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2024 Angelika Amon Young Scientist award winners announced

The Koch Institute at MIT is pleased to announce the winners of the 2024 Angelika Amon Young Scientist Award, Anna Uzonyi and Lukas Teoman Henneberg.

Koch Institute
September 3, 2024

The Koch Institute at MIT is pleased to announce the winners of the 2024 Angelika Amon Young Scientist Award, Anna Uzonyi and Lukas Teoman Henneberg.

The prize was established in 2021 to recognize graduate students in the life sciences or biomedical research from institutions outside the United States who embody Dr. Amon’s infectious enthusiasm for discovery science.

Both of this year’s winners work to unravel the fundamental biology of chromatin, the densely structured complex of DNA, RNA, and proteins that makes up a cell’s genetic material.

Uzonyi is pursuing her PhD at the Weizmann Institute of Science in Israel under the supervision of Schraga Schwartz and Yonatan Stelzer. In her thesis, Uzonyi focuses on deciphering the principles of RNA editing code via large-scale systematic probing.

Henneberg is a doctoral candidate in the Department of Molecular Machines and Signaling, at the Max Planck Institute of Biochemistry in Germany, works under the supervision of Professor Brenda Schulman and Professor Matthias Mann. For his research project, he probes active ubiquitin E3 ligase networks within cells. He works on the development of probes targeting active ubiquitin E3 ligases within cells and utilizing them in mass spectrometry-based workflows to explore the response of these ligase networks to cellular signaling pathways and therapeutics.

This fall, Anna Uzonyi and Lukas Teoman Henneberg, will visit the Koch Institute. The MIT community and Amon Lab alumni are invited to attend their scientific presentations on Thursday, November 14 at 2:00 p.m. in the Luria Auditorium, followed by a 3:30 p.m. reception in the KI Galleries.

Uzonyi will present on “Inosine and m6A: Deciphering the deposition and function of adenosine modifications” and Henneberg will present on “Capturing active cellular destroyers: Probing dynamic ubiquitin E3 ligase networks.“

Brain cell types are affected differently by Rett Syndrome mutation

New research from Jaenisch Lab postdoc Danielle Tomasello focuses on an understudied question: how Rett Syndrome affects cell types in the human brain other than neurons.

Greta Friar | Whitehead Institute
September 6, 2024

Rett Syndrome is a X-chromosome-linked neurodevelopmental disorder; it can lead to loss of coordination, mobility, ability to speak, and use of the hands, among other symptoms. The syndrome is typically caused by mutations within the gene MECP2. Researchers in Whitehead Institute Founding Member Rudolf Jaenisch’s lab have studied Rett Syndrome for many years in order to understand the biological mechanisms that cause disease symptoms, and to identify possible avenues for treatments or a cure. Jaenisch and colleagues have gained many insights into the biology of Rett syndrome and developed tools that can rescue neurons from Rett syndrome symptoms in lab models.

However, much about the biology of Rett Syndrome remains unknown. New research from Jaenisch and postdoc in his lab Danielle Tomasello focuses on an understudied question: how Rett Syndrome affects cell types in the human brain other than neurons. Specifically, Tomasello investigated the effects of Rett Syndrome on astrocytes, a type of brain cell that supports and provides energy for neurons. The work, shared in the journal Scientific Reports on September 6, details changes that occur in Rett syndrome astrocytes, in particular in relation to their mitochondria, and shows how these changes directly impact neurons. The findings provide a new framework for thinking about Rett Syndrome and possible new avenues for therapies.

“By considering Rett Syndrome from a different perspective, this project expands our understanding of a multifaceted and thus far incurable disease,” says Jaenisch, who is also a professor of biology at the Massachusetts Institute of Technology.

Energy metabolism in Rett Syndrome

Mitochondria are organelles that generate energy, which cells use to carry out their functions, and mitochondrial dysfunction was known to occur in Rett Syndrome. Jaenisch and Tomasello found that mitochondria in astrocytes are particularly affected, even more so than mitochondria in neurons. Tomasello grew human stem-cell-derived astrocytes in 2D cultures and also grew 3D organoids: mini brain-like tissues that contain multiple cell types growing in a structure that resembles actual brain anatomy. This approach allowed Tomasello to use human cells, rather than an animal model, and to study how cells behave within a brain-like environment.

When the researchers observed Rett astrocytes grown in these conditions, they found that the mitochondria were misshapen: short, small circles instead of large, long ovals. Additional studies showed evidence of the mitochondria experiencing stress and not being able to generate enough energy through their usual processes. The mitochondria did not have enough of the typical proteins they use to make energy, and so began to break down the cell’s supply of the building blocks of proteins, amino acids, for parts to make up for the missing material. Additionally, the researchers observed an increase in reactive oxygen species, byproducts of mitochondrial metabolism that are toxic to the cell.

Further experiments suggested that the cells try to compensate for this mitochondrial stress by increasing transcription of mitochondrial genes. For example, Tomasello found that regions of DNA called promoters that can increase expression of key mitochondrial genes were more open for the cell to use in Rett astrocytes. Altogether, these findings paint a picture of severe mitochondrial dysfunction in Rett astrocytes.

Although mitochondria in Rett neurons did not have such severe defects, astrocytes and neurons have a close relationship. Not only do neurons rely on astrocytes to supply them with energy, they even accept mitochondria from astrocytes to use for themselves. Jaenisch and Tomasello found that neurons take up dysfunctional mitochondria from Rett astrocytes at a higher rate than they take up mitochondria from unaffected astrocytes. This means that the effects of Rett syndrome on astrocytes have a direct effect on neurons: the dysfunctional mitochondria from the astrocytes end up in the neurons, where they cause damage. Tomasello took mitochondria from Rett astrocytes and placed them on both healthy and Rett neurons. In either case, the neurons took up the dysfunctional mitochondria in large numbers and then experienced significant problems. The neurons entered a hyperexcitable state that is ultimately toxic to the brain. The neurons also contained higher levels of reactive oxygen species, the toxic byproducts of mitochondrial metabolism, which can cause widespread damage. These effects occurred even in otherwise healthy neurons that did not themselves contain a Rett-causing MECP2 mutation.

“This shows that in order to understand Rett Syndrome, we need to look beyond what’s happening in neurons to other cell types,” Tomasello says.

Learning about the role that astrocytes play in Rett Syndrome could provide new avenues for therapies. The researchers found that supplying affected astrocytes with healthy mitochondria helped them to recover normal mitochondrial function. This suggests to Tomasello that one possibility for future Rett Syndrome therapies could be something that either targets mitochondria, or supplies additional mitochondria through the bloodstream.

Together, these insights and their possible medical implications demonstrate the importance of taking a broader look at the foundational biology underlying a disease.

Pursuing the secrets of a stealthy parasite

By unraveling the genetic pathways that help Toxoplasma gondii persist in human cells, Sebastian Lourido hopes to find new ways to treat toxoplasmosis.

Anne Trafton | MIT News
August 25, 2024

Toxoplasma gondii, the parasite that causes toxoplasmosis, is believed to infect as much as one-third of the world’s population. Many of those people have no symptoms, but the parasite can remain dormant for years and later reawaken to cause disease in anyone who becomes immunocompromised.

