Go back to class right now!” I learned the physics and biology I needed to be able to go to college more at the college prep course I took. I was doing my senior year in high school in the morning, I would go to my uncle’s general store to make bank deposits and issue invoices in the afternoon, and in the evening, I would go to college prep classes on [Luís Antônio] Brigadeiro street. When I got back home, I still had to find time to study.
So, you were a good student?
I was an excellent student. When test time came, I already knew a lot, I didn’t have to cram or get nervous. I was good at biology and I really liked genetics. I didn’t know how to major in genetics. They told me I had to study biology. But I saw the poverty around me and thought, what am I going to be, a biologist? At that time, I would have had to be a high school teacher because there were very few options. Then I thought: I’ll be a doctor, because that would broaden my financial opportunities. I took the college entrance exams for medicine and passed in 29th place. I also took the exams for biology for night classes and passed in 2nd place. Soon afterwards, I realized that I wouldn’t be able to go to school for both majors at the same time. Medical school required full-time study.
Was your family really proud of you when you got into Medical School?
I think so, but my family was never one to express feelings. But in the neighborhood, I was the dottore. There were only Italians from the south of Italy there, from the Adriatic region, from Bari, immigrants who spoke a dialect that was hard to understand. I remember that right after I graduated, a heavy rain fell and flooded everything, the water got into the stores and there were huge losses. There were some wooden planks that everybody would lay down to make a path to walk on and sandbags to keep the water from going in through the doors, but it would get in anyway. I had to walk over the flooded area, but I didn’t hesitate for a second: I took off my pants, went in the water wearing only my underwear, while the women shouted from the balconies, dottore, dottore…!
During your years at Medical School, what was your professional path like?
Starting my first year, I had a biochemistry class with Professor Isaias Raw. He could never accept the fact that there was no genetics program in the Medical School curriculum, and said that anyone who wanted to learn should go with him. He was going to teach classical genetics. First, the entire class left. In the second class, there were only seven people – they became my friends forever. Third, he called me to come and work with him. I asked what he did, and he said he was a biochemist and that was where I began. Actually, I wanted to be a scientist.
At that time, did you know what a scientist did?
Not at all. But Raw has this fantastic quality about him, which is to deeply trust people when he decides they are worthy of it. So, he would say, “let’s prove this or that.” He would give me yeast and tell me to put it in the refrigerator. I didn’t know what was going on, but when I would come in the next day, the yeast had grown and would overflow through the side of the door… It was crazy! I learned from him that research consists of questions and answers, questions and answers, always, without pressure. At the end of the day, the two or three ideas that he gave me, and I didn’t prove, were ideas that were before their time. Five or six years later, somebody proved them.
He thought that insulin was made as proinsulin; that is, as a single molecule that was later cut in half. And later, this was found to be true. But he wanted me to demonstrate this at a time when nobody knew it. He had an enzyme from the Krebs cycle that used two co-enzymes, and he would say it was not possible. But I wasn’t able to prove anything. I couldn’t even make yeast rise! Two weeks later, he said, “forget it, that’s not a good idea, let’s do something else.”
So, he just left you free to experiment?
Absolutely free. That was when he suggested we crystallize the Cytochrome b5 that he and other American researchers had described and demonstrated in 1955 and 1956. At that time, crystallizing a protein was a major accomplishment. We purified the protein, put it in a tube and it crystallized. He photographed it, sent it to Nature and they accepted it. My first work, while I was still a student, was in Nature. Because it was the tenth protein crystallized in the world, it was luck.
When your medical training ended, how did you end up going into biochemistry?
