Trypanosomatid protozoa are famous for attacking human beings, not to mention plants and animals of economic interest. Best known among them are the parasites Trypanosoma cruzi, responsible for Chagas disease, T. brucei, which causes African sleeping disease, and those of the genus Leishmania, agents of the different types of leishmaniasis. But to parasitologist Cristina Motta from the Carlos Chagas Filho Biophysics Institute at the Federal University of Rio de Janeiro (UFRJ), this is not the most interesting aspect of these single-celled beings. Some trypanosomatids harbor an intracellular bacterium without which they are unable to survive in nature. And vice versa: the bacterium is also incapable of living alone. This endosymbiosis may help elucidate the origin of eukaryotes (organisms whose genetic material is enclosed within cell nuclei). The cellular organelles – like mitochondria and chloroplasts – found in eukaryotic cells are the result of ancient associations with bacteria.
What we are seeing in trypanosomatids is an intermediate step in organelle evolution. “They are two different beings that met, cohabited in harmony and are now one, since one cannot exist without the other,” says Motta. It is intriguing that each protozoan harbors a single bacterium: the latter has a shorter generation time (interval between cell divisions) than the six hours needed by its host, meaning that numerous bacteria could potentially populate a single protozoan cell. “But this doesn’t happen, which strongly indicates that the protozoan controls the proliferation of the endosymbiont.” This rigorous control is important because, otherwise, the bacterium could become a parasite and overpower – or even kill – its host. Motta and her colleagues have been studying this control, and their results for the protozoan species Strigomonas culicis and Angomonas deanei were published in Frontiers in Microbiology in June 2015 as part of the doctoral thesis defended that same year by Carolina Catta-Preta. By using compounds that inhibit the cell cycle of the host, but normally do not affect bacteria, the researchers showed that the endosymbiont was also prevented from dividing. This is further evidence that the bacterium has lost control over the machinery that makes it divide, which is now run by the trypanosomatid host. When certain points of the protozoan cell cycle were blocked, the endosymbiont started replicating its genetic material, but was unable to complete the process and ended up forming a long filament containing multiple copies of the bacterial DNA.
Normally, when bacteria divide, they first duplicate all their content, including genetic material. A septum is then formed, followed by a constricting ring that “pinches” the parent bacterium into two daughter cells. In the symbiont studied in this case, these typical structures of bacterial division do not form. Years ago, microscopy expert Motta had already observed the coordinated action of Angomonas deanei and its endosymbiotic bacterium during replication. In the composite organism, replication starts with the endosymbiont’s DNA, which stretches out on top of the host’s nucleus until it finally splits in two. According to Motta, the nucleus serves as a topological reference and the bacterium needs to be correctly positioned to successfully divide and guarantee that each new protozoan will carry one symbiont. The next to divide is the kinetoplast, a specialized region of the mitochondrion. The kinetoplast contains the mitochondrial DNA and is associated with the structure that the protozoan uses for locomotion, known as the flagellum. When the division of its nucleus is finally complete, the protozoan is ready to split in two, each half coming away with one endosymbiotic bacterium, as described in a paper published in PLOS One in 2010. Scientists now want to scrutinize the molecular mechanisms involved in this synchronized division of structures. Motta relies on recent technological advances and scientific knowledge that permit the development of projects involving genomes, transcriptomes, and even metabolic networks. “It’s a more integrated view that will give us a deeper understanding of this symbiotic relationship,” she says.
The broad scope of the work calls for an assemblage of different specialties. In Rio de Janeiro, Motta works with colleagues from UFRJ and from the Oswaldo Cruz Institute. But she has also expanded her geographic horizons and dipped into cellular and genetic topics, with some help from collaborators in São Paulo, where she found other researchers interested in the evolutionary aspect of the project. Among them is parasitologist Erney Plessmann de Camargo, from the University of São Paulo (USP). Renowned for his research on T. cruzi (see interview in Pesquisa FAPESP Issue No. 204), Camargo first started studying endosymbiosis in trypanosomatids in the 1980s. More recently, he has sequenced the genomes of five protozoa who harbor symbiotic bacteria, in a partnership with the National Scientific Computing Laboratory (LNCC) in the city of Petrópolis, in the state of Rio de Janeiro. In a study published in 2013 in PLOS One, Motta analyzed the genomes of two of these species and showed that the bacteria had undergone gene loss. “It is a reduced, but quite functional genome, able to complete essential biosynthetic pathways for the protozoan host,” she remarks, comparing it to a bonsai tree.
These results help explain something that caught Motta’s attention when she was 18 years old and beginning an internship at the Carlos Chagas Filho Biophysics Institute: the extremely low nutritional requirements of a trypanosomatid parasite of insects, when compared to other organisms of the same type. Then, as now, an electron microscope helped reveal the symbiotic bacterium. Later, the genomes of protozoa and their respective endosymbionts confirmed the data previously obtained through nutritional and biochemical studies, indicating an intense metabolic exchange between the two organisms.
Thanks to the bacteria, the protozoa can produce practically all the amino acids they need, whereas symbiont-free trypanosomatids need a supplemented culture medium. This also applies to heme, an iron-based compound that is a component of some proteins, like the hemoglobin in blood. “The bacteria synthesize heme, which ends up being important for the protozoan’s growth,” says biologist Sergio Schenkman from the Federal University of São Paulo (Unifesp), co-author of the paper. “The protozoa that cause diseases do not manufacture heme, and that’s why they need to live as parasites.” According to the researcher, this gap gives them a weakness that could be exploited as a weapon to fight, or at least understand, diseases caused by trypanosomatids.