Why this single-celled parasite is so widespread, and what triggers it to reemerge, are questions that intrigue Sebastian Lourido, an associate professor of biology at MIT and member of the Whitehead Institute for Biomedical Research. In his lab, research is unraveling the genetic pathways that help to keep the parasite in a dormant state, and the factors that lead it to burst free from that state.

“One of the missions of my lab to improve our ability to manipulate the parasite genome, and to do that at a scale that allows us to ask questions about the functions of many genes, or even the entire genome, in a variety of contexts,” Lourido says.

There are drugs that can treat the acute symptoms of Toxoplasma infection, which include headache, fever, and inflammation of the heart and lungs. However, once the parasite enters the dormant stage, those drugs don’t affect it. Lourido hopes that his lab’s work will lead to potential new treatments for this stage, as well as drugs that could combat similar parasites such as a tickborne parasite known as Babesia, which is becoming more common in New England.

“There are a lot of people who are affected by these parasites, and parasitology often doesn’t get the attention that it deserves at the highest levels of research. It’s really important to bring the latest scientific advances, the latest tools, and NetBet sportthe latest concepts to the field of parasitology,” Lourido says.

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As a child in Cali, Colombia, Lourido was enthralled by what he could see through the microscopes at his mother’s medical genetics lab at the University of Valle del Cauca. His father ran the family’s farm and also worked in government, at one point serving as interim governor of the state.

“From my mom, I was exposed to the ideas of gene expression and the influence of genetics on biology, and I think that really sparked an early interest in understanding biology at a fundamental level,” Lourido says. “On the other hand, my dad was in agriculture, and so there were other influences there around how the environment shapes biology.”

Lourido decided to go to college in the United States, in part because at the time, in the early 2000s, Colombia was experiencing a surge in violence. He was also drawn to the idea of attending a liberal arts college, where he could study both science and art. He ended up going to Tulane University, where he double-majored in fine arts and cell and molecular biology.

As an artist, Lourido focused on printmaking and painting. One area he especially enjoyed was stone lithography, which involves etching images on large blocks of limestone with oil-based inks, treating the images with chemicals, and then transferring the images onto paper using a large press.

“I ended up doing a lot of printmaking, which I think attracted me because it felt like a mode of expression that leveraged different techniques and technical elements,” he says.

At the same time, he worked in a biology lab that studied Daphnia, tiny crustaceans found in fresh water that have helped scientists learn about how organisms can develop new traits in response to changes to their environment. As an undergraduate, he helped develop ways to use viruses to introduce new genes into Daphnia. By the time he graduated from Tulane, Lourido had decided to go into science rather than art.

“I had really fallen in love with lab science as an undergrad. I loved the freedom and the creativity that came from it, the ability to work in teams and to build on ideas, to not have to completely reinvent the entire system, but really be able to develop it over a longer period of time,” he says.

After graduating from college, Lourido spent two years in Germany, working at the Max Planck Institute for Infection Biology. In Arturo Zychlinksy’s lab, Lourido studied two bacteria known as Shigella and Salmonella, which can cause severe illnesses, including diarrhea. His studies there helped to reveal how these bacteria get into cells and how they modify the host cells’ own pathways to help them replicate inside cells.

As a graduate student at Washington University in St. Louis, Lourido worked in several labs focusing on different aspects of microbiology, including virology and bacteriology, but eventually ended up working with David Sibley, a prominent researcher specializing in Toxoplasma.

“I had not thought much about Toxoplasma before going to graduate school,” Lourido recalls. “I was pretty unaware of parasitology in general, despite some undergrad courses, which honestly very superficially treated the subject. What I liked about it was here was a system where we knew so little — organisms that are so different from the textbook models of eukaryotic cells.”

Toxoplasma gondii belongs to a group of parasites known as apicomplexans — a type of protozoans that can cause a variety of diseases. After infecting a human host, Toxoplasma gondii can hide from the immune system for decades, usually in cysts found in the brain or muscles. Lourido found the organism especially intriguing because as a 17-year-old, he had been diagnosed with toxoplasmosis. His only symptom was swollen glands, but doctors found that his blood contained antibodies against Toxoplasma.

“It is really fascinating that in all of these people, about a quarter to a third of the world’s population, the parasite persists. Chances are I still have live parasites somewhere in my body, and if I became immunocompromised, it would become a big problem. They would start replicating in an uncontrolled fashion,” he says.

A transformative approach

One of the challenges in studying Toxoplasma is that the organism’s genetics are very different from those of either bacteria or other eukaryotes such as yeast and mammals. That makes it harder to study parasitic gene functions by mutating or knocking out the genes.

Because of that difficulty, it took Lourido his entire graduate career to study the functions of just a couple of Toxoplasma genes. After finishing his PhD, he started his own lab as a fellow at the Whitehead Institute and began working on ways to study the Toxoplasma genome at a larger scale, using the CRISPR genome-editing technique.

With CRISPR, scientists can systematically knock out every gene in the genome and then study how each missing gene affects parasite function and survival.

“Through the adaptation of CRISPR to Toxoplasma, we’ve been able to survey the entire parasite genome. That has been transformative,” says Lourido, who became a Whitehead member and MIT faculty member in 2017. “Since its original application in 2016, we’ve been able to uncover mechanisms of drug resistance and susceptibility, trace metabolic pathways, and explore many other aspects of parasite biology.”

Using CRISPR-based screens, Lourido’s lab has identified a regulatory gene called BFD1 that appears to drive the expression of genes that the parasite needs for long-term survival within a host. His lab has also revealed many of the molecular steps required for the parasite to shift between active and dormant states.

“We’re actively working to understand how environmental inputs end up guiding the parasite in one direction or another,” Lourido says. “They seem to preferentially go into those chronic stages in certain cells like neurons or muscle cells, and they proliferate more exuberantly in the acute phase when nutrient conditions are appropriate or when there are low levels of immunity in the host.”

Talented high schoolers excel while they explore the brain

Over six years of operation, pre-college outreach programs administered by Mandana Sassanfar, Senior Lecturer and Director of Diversity and Outreach, have placed seven exceptional pre-college students, often from underserved or underrepresented backgrounds, with research groups in The Picower Institute.

David Orenstein | The Picower Institute for Learning and Memory
August 14, 2024

During the pandemic, when many classes delivered online could barely hold students’ attention, Presley Simelus became captivated by the subject of biology thanks to their boundless curiosity and their uncommonly engaging teacher at Prospect Hill Academy Charter School in Cambridge. Meanwhile for Eli Hanechak, the science bug must have bit her very early. She’s wanted to be a doctor for as long as she can remember and in fifth grade built a model of a space station the size of a car out of duct tape, cardboard and broomsticks.

Not every teenager is expected to want to spend their summer breaks exploring science at a bench in an MIT lab, but each year students like Simelus and Hanechak, who have a distinct passion for research, can bring that to The Picower Institute and other research entities around MIT. Over six years of operation, pre-college outreach programs administered by Mandana Sassanfar, Director of Diversity and Outreach, have placed seven exceptional pre-college students, often from underserved or underrepresented backgrounds, with research groups in The Picower Institute. Despite their relative lack of experience compared to the technicians, graduate students, postdocs and professors around them, the students typically thrive.