In the 6th year, we had to be on call at the hospital, but the school understood that some students would do what they called a basic chair, which meant that instead of caring for the patients, they would go back to the laboratory. Ricardo Brentani and I did this. But we also took the exam for the SAMDU, the Home and Urgent Medical Care Service that Brazil had. And then I was on call once a week in Santos, where 250 people on average were seen in a day. When I got there, I said I was a student, not a doctor, and that I was there for a type of internship. The two doctors there responded: “That’s what you think! We’ll divide the patients, 80 for each of us.” I had no choice but to practice a little medicine. In 1963, Raw said that Governor Carvalho Pinto had instituted the full time system, under which Brentani and I wouldn’t get rich, but also wouldn’t starve. “Do you want to be my assistants?” he asked. We accepted on the spot. I went to study biochemistry. Genetics got a little lost in the shuffle and appears in a later story.
When did you go to the United States?
In mid-1963, Raw brought Maynard Pullman to Brazil. Pullman had made a very important discovery in the United States, and had the right to be away for a year. He came with his wife and three daughters and stayed until mid-1964. So, I worked with him; my doctorate was about the new questions he was working on, and which I continued to work on with Raw after Pullman went back. I defended my doctoral dissertation in August 1966. Pullman had said that I should do post-doctoral work with him in New York when I finished my doctorate, so that’s what I did.
What were your doctoral and post-doctoral studies on?
Researchers questioned whether protein synthesis occurs in cell mitochondria, and I showed that it did. But Maynard was very cautious and said that Brazil was a tropical country, so perhaps I was measuring protein synthesis in contaminated bacteria. I bent over backwards to show that there was no contamination, but he never believed it. I defended my doctoral dissertation, but never published the study, and at the end, we lost our status as the first, because others proved what I had shown. This 1967 study was only cited in one paragraph, in one review. But even so, I went to work with Maynard, who is an excellent person, just very cautious. He would only publish in the best journals. I did have some problems with him, but I stayed with it, and we published two excellent studies. Being there, I also connected with a Japanese scientist, Michio Oishi. We would have lunch together every day and planned all the experiments together at lunch. After two years, I asked for a leave of absence from USP and I went to work with him. He was studying modern genetics; he looked at DNA from a chemical standpoint. We published four studies.
Which studies did you publish with Pullman?
In Journal of Biological Chemistry, the best at that time, we published one study on the synthesis of fatty acids in mitochondria and another that showed that ATP [adenosine triphosphate] was the first product made by mitochondria. Was it original? No. But there was a school of thought at that time that said that it wasn’t ADP [adenosine diphosphate] that produced ATP, but AMP [adenosine monophosphate], so we looked at it more closely, and showed that they were wrong. We proved what had already been proven, but it was important at that time.
And with Oishi?
We isolated a gene, the first to be isolated, but not the way isolation is done today. It was 1968 and the restriction enzymes used for cloning only appeared on the circuit in 1974. We broke the DNA up by force. It was put in the ultrasound, broken into pieces, separated by columns, and since the ribosome gene has a different makeup – more GC than AT bases –, the column held more of these genes so we purified them. Depending on the size, and since these are repetitive genes, there is one 23S ribosome gene, one 16S and one 5S, and then there is a spacer, whose size we determined. We showed the topological relationship between the rRNA genes. It was very good. The study was published in 1969, was highly cited at the time, and repeated in books. That’s how I ended up finding genetics in a round-about way. Then there was a study in 1970 and two more in 1971. Between these is the one that is most often cited, which includes another author and which deals with the physical connection of ribosome genes of Bacillus subtilis, published in the Journal of Molecular Biology. We showed that there was a connection between the genes.
In your years in the US, things were not easy in Brazil. That was when Isaias Raw left the country.
That’s true. He was very politically oriented, although he wasn’t connected with any party. He had a lot of friends who I later learned were in the Communist Party. They were serious, they knew what the university should be, and they never thought it was a union. They were very good, and I don’t say that about the entire left. I went back at Christmas 1969. I knew there were problems here, because Raw called me in April 1969 and said he’d been removed from his duties. From my reading, I knew nothing. My degree of awareness about the size of the problems in Brazil was almost non-existent. When I arrived at the airport here, I saw soldiers with bayonets, and it was then I realized the situation was very bad. I was really quite unaware.
But when Isaias Raw arrived in the United States, after being removed from his position, didn’t you talk?