Nutritional self-sufficiency could be essential for the seven species known to harbor endosymbionts. These trypanosomatids infect only insects, which provide inconsistent nutrition. “Protozoa that infest vertebrates find a richer environment, either in the host’s blood or within cells,” Motta compares. Her association with the groups led by Schenkman and by biologist Carolina Elias, at the Butantan Institute, has been ongoing for about a decade and aims to elucidate aspects of the protozoan’s cell cycle, as well as understand how a bacterium can co-evolve with its host cell. “Only T. brucei, which does not have an endosymbiont, had a known cell cycle,” says Schenkman. In his view, an organism can only be understood when its cell division process and the molecules that regulate it are known in detail. This process, known as mitosis, is how these unicellular organisms reproduce. In the case of protozoa and their endosymbionts, scientists aren’t quite there yet. “We don’t know how the host controls the formation of the ring that divides the bacterial cells.”
The goal of the researchers is to progressively refine their understanding of this integrated system. The partnership with Schenkman, which is one facet of the work of Carolina Catta-Preta, focuses among other things on using the RNA interference system – known as RNAi – to influence the cell cycle of A. deanei. This method provides a more precise tool for manipulating specific points of the system that controls cell division, once the target sequences have been identified. The researchers’ as yet unpublished results show that this is a promising route towards supporting and expanding upon the descriptions made earlier using drugs that block bacterial division, among other aspects.
As part of a collaboration with colleagues at the French universities of Lyon and Bordeaux, in addition to the LNCC, Motta has also been investigating the details of the metabolic network and energy metabolism of these organisms, by using a computer to analyze the genetic sequences that have been identified.
A full understanding of these composite organisms is still a long way off, but Motta has her sights set on a much loftier goal. “We use endosymbiosis in trypanosomatids to understand the origin of organelles in eukaryotic cells, as well as their optimized structure and operation,” she reveals. “Determining how a protozoan controls bacterial division has a direct connection to the origin of mitochondria in eukaryotic cells.”
Evolutionary studies show that the symbiotic bacteria of different species of trypanosomatids share a single ancestor, according to a study published in 2013 in BMC Evolutionary Biology, with João Alves from the Institute of Biomedical Sciences (ICB) at USP as first author. The study also indicates a transfer of bacterial genes into the nucleus of the host cell – originating both from endosymbionts and from other species that have since been lost. Some of these gene transfers allowed protozoa to complete the synthesis pathways for amino acids that are essential to their survival.
The single origin of trypanosomatid endosymbionts can also be correlated with the advent of organelles like the mitochondrion, which derives from a single encounter between microorganisms. Endosymbiosis theory, which describes this event, became popular in the 1970s with the publication of The Origin of Eukaryotic Cells by American evolutionist Lynn Margulis. But the theory was first proposed far earlier. In 1905, Russian biologist Konstantin Mereschkowski proposed that plant cell structures could have emerged from a cyanobacterium.
An infinity of studies have corroborated that idea since then, but investigating and discovering how this process could have happened is a privilege of few. Endosymbionts of trypanosomatids are somewhere along the evolutionary path leading from free-living bacteria to intracellular organelles. Having lost most of their genetic material and cell wall, they cannot exist independently in nature. In practice, the association already operates as a single organism, although the protozoan can be “cured” with antibiotics. This so-called cure may be useful to science, but not so much to the patient, which ends up sentenced to a life in the laboratory, supported by nutrients provided by the researchers.
“I see endosymbiosis in trypanosomatids as a case of eternal love, and this has always encouraged me to study that story,” says Motta, comparing this marriage of two microorganisms to the interest that drives her.
Although much of her time is spent at the microscope, in the laboratory, or performing computer analyses, Motta says that her main tool of the trade is thought. So, for over 10 years, she has also dedicated her time to taking specialization courses in philosophy, and she even teaches philosophy to graduate students in the biophysics program at UFRJ. This multidisciplinary view takes her beyond laboratory science and gives her a broad perspective. “Parasitism is also a form of symbiosis, as the term symbiosis means living together,” she says, widening the scope of fascination for her study subject. “It is both sides of a coin in a single being: the protozoan is simultaneously a parasite to the insect and host to the endosymbiont.”
CATTA-PRETA, C. M. et al. Endosymbiosis in trypanosomatid protozoa: the bacterium division is controlled during the host cell cycle. Frontiers in Microbiology. V. 6, article 520. June 2, 2015.
ALVES, J. M. P. et al. Endosymbiosis in trypanosomatids: the genomic cooperation between bacterium and host in the synthesis of essential amino acids is heavily influenced by multiple horizontal gene transfers. BMC Evolutionary Biology. V. 13, p. 190. Sep. 9, 2013.
MOTTA, M. C. M. et al. Predicting the proteins of Angomonas deanei, Strigomonas culicis and their respective endosymbionts reveals new aspects of the Trypanosomatidae family. PLOS One. V. 8, No. 4, e60209. June 2, 2013.
MOTTA, M. C. M. et al. The bacterium endosymbiont of Crithidia deanei undergoes coordinated division with the host cell nucleus. PLOS One. V. 5, No. 8, e12415. Aug. 26, 2010.