“Eli has been a wonderful addition to our lab for the summer,” said Kendyll Burnell, the graduate student in the lab of Professor Elly Nedivi who has been working closely with Hanechak. “She is a hard worker, has caught on to techniques quickly, and is constantly asking excellent questions about science and doing research.”

Simelus, too, has been not only learning but also contributing, said their summer host, Yire Jeong, a postdoc in the lab of Associate Professor Gloria Choi.

“Presley has been amazing in our lab, and I was impressed by Presley’s eagerness to learn so much about neuroscience,” Jeong said. “Even when facing technical difficulties, Presley diligently worked to overcome them and achieved meaningful results.”

‘Dive into it’

Simelus, who hails from Everett, Mass., and will be enrolling in Swarthmore College this fall to study biochemistry, first came to MIT through the Leah Knox Scholars Program. Friends who’d been in the program before encouraged them to apply and they got in. During five weeks last summer Simelus and their cohort of fellow Leah Knox high-schoolers had the geeky pleasure of extracting bacteria out of the Charles River and performing a battery of tests to genetically characterize the novel organisms they found. Sassanfar noted that Simelus did the lab work exceptionally well, which is something she looks for when determining whom she might invite back the next summer to do research in an MIT Brain and Cognitive Sciences or Biology lab.

This spring when it came time for Simelus to decide where they might like to take that opportunity, they chose the Choi lab, which studies how the central nervous systems and immune systems interact, sometimes with consequences relevant to disorders including autism. Those keywords intrigued Simelus but really they made the choice because of the potential to learn something entirely new.

“It was all this stuff I just simply wasn’t familiar with and I wanted to learn more about it,” Simelus said. “With Gloria’s lab I was truly mystified and I wanted to dive into it. That’s the reason I chose it.”

This summer Simelus has been working with Jeong on a study of how brain cell activity differs when mice are sick vs. when they are well. The project has involved imaging neurons in the brain to detect telltale signs of recent activation, expression of a protein called c-fos. Learning about neuroscience and gaining skills like preparing, staining and imaging tissue have been a very fulfilling outcome of the internship, Simelus said.

“I truly have learned so much about neuroscience,” they said. “I feel like the field, anything related to the brain or neuroscience, is always under this sort of veil and nobody really knows what’s going on. But I feel like my time at the Choi lab has really allowed me to see what neuroscience is about. It’s taught be more about the brain itself and also more about different biology techniques and skills I might need.”

Now the only problem, Simelus said, is that there are even more things to be deeply curious about. Simelus feels committed to harnessing the life sciences in some way in the future to sustain human life and experience. And as someone who not only plays the viola but also composes, they’ve begun thinking more about how the brain responds to music.

There will no doubt be many chances to continue exploring these interests at Swarthmore, but during the summer at MIT, Simelus said they’ve expanded their horizons while still hanging out with friends, some of whom have been working in other nearby labs.

“I don’t think I would have changed my summer,” Simelus said.

‘The perfect opportunity’

Hanechak lives in the tiny Western Massachusetts town of Russell (population: 1,643) and commutes 45 minutes to Pope Francis Preparatory School in Springfield, where she is a rising senior.

In her freshman year at a different school, she yearned for an extra challenge so she got involved netbet online sports bettingin science fair. Interested in medicine, but eager for a project in which she could make a difference without having clinical credentials, she chose to work on reducing pollution by developing a microbe-derived enzyme that could biodegrade plastics. She had read about such enzymes in the research literature and learned that they don’t work as well as engineers have hoped. In successive years she has scrounged lab space and general supervision in labs at Westfield State University and UMass Amherst to create and screen beneficial mutations in the enzyme and to synthesize structures that might help the enzyme work better. The enzyme she presented at the International Science and Engineering Fair last year can degrade plastics in 24 hours.

Sasssanfar, who also directs the Massachusetts Junior Academy of Science (MassJAS), learned of Hanechak’s award-winning science fair presentation and invited her to present at the MassJAS symposium, held at MIT last October. Hanechak did so well, Sassanfar said, she earned a spot present at the American Junior Academy of Science meeting (adjacent to the American Association for the Advancement of Science Annual Meeting) in Denver in February. She also earned Sassanfar’s invitation to join a lab this summer at MIT.

Hanechak has long had an MIT pennant on her wall at home and has admired MIT as a place where regardless of one’s background, if one has a passion for science and technology, that’s what matters.

“No one in my family has gone to college and no one has been involved in a science-related career of any kind,” she said. “One of the reasons MIT has always stood out to me is that there are especially great minds here, but they didn’t all come from established families or super prestigious backgrounds or anything like that. They kind of just were able to make their own way.”

Moreover, the chance to come to MIT to learn about the brain in the Nedivi lab seemed like a great step to take toward that longer-term goal of medicine.

“It seemed like the perfect opportunity to start transitioning into what I want my career to look like and to get some experience doing neuroscience research,” Hanechak said. “I’m very glad I’m able to have this summer experience, like learning the techniques. When I go into my college major of neuroscience, I will have a good background of what I’m doing, besides just my environmental research.”

With Burnell, Hanechak is working on finding a DNA promoter specific for a rare but interesting kind of neuron in the visual cortex, where the brain processes what the eyes see. Finding this genetic signature would allow the lab to label these cells and image them under the microscope, so that they could see how the cells contribute to visual processing.

Hanechak acknowledged she was anxious at first about joining a bigger lab with scientists who have much more experience.

“But my entire summer has been incredibly gratifying and exciting—just being able to work in Cambridge, and live in this area, and experience city life, and then also be in a lab environment where it’s so collaborative and everyone’s very friendly,” she said.

For many teens, summer provides a chance to do what they want to do. Simelus and Hanechak chose the opportunity to explore the brain at The Picower Institute and have made the most of it.

Two Whitehead Institute graduate researchers awarded the 2024 Regeneron Prize for Creative Innovation

Whitehead Institute graduate student researchers Christopher Giuliano (Lourido Lab) and Julian Roessler (Hrvatin Lab) have been awarded the 2024 Regeneron Prize for Creative Innovation.

Merrill Meadow | Whitehead Institute
July 30, 2024

Whitehead Institute graduate student researchers Christopher Giuliano and Julian Roessler have been awarded the 2024 Regeneron Prize for Creative Innovation. In addition, postdoctoral researcher Chen Weng was selected as a finalist in the postdoctoral fellows competition.

The Regeneron Prize, sponsored by global biotechnology company Regeneron Pharmaceuticals, Inc., is a competitive award designed to recognize and honor exceptional talent and originality in biomedical research. Individual graduate students and postdoctoral fellows in the biomedical sciences are nominated by the nation’s top research universities. Then, nominees outline their “Dream Projects” — potentially groundbreaking research projects that they would pursue given unrestricted access to resources and state-of-the-art technology.

The “Dream Project” proposals, presented by the nominees to a selection committee comprised of Regeneron’s leading scientists, are used to evaluate a trainee’s scientific merit, elegance, precision, and creativity. Novel research ideas and out-of-the-box thinking is encouraged — although the proposal must include a strong rationale, basic methodology and design for the project, and a discussion of how its results could advance the field. Both Giuliano and Roessler have been awarded $50,000 for their proposals, which can be used in any way the winners choose. In addition, Weng was awarded $5,000 as a finalist, and Regeneron has made a $10,000 grant to the Whitehead Institute as the home institute of the winners to support its seminar series.