We did. He came to my house, we had dinner, he told me everything, but he did it in the Raw way, which is without any analysis. I only got analyses when Luiz Hildebrando went there, but then what I was most interested in was knowing what had happed in Paris in 1968. Raw said that they had taken over the medical school and they had removed him. It was the medical school that had done this, someone had turned him in, so-and-so had accused him and they had all been kicked out. I didn’t get the full story about what was happening in the country. Not that he didn’t know; he just didn’t talk about it, his attitude was just to move forward.
When you returned, you went back to the Medical School, right?
No. The Chemistry Institute was being built, and there was the notion that we should evolve towards a college. This notion was very strong at the universities, even though we were in a period of dictatorship. This made it possible in 1965 for Newton Sucupira to give his famous opinion on graduate studies. There were people of a very high caliber who fought for reform, such as Anísio Teixeira, and this same reform was supported by Fernando Henrique Cardoso, Arthur Giannotti, Isaias Raw, Alberto Carvalho da Silva, etc. It won out. In 1968 the National Educational Bases and Guidelines Law was implemented, and we were already building the basic institutes to implement the college concept here. Raw was so fanatical about university reform that at the Medical School, he was seen as a traitor to the faculty and to the system of chairs. One night, in August 1965, he had a truck brought to the school and we moved the entire department here. By morning, the 4th floor was empty.
Was it the Physiology Department?
Physiological Chemistry. For example, he brought a Cary 14 spectrophotometer, which was something really new at that time, it was very good. They were heavy and we put them in the truck. I know – it was crazy, but he knew that if we hadn’t done that, it wouldn’t have been allowed. He was the first to arrive and he set himself up in block 10. In 1966, other groups began to come: all the chemists from the Faculty of Philosophy, the biochemists from Odontology, from the Veterinary School, etc. When I returned, I was still with the Medical School, but on January 1, 1970 I became a professor at the Chemistry Institute, in the Biochemistry Department. From that point on, I’ve been going there every day for 44 years, and I’ve never wanted to leave.
Between 1970 and 1995, your laboratory made important scientific discoveries. I’d like to hear more about this.
When I arrived, some young people who were part of Raw’s team were waiting for me. Among them were Maria Julia Manso Alves, Bianca Zingales, Marinei Ferreira Gonçalves, Clara Carniol and Anita Marzzoco, who is the author of biochemistry books. There were three or four experienced people in the laboratory and I decided to work on three lines of research. The first was to continue with what had been developed in the United States. I demonstrated that there was a physical link between 10 genes that were separated by spaces; this raised the question: are these genes co-transcribed or transcribed individually? Until I could prove this, my work led to a series of American groups trying to verify whether this was the same as in Escherichia coli. And science has its favorite bugs. If you work with E. coli, hepatic cells or mice, you are in business. If you work with something like a bacillus, it’s no good, it’s not universal. It is a psychological issue. We published Bianca’s thesis in 1977 (look how long it took!), which proved that the ribosome genes in Bacillus subtilis had a co-transcription mechanism; they were all copied together by a single enzyme. But this wasn’t anything new: the same thing had been demonstrated in 1975 or 1976 for E. coli. So I learned that there was no point in bringing very valuable things from the United States, because if they are important, they would have gotten to them first.
And what about the other two lines of research?
The second line was to study the DNA of Acanthamoeba castellanii, which I did with Anita. It is a huge amoeba, quite beautiful! You look in the microscope and it’s spectacular, it spreads out, and it’s a marvelous thing. It is a free-standing organism, but if it goes up somebody’s nose, it will end up in their brain, and then they will have serious problems. But it’s an opportunistic pathogen. We found three or four types of DNA in this amoeba and we couldn’t understand why, because with regard to the nuclear and mitochondrial DNA it was okay, but what about the other two? Where did they come from? Now we know that these amoeba carry enormous viruses inside them and are used to colonize ocean viruses. We were probably looking at a virus and had no idea.
And the third line?