This year’s awards are distinctive in that the two winners are from the same institution: Both Giuliano and Roessler are pursuing their PhDs at Massachusetts Institute of Technology (MIT) and conducting their doctoral research at Whitehead Institute.

Giuliano is a researcher in the lab of Whitehead Institute Member Sebastian Lourido, who is also an associate professor of biology at MIT and holds the Landon Clay Career Development Chair at Whitehead Institute. Giuliano’s Dream Project seeks to address the unique challenges posed by genetically based muscle disorders. “An obstacle in using current gene therapies to treat these conditions,” he explains, “is that muscle tissue comprises large syncytial cells, which contain hundreds of nuclei in a shared cytoplasm. Even when a gene therapy is able to reach an individual muscle cell, it often isn’t able to spread to every nucleus within that cell.” However, certain parasites, like Toxoplasma gondii, thrive because they have the capacity to successfully gain access to and manipulate muscle cells. T. gondii, the primary focus of the Lourido lab’s work, may infect nearly one third of all humans. “My project,” Giuliano says, “would identify the specific biological mechanisms used by the parasites to spread their virulence factor proteins throughout the cell. Using genetic screens for protein spread, we would work toward applying these protein features to improve the efficiency of muscle-directed gene therapies, and ultimately test our system in a mouse model of Duchenne muscular dystrophy.”

Roessler is a researcher in the lab of Whitehead Institute Member Siniša Hrvatin, who is also an assistant professor of biology at MIT. While Roessler’s doctoral research focuses on the neuronal circuitry underlying torpor and hibernation in small mammals, his Dream Project seeks to identify the sensory circuitry regulating the “diving reflex” displayed in land- and sea-dwelling mammals, including humans. The diving reflex occurs when an animal’s face is immersed in cold water, prompting an array of organs to reduce their function in ways that, scientists believe, privileges the flow of oxygen to the brain and muscles. “That this reflex has been conserved across millions of years of mammalian evolution suggests an extraordinary genetic advantage,” Roessler says. “Yet, researchers have given comparatively little attention to the neuronal circuits underlying this reflex, and we don’t understand even the fundamental mechanisms by which the nervous system coincidently detects both cold temperature and the presence of water.” Beyond elucidating a foundational aspect of mammalian biology, Roessler’s projects could, if pursued, underpin new interventions for conditions ranging from migraine headaches to cardiac arrhythmia that might be ameliorated by artificial stimulation or inhibition of the diving response.

Weng is a postdoctoral researcher in the lab of Whitehead Institute Member Jonathan Weissman, who is also a professor of biology at MIT, the Landon T. Clay Professor of Biology at Whitehead Institute, and an Investigator of the Howard Hughes Medical Institute. His Dream Project — which proposes a new approach to using single-cell genealogy to understand factors driving cell line evolution — is an extension of his current work. Indeed, this past year he co-developed a technology that details the family trees of human blood cells and provides new insights into the differences between lineages of hematopoietic stem cells. The technology gives researchers unprecedented access to any human cells’ histories — and a path to resolving previously unanswerable questions.

News Brief: Lamason Lab uncovers seven novel effectors in Rickettsia parkeri infection

The enemy within: new research reveals insights into the arsenal Rickettsia parkeri uses against its host

Lillian Eden | Department of Biology
July 29, 2024

Identifying secreted proteins is critical to understanding how obligately intracellular pathogens hijack host machinery during infection, but identifying them is akin to finding a needle in a haystack.

For then-graduate student Allen Sanderlin, PhD ’24, the first indication that a risky, unlikely project might work was cyan, tic tac-shaped structures seen through a microscope — proof that his bacterial pathogen of interest was labeling its own proteins.  

Sanderlin, a member of the Lamason Lab in the Department of Biology at MIT, studies Rickettsia parkeri, a less virulent relative of the bacterial pathogen that causes Rocky Mountain Spotted Fever, a sometimes severe tickborne illness. No vaccine exists and definitive tests to diagnose an infection by Rickettsia are limited.

Rickettsia species are tricky to work with because they are obligately intracellular pathogens whose entire life cycles occur exclusively inside cells. Many approaches that have advanced our understanding of other bacterial infections and how those pathogens interact with their host aren’t applicable to Rickettsia because they can’t be grown on a plate in a lab setting. 

In a paper recently published in Nature Communications, the Lamason Lab outlines an approach for labeling and isolating R. parkeri proteins released during infection. This research reveals seven previously unknown secreted factors, known as effectors, more than doubling the number of known effectors in R. parkeri. 

Better-studied bacteria are known to hijack the host’s machinery via dozens or hundreds of secreted effectors, whose roles include manipulating the host cell to make it more susceptible to infection. However, finding those effectors in the soup of all other materials within the host cell is akin to looking for a needle in a haystack, with an added twist that researchers aren’t even sure what those needles look like for Rickettsia.  

Approaches that worked to identify the six previously known secreted effectors are limited in their scope. For example, some were found by comparing pathogenic Rickettsia to nonpathogenic strains of the bacteria, or by searching for proteins with domains that overlap with effectors from better-studied bacteria. Predictive modeling, however, relies on proteins netbet online sports bettingbeing evolutionarily conserved. 

“Time and time again, we keep finding that Rickettsia are just weird — or, at least, weird compared to our understanding of other bacteria,” says Sanderlin, the paper’s first author. “This labeling tool allows us to answer some really exciting questions about rickettsial biology that weren’t possible before.”

The cyan tic tacs

To selectively label R. parkeri proteins, Sanderlin used a method called cell-selective bioorthogonal non-canonical amino acid tagging. BONCAT was first described in research from the Tirrell Lab at Caltech. The Lamason Lab, however, is the first group to use the tool successfully in an obligate intracellular bacterial pathogen; the thrilling moment when Sanderlin saw cyan tic-tac shapes indicated successfully labeling only the pathogen, not the host. 

Sanderlin next used an approach called selective lysis, carefully breaking open the host cell while leaving the pathogen, filled with labeled proteins, intact. This allowed him to extract proteins that R. parkeri had released into its host because the only labeled proteins amid other host cell material were effectors the pathogen had secreted. 

Sanderlin had successfully isolated and identified seven needles in the haystack, effectors never before identified in Rickettsia biology. The novel secreted rickettsial factors are dubbed SrfA, SrfB, SrfC, SrfD, SrfE, SrfF, and SrfG. 

“Every grad student wants to be able to name something,” Sanderlin says. “The most exciting — but frustrating — thing was that these proteins don’t look like anything we’ve seen before.”

Special delivery

Theoretically, Sanderlin says, once the effectors are secreted, they work independently from the bacteria — a driver delivering a pizza does not need to check back in with the store at every merge or turn.

Since SrfA-G didn’t resemble other known effectors or host proteins the pathogen could be mimicking during infection, Sanderlin then tried to answer some basic questions about their behavior. Where the effectors localize, meaning where in the cell they go, could hint at their purpose and what further experiments could be used to investigate it. 

To determine where the effectors were going, Sanderlin added the effectors he’d found to uninfected cells by introducing DNA that caused human cell lines to express those proteins. The experiment succeeded: he discovered that different Srfs went to different places throughout the host cells.  

SrfF and SrfG are found throughout the cytoplasm, whereas SrfB localizes to the mitochondria. That was especially intriguing because its structure is not predicted to interact with or find its way to the mitochondria, and the organelle appears unchanged despite the presence of the effector. 

Further, SrfC and SrfD found their way to the endoplasmic reticulum. The ER would be especially useful for a pathogen to appropriate, given that it is a dynamic organelle present throughout the cell and has many essential roles, including synthesizing proteins and metabolizing lipids. 

Aside from where effectors localize, knowing what they may interact with is critical. Sanderlin showed that SrfD interacts with Sec61, a protein complex that delivers proteins across the ER membrane. In keeping with the theme of the novelty of Sanderlin’s findings, SrfD does not resemble any proteins known to interact with the ER or Sec61. 

With this tool, Sanderlin identified novel proteins whose binding partners and role during infection can now be studied further. 

“These results are exciting but tantalizing,” Sanderlin says. “What Rickettsia secrete — the effectors, what they are, and what they do is, by and large, still a black box.” 

There are very likely other effectors in the proverbial cellular haystack. Sanderlin found that SrfA-G are not found in every species of Rickettsia, and his experiments were solely conducted with Rickettsia at late stages of infection — earlier windows of time may make use of different effectors. This research was also carried out in human cell lines, so there may be an entirely separate repertoire of effectors in ticks, which are responsible for spreading the pathogen.

Expanding Tool Development

Becky Lamason, the senior author of the Nature Communications paper, noted that this tool is one of a few avenues the lab is exploring to investigate R. parkeri, including a paper in the Journal of Bacteriology on conditional genetic manipulation. Characterizing how the pathogen behaves with or without a particular effector is leaps and bounds ahead of where the field was just a few years ago when Sanderlin was Lamason’s first graduate student to join the lab.

“What I always hoped for in the lab is to push the technology, but also get to the biology. These are two of what will hopefully be a suite of ways to attack this problem of understanding how these bacteria rewire and manipulate the host cell,” Lamason says. “We’re excited, but we’ve only scratched the surface.”

Unusual Labmates: Meet tardigrades, the crafters of nature’s ultimate survival kit

Whitehead Institute Member Siniša Hrvatin is studying tardigrades to decode the mechanisms enabling their survival in extreme environmental conditions. Learn about the biology of these microscopic “water bears” and what makes them a particularly fascinating model organism.

Shafaq Zia | Whitehead Institute
July 23, 2024

Tardigrades, also affectionately known as “water bears” or “moss piglets”, are remarkable microscopic organisms that have captured the imagination of scientists and nature enthusiasts alike.

With adults measuring anywhere from 0.2 to 1.2 millimeters in length — as big as a grain of salt — tardigrades possess the astounding ability to survive harsh environmental conditions. These resilient creatures have been found in habitats ranging from the depths of oceans and hot radioactive springs to the frigid expanses of Antarctica. It is their unparalleled adaptability that makes them invaluable as a model organism for researchers like Whitehead Institute Member Siniša Hrvatin, who’s studying physiological adaptation in animals with a focus on states that can slow down tissue damage, disease progression, and even aging.

Follow along to learn what’s behind tardigrades’ nearly indestructible nature, how researchers at Whitehead Institute — and beyond — are studying them, and what insights this work can offer into long-term organ preservation, space exploration, and more.

Big discovery of a tiny creature

In 1773, German naturalist Johann August Ephraim Goeze was analyzing moss samples under a microscope when he stumbled upon an unusual creature. Captivated by its peculiar appearance, he continued his observations and documented the discovery of Kleiner Wasserbär, translating to “little water bear”, in his publication. This work also featured the first-ever drawing of a tardigrade.

Since then, researchers’ understanding of this remarkable organism has evolved alongside advancements in imaging technology. Today, tardigrades are recognized as bilaterally symmetrical invertebrates with two eyes and eight chubby legs adorned with hook-like claws. Often described as a mix between nematodes and insects, these extremophiles are able to withstand freezing, intense radiation, vacuum of outer space, desiccation, chemical treatments, and possibly more.

And the best part? Despite their otherworldly appearance and surprising capabilities, tardigrades share plenty of similarities with larger, more complex organisms, including possessing a primordial brain, muscles, and even a digestive system.

The biology of an extremophile

Researchers trace the evolutionary origins of tardigrades back to panarthropods, a group that includes now-extinct worm-like organisms called lobopodians. To date, over a thousand species of tardigrades have been identified, with terrestrial species inhabiting environments like moss, leaf litter, and lichen, grassland, and deserts while aquatic ones are found in both fresh and saltwater.

Little is known about tardigrades’ diet but researchers are particularly drawn to herbivorous ones that like to munch on single-celled algae and thrive in water. There’s good reason for it: algae are inexpensive to grow in the lab with just light and basic nutrients. But it’s not just their diet that makes tardigrades an attractive model organism — they also have a short generation time (11 to 14 days), with eggs hatching within a four-day span. In fact, some species are able to reproduce without sexual reproduction through a process called parthenogenesis, during which the female egg undergoes cell division without fertilization by a male gamete.

Although genomic resources for studying tardigrades are limited to only a few species, researchers from Keio University and University of Edinburgh have successfully sequenced the genome of a moss-residing tardigrade commonly used in research called Hypsibius exemplaris. Its genome is less than half the size of a Drosophila melanogaster genome, consisting of 105 million base pairs that serve as the building blocks of DNA.

In spite of their small genome — and only a few thousand cells in the body — tardigrades have a well-defined miniaturized body plan, consisting of a head and four segments, that holds valuable insights for researchers looking to decode their adaptation prowess.

Inside tardigrade research at Whitehead Institute

In 2022, as Hrvatin was setting up his lab at Whitehead Institute, a question lingered in his mind. “I was trying to find animals that can survive being frozen for long periods of time and then continue living,” he says. “But there are not that many that fit the bill.”

Then, an undergraduate student at Massachusetts Institute of Technology (MIT) expressed her enthusiasm for astrobiology — the study of life across the universe — and highlighted tardigrades as a favorite among space researchers. Hrvatin was intrigued.

Up until this point, his research had centered upon two states of dormancy, or reduced metabolic activity, in animals: hibernation and a shorter, less intense torpor. But tardigrades possessed a survival mechanism unlike any other. When faced with harsh conditions like dehydration, they would expel water, retract their head and legs, and curl up in a small, dry ball, entering a state of suspended animation called crytobiosis or tun formation.

For decades, researchers hypothesized that the tun state might be responsible for tardigrades’ unparalleled ability to withstand a myriad of environmental assaults, including extremely low temperature. However, recent work has revealed that these animals utilize a separate and unique adaptation, distinct from the tun state, to survive being frozen for extended periods. In fact, preliminary evidence from a preprint by a team of scientists at UC Berkeley and UC San Francisco illustrates unique patterns of how tardigrades survive freezing while hydrated in water.