Julia had learned to work with Trypanosoma cruzi at Professor José Ferreira Fernandes’ laboratory, and I thought: why not Trypanosoma? It’s a national bug that has everything the others have, but without the charm. It has mitochondria, flagellum. And it was my third line.
Let’s begin with your fundamental discovery about the surface of the Trypanosoma membrane.
When I came into contact with Trypanosoma, I reasoned that if it enters a cell, it’s because it sees the cell and the cell sees it. So what we should be studying is the membrane, the structure that sees the exterior. Of course I began with non-infectious Trypanosoma, in the phase in which it is found in the insect. First, because it’s easy to grow in a laboratory, and second, because I was very worried about having problems with the infectious form. I wondered what it would have on the surface: sugar, glycoproteins? At that time, it had already been shown that some plant lectins were capable of recognizing sugar. The most common lectin is concanavalin, which comes from beans. In 1971 I went to the United States to take a course in yeast genetics and stopped in New York, where I met up with a friend who I told about the trouble I was having in purchasing concanavalin. He had some and gave me a bottle of it. I suggested to Julia that we make a solution and put it on top of the Trypanosoma. It did exactly what we expected it to do; they all agglutinated instantly, which was proof that there was a large quantity of sugars on the surface.
Was the solution put in a dish?
No, it was put in a tube in which Trypanosoma was present in a culture, which it needs to live. Bacteria can live without it, but Trypanosoma is a protozoan and only lives in complex undefined medium. That is, you put in salt, sugar; you put everything in and 10% serum, which is undefined. It needs that to live, you don’t know everything that is in there. You study it, analyze it, but can’t come up with a good definition for cruzi. It needs serum so what can you do?
Then what happened?
I published the study saying that T. cruzi had proteins, probably glycoproteins, on the surface, and then I tried to isolate them. I used traditional methods and subjected Trypanosoma to isolation. I ran it under electrophoresis, colored it with sugar dyes and saw that there were four clear bands of sugar-containing molecules. I took one of them, the one that ran the fastest and was the smallest, and in the preliminary studies it seemed to have lipids, sugar and protein. But I thought, this molecule has everything, nobody is going to believe me! That was when an Argentine, Rosa Lederkremer, came to São Paulo, and by luck, ended up at my laboratory. It was great because she’s a chemist and a very good one. We planned that I would do the experiment. I did a gas chromatography, mass spectrometry, a whole series of things that had never been done. And we showed that there was a molecule there, which we named LPPG, lipopeptide phosphoglycan.
Did you publish that right away?
The first study with Julia was published in a very short article in Febs Letters,. Later, with Rosa and Julia, we published in several other journals. We began studying the structure, and maybe that’s how we lost our primacy. As we learned about the structure, we saw that it contained components of molecules that are found in our brain or nervous system. These are the so-called gangliosides and cerebrosides. The structure was similar, but it had a molecule called inositol that is not found in any of these known structures. What we had in fact was a family of new molecules, but we just didn’t realize it because we thought they belonged to the gangliosides. We published several studies on things, part of the lipid structure, then the inositol, but the structure of the sugars gave us a lot of work, and other groups of Brazilians joined in the research. From Rio, Lúcia and José Osvaldo Previato joined, because they had something similar in their hands, but we didn’t realize that it was the same thing. Much later, Lúcia went to France to study the structure of the molecule, using atomic spectrometry devices, and reached the conclusion that it was a new molecule. A short time later, Rosa, already back in Argentina, went to work with Michael Ferguson, in Scotland, and he also determined the structure of the molecule. Those who work in this area know that our discovery was the original molecule. And Michael Ferguson knew it better than anyone. He was doing post-doc studies in New York, working with George Cross. He met Julia and said he couldn’t believe what we were proposing until he could repeat everything and see that we were right. He went to Scotland to study something new, protein anchors, which had been discovered by an Englishman and a Brazilian, Maria Lúcia Cardoso de Almeida, at the Paulista School of Medicine [Unifesp], in African Trypanosoma brucei. And it was by studying these anchors in T. brucei that Ferguson saw that they were similar to the things we had described. They’re practically the same thing.