This phenomenon is markedly different from hibernation and its cousin torpor. “Unlike animals lowering their body temperature, we’re talking about putting tardigrades at minus 180 degrees Celsius, and then thawing them,” says Hrvatin. In fact, cryobiosis is so intense that tardigrades’ metabolic activity drops to undetectable levels, rendering them virtually, but not quite, dead. The organisms can then remain in this state from months to years, only to revive as healthy when conditions become favorable once again.

netbet sports bettingFrozen in time

In 2014, a group of Japanese researchers at Tokyo’s National Institute for Polar Research undertook an intriguing experiment. They began by thawing moss samples collected from East Antarctica in November 1983. Then, they carefully teased apart each sample using tweezers to retrieve tardigrades that might be nestled within. Among the tardigrades the researchers found, two stood out: Sleeping Beauty 1 and Sleeping Beauty 2 who were believed to be undergoing cold induced-dormancy. Turns out, the researchers were right — within the first day of being placed in the Petri dish with water, the tardigrades began exhibiting slow movements despite having been frozen for over 30 years.

The Swiss army knife in tardigrades’ toolbox

Yet, the remarkable resilience of tardigrades continues to baffle scientists. Recently, they’ve uncovered what could be another potential weapon in the creatures’ arsenal: intrinsically disordered proteins or IDPs. Picture them as putty — a group of proteins that do not have a well-defined three-dimensional structure and can interact with other molecules to produce a range of different outcomes. Some researchers have linked these tardigrade-specific IDPs to the animals extraordinary resilience: under extreme heat, these proteins remain stable. And when desiccated, they form protective glasses that shield cells and vital enzymes from dehydration.

If confirmed, the implications of this work would extend beyond tardigrades’ survival, potentially revolutionizing dry vaccine storage and the development of drought-resistant crops.

MIT affiliates named 2024 HHMI Investigators

Four faculty members and four others with MIT ties are recognized for pushing the boundaries of science and for creating highly inclusive and collaborative research environments.

School of Science
July 23, 2024

The Howard Hughes Medical Institute (HHMI) today announced its 2024 investigators, four of whom hail from the School of Science at MIT: Steven Flavell, Mary Gehring, Mehrad Jazayeri, and Gene-Wei Li.

Four others with MIT ties were also honored: Jonathan Abraham, graduate of the Harvard/MIT MD-PhD Program; Dmitriy Aronov PhD ’10; Vijay Sankaran, graduate of the Harvard/MIT MD-PhD Program; and Steven McCarroll, institute member of the Broad Institute of MIT and Harvard.

Every three years, HHMI selects roughly two dozen new investigators who have significantly impacted their chosen disciplines to receive a substantial and completely discretionary grant. This funding can be reviewed and renewed indefinitely. The award, which totals roughly $11 million per investigator over the next seven years, enables scientists to continue working at their current institution, paying their full salary while providing financial support for researchers to be flexible enough to go wherever their scientific inquiries take them.

Of the almost 1,000 applicants this year, 26 investigators were selected for their ability to push the boundaries of science and for their efforts to create highly inclusive and collaborative research environments.

“When scientists create environments in which others can thrive, we all benefit,” says HHMI president Erin O’Shea. “These newest HHMI Investigators are extraordinary, not only because of their outstanding research endeavors but also because they mentor and empower the next generation of scientists to work alongside them at the cutting edge.”

Steven Flavell

Steven Flavell, associate professor of brain and cognitive sciences and investigator in the Picower Institute for Learning and Memory, seeks to uncover the neural mechanisms that generate the internal states of the brain, for example, different motivational and arousal states. Working in the model organism, the C. elegans worm, the lab has used genetic, systems, and computational approaches to relate neural activity across the brain to precise features of the animal’s behavior. In addition, they have mapped out the anatomical and functional organization of the serotonin system, mapping out how it modulates the internal state of C. elegans. As a newly named HHMI Investigator, Flavell will pursue research that he hopes will build a foundational understanding of how internal states arise and influence behavior in nervous systems in general. The work will employ brain-wide neural recordings, computational modeling, expansive research on neuromodulatory system organization, and studies of how the synaptic wiring of the nervous system constrains an animal’s ability to generate different internal states.

“I think that it should be possible to define the basis of internal states in C. elegans in concrete terms,” Flavell says. “If we can build a thread of understanding from the molecular architecture of neuromodulatory systems, to changes in brain-wide activity, to state-dependent changes in behavior, then I think we’ll be in a much better place as a field to think about the basis of brain states in more complex animals.”

Mary Gehring

Mary Gehring, professor of biology and core member and David Baltimore Chair in Biomedical Research at the Whitehead Institute for Biomedical Research, studies how plant epigenetics modulates plant growth and development, with a long-term goal of uncovering the essential genetic and epigenetic elements of plant seed biology. Ultimately, the Gehring Lab’s work provides the scientific foundations for engineering alternative modes of seed development and improving plant resiliency at a time when worldwide agriculture is in a uniquely precarious position due to climate changes.

The Gehring Lab uses genetic, genomic, computational, synthetic, and evolutionary approaches to explore heritable traits by investigating repetitive sequences, DNA methylation, and chromatin structure. The lab primarily uses the model plant A. thaliana, a member of the mustard family and the first plant to have its genome sequenced.

“I’m pleased that HHMI has been expanding its support for plant biology, and gratified that our lab will benefit from its generous support,” Gehring says. “The appointment gives us the freedom to step back, take a fresh look at the scientific opportunities before us, and pursue the ones that most interest us. And that’s a very exciting prospect.”

Mehrad Jazayeri

Mehrdad Jazayeri, a professor of brain and cognitive sciences and an investigator at the McGovern Institute for Brain Research, studies how physiological processes in the brain give rise to the abilities of the mind. Work in the Jazayeri Lab brings together ideas from cognitive science, neuroscience, and machine learning with experimental data in humans, animals, and computer models to develop a computational understanding of how the brain creates internal representations, or models, of the external world.

Before coming to MIT in 2013, Jazayeri received his BS in electrical engineering, majoring in telecommunications, from Sharif University of Technology in Tehran, Iran. He completed his MS in physiology at the University of Toronto and his PhD in neuroscience at New York University.

With his appointment to HHMI, Jazayeri plans to explore how the brain enables rapid learning and flexible behavior — central aspects of intelligence that have been difficult to study using traditional neuroscience approaches.

“This is a recognition of my lab’s past accomplishments and the promise of the exciting research we want to embark on,” he says. “I am looking forward to engaging with this wonderful community and making new friends and colleagues while we elevate our science to the next level.”

Gene-Wei Li,

Gene-Wei Li, associate professor of biology, has been working on quantifying the amount of proteins cells produce and how protein synthesis is orchestrated within the cell since opening his lab at MIT in 2015.

Li, whose background is in physics, credits the lab’s findings to the skills and communication among his research team, allowing them to explore the unexpected questions that arise in the lab.

For example, two of his graduate student researchers found that the coordination between transcription and translation fundamentally differs between the model organisms E. coli and B. subtilis. In B. subtilis, the ribosome lags far behind RNA polymerase, a process the lab termed “runaway transcription.” The discovery revealed that this kind of uncoupling between transcription and translation is widespread across many species of bacteria, a study that contradicted the long-standing dogma of molecular biology that the machinery of protein synthesis and RNA polymerase work side-by-side in all bacteria.

The support from HHMI enables Li and his team the flexibility to pursue the basic research that leads to discoveries at their discretion.

“Having this award allows us to be bold and to do things at a scale that wasn’t possible before,” Li says. “The discovery of runaway transcription is a great example. We didn’t have a traditional grant for that.”

Gene silencing tool has a need for speed

Small changes in the molecular machines that carry out RNA interference can lead to big differences in the efficacy of gene silencing. These new findings from the Bartel Lab have implications for the design of gene-silencing therapeutics.

Greta Friar | Whitehead Institute
July 17, 2024

RNA interference (RNAi) is a process that many NetBet live casinoorganisms, including humans, use to decrease the activity of target RNAs in cells by triggering their degradation or slicing them in half. If the target is a messenger RNA, the intermediary between gene and protein, then RNAi can decrease or completely silence expression of the gene. Researchers figured out how to tailor RNAi to target different RNAs, and since then it has been used as a research tool to silence genes of interest. RNAi is also used in a growing number of therapeutics to silence genes that contribute to disease.

However, researchers still do not understand some of the biochemistry underlying RNAi. Slight differences in the design of the RNAi machinery can lead to big differences in how effective it is at decreasing gene expression. Through trial and error, researchers have worked out guidelines for making the most effective RNAi tools without understanding exactly why they work. However, Whitehead Institute Member David Bartel and graduate student in his lab Peter Wang have now dug deeper to figure out the mechanics of the main cellular machine involved in RNAi. The researchers’ findings, shared in Molecular Cell on July 17, not only provide explanations for some of the known rules for RNAi tool design, but also provide new insights that could improve future designs.

Slicing speed is highly variable

The cellular machine that carries out RNAi has two main parts. One is a guide RNA, a tiny RNA typically only 22 bases or nucleotides long. RNA, like DNA, is made of four possible bases, although RNA has the base uracil (U) instead of the DNA base thymine (T). RNA bases bind to each other in certain pairings—guanines (G) pair to cytosines (C) and adenines (A) pair to U’s—and the sequence of bases in the guide RNA corresponds to a complementary sequence within the target RNA. When the guide RNA comes across a target, the corresponding bases pair up, binding the RNAs. Then the other part of the RNAi machine, an Argonaute protein bound to the guide RNA, can slice the target RNA in half or trigger the cell to break it down more gradually.

In humans, AGO2 is the Argonaute protein that is best at slicing. Only a couple dozen RNA targets actually get sliced, but these few targets play essential roles in processes such as neuron signal control and accurate body shape formation. Slicing is also important for RNAi tools and therapeutics.

In order for AGO2 to slice its target, the target must be in the exact right position. As the guide and target RNAs bind together, they go through a series of motions to ultimately form a double helix. Only in that configuration can AGO2 slice the target.

Researchers had assumed that AGO2 slices through different target RNAs at roughly the same rate, because most research into this process used the same few guide RNAs. These guide RNAs happen to have similar features, and so similar slicing kinetics—but they turn out not to be representative of most guide RNAs.

Wang paired AGO2 with a larger variety of guide RNAs and measured the rate at which each AGO2-guide RNA complex sliced its targets. He found big differences. Whereas the commonly used guide RNAs might differ in their slicing rate by 2-fold, the larger pool of guide RNAs differed by as much as 250-fold. The slicing rates were often much slower than the researchers expected. Previously, researchers thought that all targets could be sliced relatively quickly, so the rate wasn’t considered as a limiting factor – other parts of the process were thought to determine the overall pace – but Wang found that slicing can sometimes be the slowest step.

“The important consideration is whether the slicing rate is faster or slower than other processes in the cell,” Wang says. “We found that for many guide RNAs, the slicing rate was the limiting factor. As such, it impacted the efficacy.”

The slower AGO2 is to slice targets, the more messenger RNAs will remain intact to be made into protein, meaning that the corresponding gene will continue being expressed. The researchers observed this in action: the guide RNAs with slower slicing rates decreased target gene expression by less than the faster ones.

Small changes lead to big differences in slicing rate

Next, the researchers explored what could be causing such big differences in slicing rate between guide RNAs. They mutated guide RNAs to swap out single bases along the guide RNA’s sequence—say, switching the 10th base in the sequence from a C to an A—and measured how this changed the slicing rate.

“The important consideration is whether the slicing rate is faster or slower than other processes in the cell,” Wang says. “We found that for many guide RNAs, the slicing rate was the limiting factor. As such, it impacted the efficacy.”

The researchers found that slicing rate increased when the base at position 7 was an A or a U. The bases A and U pair more weakly than C and G. The researchers found that having a weak A-U pair at that position, or a fully mismatched pair at position 6 or 7, may allow a kink to form in the double helix shape that actually makes the target easier to slice. Wang also found that slicing rate increases with certain substitutions at the 10th and the 17th base positions, although the researchers could not yet determine possible underlying mechanisms.

These observations correspond to existing recommendations for RNAi design, such as not using a G at position 7. The new work demonstrates that the reason these recommendations work is because they affect the slicing rate, and, in the case of position 7, the new work further identifies the specific mechanism at play.

Interplay between regions matters

People designing synthetic guide RNAs thought that the bases at the tail end, past the 16th position, were not very important. This is because in the case of the most commonly used guide RNAs, the target will be rapidly cleaved even if all of the tail end positions are mismatches that cannot pair.

However, Wang and Bartel found that the identity of the tail end bases are only irrelevant in a specific scenario that happens to be true of the most commonly used guide RNAs: when the bases in the center of the guide RNA (positions 9-12) are strong-pairing Cs and Gs. When the center pairings are weak, then the tail end bases need to be perfect matches to the target RNA. The researchers found that guide RNAs could have up to a 600-fold difference in tolerance for tail end mismatches based on the strength of their central pairings.

The reason for this difference has to do with the final set of motions that the two RNAs must perform in order to assume their final double helix shape. A perfectly paired tail end makes it easier for the RNAs to complete these motions. However, a strong enough center can pull the RNAs into the double helix even if the tail ends are not ideally suited for doing so.

The observation that weak central pairing requires perfect or near perfect tail end matches could provide a useful new guideline for designing synthetic RNAs. Any guide RNA runs the risk of sometimes binding other messenger RNAs that are similar enough to the intended target RNA. In the case of a therapy, this off-target binding can lead to negative side effects. Bartel and Wang suggest that researchers could design guide RNAs with weak centers, which would require more perfect pairing in the tail end, so that the guide RNA will be less likely to bind non-target RNAs; only the perfect pairing of the target’s RNA sequence would suffice.

Altogether, Wang and Bartel’s findings explain how small differences between guide RNAs can make such large differences in the efficacy of RNAi, providing a rationale for the long-standing RNAi design guidelines. Some of the findings even suggest new guidelines that could help with future synthetic guide RNA designs.

“Discovering the interplay between the center and tail end of the guide RNA was unexpected and satisfying,” says Bartel, who is also a professor at the Massachusetts Institute of Technology and a Howard Hughes Medical Investigator. “It explains why, even though the guidelines suggested that tail-end sequence doesn’t matter, the target RNAs that are sliced in our cells do have pairing to the tail end. This observation could prove useful to reduce off-target effects in RNAi therapeutics.”