Is it called the anchor because it really allows the proteins to attach themselves to the surface of the cells?
That’s right; there are two ways for proteins to anchor themselves. They can be superficial and swim in the membrane or they can be transmembranic. A membrane is composed of lipids and they hate water, they are hydrophobic. They have a polar head turned towards the environment where there is water, and another polar head turned inwards where there is also water. But between one extremity and the other, the apolar body repels water and everything that is hydrosoluble. So, in order to keep a protein in the membrane, if it is transmembranic, either a part of the protein is composed of hydrophobic amino acids, and that is the part that is in the membrane, or it is linked to an anchor of lipids, which is what we saw. Actually, we didn’t see the anchor, but rather a structure that is found on all anchors. Ferguson named these structures GIPLs, which stands for glycoinositol phospholipids. The inositol is in there, and it’s the thing that I didn’t pay much attention to that ended up being important.
Does that make you mad at all?
A little, sometimes. Some Argentine colleagues say that we had the anchor in our hand and we threw it overboard. No, what we had was a molecule that later looked like the anchor. We could have said that it was a new molecule in the literature, but we didn’t. This tells me that if I had been in the United States, I would have had a different future.
In civilized countries, there is a lot more critical mass. During the discovery of the DNA helix, when Watson and Crick were building models, one used biological intuition and the other did the calculations to see if it was possible. They arrived at the double helix structure and said it was not possible because it would not stay in space, because where there was an oxygen molecule, there should have been an OH molecule. There had to be a hydrogen molecule there in order to have a hydrogen bridge, otherwise the structure would collapse. Then a chemist, Jerry Donahue, came by and said that at the pH the oxygen was at, it actually wasn’t O, but OH. This type of suggestion happens.
So your complaint is about the poverty of the scientific environment, even in São Paulo?
Remember São Paulo is underdeveloped! Look, I’ve worked in this for so long that I’ve had to learn the chemistry of sugars and lipids. But I’m not an expert, because knowing the chemistry of sugars as Rosa and other Argentines do, because they have a tradition in this, is impossible for me. Now they’ve invited me to give a four-hour class on this at the USP Chemistry Institute because nobody else is able to explain it well. We were alone and we still are.
Once the anchor was discovered, how did your work progress?
In 1980, a paper reviewer returned a study to us because he thought it was time to move on to the forms of T. cruzi that cause the disease. I thought so, too. These forms either come from the patients, in which case the quantities are very small, from the mouse that we have to infect, which is very complicated, or from growing tissue. I didn’t know how to grow tissue, but then there was another coincidence. An advisee of Professor Hugo Armelin, Norma Andrews, today at the University of Maryland, said that she wanted to work with me and knew how to grow tissue. We studied the best ways to obtain the best yield, and this study, “Andrews and Colli, 1982 – Adhesion and interiorization of Trypanosoma cruzi in mammalian cells” is often cited. We were able to find an infectious form of trypomastigotes and we had to convert the laboratory to an NB2 security standard. At that point I began to wonder what was in the membrane of this infectious Trypanosoma, why it is the one that does the invading and not the other. And we saw that it had 10 times fewer GIPLs than the non-infectious form. It down regulates; that is, when it becomes infectious, the production of GIPLs is greatly reduced.
And why is that?
I don’t know, maybe because it doesn’t need them. With some colleagues from Rio de Janeiro, we decided to find out what GIPLs do in the non-infectious form. We saw that they are used by T. cruzi to stick to the intestinal walls of the barbeiro insect. They have to attach themselves in order to become infectious. This is one of the mysteries of the Trypanosoma. It took a long time for us to publish anything about this. It was only in 2007 that “Trypanosoma cruzi: involvement of glycoinositolphospholipids in the attachment to the luminal midgut surface of Rhodnius prolixus” came out. And it’s not an important study, because something similar had already been published about Leishmania.