A genome-wide screen in live hosts reveals new secrets of parasite infection

Researchers in the Lourido Lab performed the first genome-wide screen of Toxoplasma gondii in live hosts, revealing genes that are important for infection but previously undetected in cell culture experiments. 

Greta Friar | Whitehead Institute
July 8, 2024

Apicomplexan parasites are a common cause of disease, infecting hundreds of millions of people each year. They are responsible for spreading malaria; cryptosporidiosis – a severe childhood diarrheal disease; and toxoplasmosis – a disease that endangers immune compromised people and fetuses, and is the reason why pregnant women are told to avoid changing cat litter. Apicomplexan parasites are very good at infecting humans and many other animals, and persisting inside of them. The more that researchers can learn about how apicomplexans infect hosts, the better they will be able to develop effective treatments against the parasites.

To this end, researchers in Whitehead Institute Member Sebastian Lourido’s lab, led by graduate student Christopher Giuliano, have now completed a genome-wide screen of the apicomplexan parasite Toxoplasma gondii (T. gondii), which causes toxoplasmosis, during its infection of mice. This screen shows how important each gene is for the parasite’s ability to infect a host, providing clues to genes’ functions. In the journal Nature Microbiology on July 8, the researchers share their approach for tracing lineages of parasites in a live host, and some specific findings of interest—including a possible anti-parasitic drug target.

From dish to animal

Researchers in Lourido’s lab previously developed a screen to test the function of every T. gondii gene in cells in a dish in 2016. They used CRISPR gene editing technology to make mutant parasites in which each lineage had one gene inactivated. The researchers could then assess the importance of each gene to a parasite’s fitness, or ability to thrive, based on how well the mutants missing that gene did. If a mutant died off, this implied that its inactivated gene is essential for the parasite’s survival.

This screen taught the researchers a lot about T. gondii’s biology but faced a common limitation: the parasites were studied in a dish rather than a live host. Cell culture provides an easier way to study parasites, but the conditions are not the same as what parasites face in an animal host. A host’s body is a more complex and dynamic environment, so it may require parasites to rely on genes that they don’t need in the artificial setting of cell culture.

To overcome this limitation, researchers in Lourido’s lab figured out how to repeat the T. gondii genome-wide screen, which their colleagues in the lab had previously done in cell culture, in live mice. This was a massive undertaking, which required solving various technical challenges and running a large number of parallel experiments. T. gondii has around eight thousand genes, so the researchers performed pooled experiments, with each mouse getting infected by many different mutants—but not so many as to overwhelm the mouse. This meant that the researchers needed a way to more closely monitor the trajectories of mutants in the mouse. They needed to track the lineages of parasites that carried the same mutation over time, as this would allow them to see how different replicate lineages of a particular mutant performed.

“This is an outstanding resource,” says Lourido. “The results of the screen reveal such a broader spectrum of ways in which the parasites are interacting with hosts, and enrich our perception of the parasites’ abilities and vulnerabilities.”
netbet online sports bettingThe researchers added barcodes to the CRISPR tools that inactivated a gene of interest in the parasite. When they harvested the parasites’ descendants, the barcode would identify the lineage, distinguishing replicate parasites that had been mutated in the same way. This allowed the researchers to use a population-based analytical approach to rule out false results and decrease experimental noise. Then they could draw conclusions about how well each lineage did. Lineage tracing allowed them to map how different populations of parasites traveled throughout the host’s body, and whether some populations grew better in one organ versus another.

The researchers found 237 genes that contribute to the parasite’s fitness more in a live host than in cell culture. Many of these were not previously known to be important for the parasite’s fitness. The genes identified in the current screen are active in different parts of the parasite, and affect diverse aspects of its interactions with a host. The researchers also found instances in which parasite fitness in a live host increased when a gene was inactivated; these genes may be, for example, related to signals that the host immune system uses to detect the parasites. Next, the researchers followed up on several of the fitness-improving genes that stuck out as of particular interest.

Genes that make the difference in a live host

One gene that stuck out was GTP cyclohydrolase I (GCH), which codes for an enzyme involved in the production of the essential nutrient folate. Apicomplexans rely on folate, and so the researchers wanted to understand GCH’s role in securing it for the parasite. Cell culture media contains high levels of folate, and in this nutrient-rich environment, GCH is not essential. However, in a live mouse, the parasite must both scavenge folate and synthesize it using the metabolic pathway containing GCH. Lourido and Giuliano uncovered new details of how that pathway works.

Although previously GCH’s role was not fully understood, the importance of folate for apicomplexans is a well-known vulnerability that has been used to design anti-parasitic therapies. The anti-folate drug pyrimethamine was commonly used to treat malaria, but many parasites have developed resistance to it.

Some drug-resistant apicomplexans have increased the number of GCH gene copies that they have, suggesting that they may be using GCH-mediated folate synthesis to overcome pyrimethamine. The researchers found that combining a GCH inhibitor with pyrimethamine increased the efficacy of the drug against the parasites. The GCH inhibitor was also effective on its own. Unfortunately, the currently available GCH inhibitor targets mammalian as well as parasitic folate pathways, and so is not safe for use in animals. Giuliano and colleagues are working on developing a GCH inhibitor that is parasite-specific as a possible therapy.

“There was an entire half of the folate metabolism pathway that previously looked like it wasn’t important for parasites, simply because people add so much folate to cell culture media,” Giuliano says. “This is a good example of what can be missed in cell culture experiments, and what’s particularly exciting is that the finding has led us to a new drug candidate.”

Another gene of interest was RASP1. The researchers determined that RASP1 is not involved in initial infection attempts, but is needed if the parasites fail and need to mount a second attempt. They found that RASP1 is needed to reload an organelle of the parasites called a rhoptry that the parasites use to breach and reprogram host cells. Without RASP1, the parasites could only deploy one set of rhoptries, and so could only attempt one invasion.

Identifying the function of RASP1 in infection also demonstrated the importance of studying how parasites interact with different cell types. In cell culture, researchers typically culture parasites in fibroblasts, a connective tissue cell. The researchers found that parasites could invade fibroblasts with or without RASP1, suggesting that this cell type is easy for them to invade. However, when the parasites tried to invade macrophages, an immune cell, those without RASP1 often failed, suggesting that macrophages present the parasites with more of a challenge, requiring multiple attempts. The screen uncovered other probable cell-type specific pathways, which would not have been found using only model cell types in a dish.

The screen also highlighted a previously unnamed gene that the researchers are calling GRA72. Previous studies suggested that this gene plays a role in the vacuole or protective envelope that the parasite forms around itself. The Lourido lab researchers confirmed this, and discovered additional details of how the absence of GRA72 disrupts the parasite vacuole.

A rich resource for the future

Lourido, Giuliano, and colleagues hope that their findings will provide new insights into parasite biology and, especially in the case of GCH, lead to new therapies. They intend to continue pulling from the treasure trove of results—their screen identified many other genes of interest that require follow-up—to learn more about apicomplexan parasites and their interactions with mammalian hosts. Lourido says that other researchers in his lab have already used the results of the screen to guide them towards relevant genes and pathways in their own projects.

“This is an outstanding resource,” says Lourido, who is also an associate professor of biology at MIT. “The results of the screen reveal such a broader spectrum of ways in which the parasites are interacting with hosts, and enrich our perception of the parasites’ abilities and vulnerabilities.”