But going back to the development of your work in your laboratory…
In 1980 or 1981 I received a post-doctoral student from Argentina who had done his doctorate in parasitology and I suggested that we use a lectin that recognizes sialic acid and glucosamine. We made an extract of trypanosoma on a column that connected these two sugars, and all the glycoproteins they contained became attached to the column. From that, all we had to do was to elute the attached material with N-Acetylglucosamine and we were able to isolate a compound that produced a beautiful and marvelous band in the electrophoresis. We called it TC85, and we said it was specific to infectious Trypanosoma cruzi, and therefore was important in the infection. After that, I conducted other studies with Norma and Bianca. Then Julia, who had been abroad, came, and made a monoclonal antibody against this protein. We did a study and demonstrated that it was part of a family, because they were proteins recognized by the same antibody. In the electrophoresis they migrated to different places, but they were all cousins to each other. We called it the TC85 family. With the entry of other researchers, principally Americans and Argentines, the name was changed. These molecules began to be called GP85 because they determined that it was a much more complex and extensive family, they saw how many genes it had, and they did not know if mine was the same as theirs. That’s how things happen in science, names of things get changed. I’m not complaining. Now, when we publish, we use “TC85, an element of the GP85 family.” At the same time, there was another discovery: José Osvaldo and Lúcia Previato suspected that the sialic acid was going directly from a large molecule to the surface of Trypanosoma and in experiments in just non-infectious forms, they determined this indirectly. They published the study in 1985 and they suggested that I investigate this in the infectious form. And, together with Bianca, we demonstrated the existence of this enzyme directly. We gave it a name, sialic acid glycosyltransferase, and we published a study in 1987, showing that the sialic acid jumped directly from a protein to the Trypanosoma. A student who attended our classes told Victor Nussenzweig about this and they began to investigate. They described the gene in a 1994 article, but the name of the enzyme changed, and today it’s called transialidase. And when the name changes, those who were there at the beginning are no longer relevant. That’s my story. I discovered things that changed, perhaps because I didn’t explore them all the way, perhaps because I was not capable of this. It doesn’t matter. The Argentine and American groups, including George Cross, went after this using some important genetic tools, which I don’t fully know how to use, showed that the GP85, including the TC85, and the transialidase were all part of the same family; they were all similar. I mean, you can see that I’m in this story, right?
How about the vesicles of the T. cruzi that you studied?
In 1991 we demonstrated that T. cruzi releases vesicles into the culture medium. Some time later, we analyzed the vesicles and saw that in a culture of mammalian cells, between 3 and 24 hours later, they begin to enter the cytoplasm of the host cell. Within 24 hours, all the vesicles are formed around the cell nucleus. These are pieces of Trypanosoma, but they get to the cell and go into it, they have the same capability as the Trypanosomas. So our hypothesis in the literature is that the vesicles are a message sent by the Trypanosoma to say that it is coming. And something happens that opens the doors of the cells.
And in relation to TC85?
Based on how the other groups have defined the family, my story is on firmer ground. Since then, we’ve been trying to show, with Julia and students, that there are pieces of this glycoprotein – not sugar, but protein – that recognize the host cell. We have published many studies. But other groups have also shown that other molecules are equally important for Trypanosoma penetration. So currently Trypanosoma cruzi enters in several ways. And it may be true. When we make antibodies for TC85, we are able to prevent 70% of Trypanosoma from entering, but others are able to inhibit 70% from entering by using antibodies for other molecules. Everything is able to inhibit 70%, but 30% to 40% still end up entering. So we don’t know how it gets in. Period.
In other words, Trypanosoma cruzi continues to be a topic that is full of mystery.
Although it is very primitive, it evolved in a way that allows it to enter anywhere, into any cell. This is another cruel mystery. If you use any type of cell in a laboratory, it will enter. If you produce a super-infection in a model animal, it will enter the spleen or the liver; but it will not go to the brain, because there is a barrier. In the end though, it is found in the heart and the muscles of the gastrointestinal tract, but disappears from everything else. This is the mystery. It hides.