Research Area: Microbiology

A sum of their parts

Researchers in the Department of Biology at MIT use an AI-driven approach to computationally predict short amino acid sequences that can bind to or inhibit a target, with a potential for great impact on fundamental biological research and therapeutic applications.

Lillian Eden | Department of Biology
February 6, 2025

All biological function is dependent on how different proteins interact with each other. Protein-protein interactions facilitate everything from transcribing DNA and controlling cell division to higher-level functions in complex organisms.

Much remains unclear about how these functions are orchestrated on the molecular level, however, and how proteins interact with each other — either with other proteins or with copies of themselves. 

Recent findings have revealed that small protein fragments have a lot of functional potential. Even though they are incomplete pieces, short stretches of amino acids can still bind to interfaces of a target protein, recapitulating native interactions. Through this process, they can alter that protein’s function or disrupt its interactions with other proteins. 

Protein fragments could therefore empower both basic research on protein interactions and cellular processes and could potentially have therapeutic applications. 

Recently published in Proceedings of the National Academy of Sciences, a new computational method developed in the Department of Biology at MIT builds on existing AI models to computationally predict protein fragments that can bind to and inhibit full-length proteins in E. coli. Theoretically, this tool could lead to genetically encodable inhibitors against any protein. 

The work was done in the lab of Associate Professor of Biology and HHMI Investigator Gene-Wei Li in collaboration with the lab of Jay A. Stein (1968) Professor of Biology, Professor of Biological Engineering and Department Head Amy Keating.

Leveraging Machine Learning

The program, called FragFold, leverages AlphaFold, an AI model that has led to phenomenal advancements in biology in recent years due to its ability to predict protein folding and protein interactions. 

The goal of the project was to predict fragment inhibitors, which is a novel application of AlphaFold. The researchers on this project confirmed experimentally that more than half of FragFold’s predictions for binding or inhibition were accurate, even when researchers had no previous structural data on the mechanisms of those interactions. 

“Our results suggest that this is a generalizable approach to find binding modes that are likely to inhibit protein function, including for novel protein targets, and you can use these predictions as a starting point for further experiments,” says co-first and corresponding author Andrew Savinov, a postdoc in the Li Lab. “We can really apply this to proteins without known functions, without known interactions, without even known structures, and we can put some credence in these models we’re developing.”

One example is FtsZ, a protein that is key for cell division. It is well-studied but contains a region that is intrinsically disordered and, therefore, especially challenging to study. Disordered proteins are dynamic, and their functional interactions are very likely fleeting — occurring so briefly that current structural biology tools can’t capture a single structure or interaction. 

The researchers leveraged FragFold to explore the activity of fragments of FtsZ, including fragments of the intrinsically disordered region, to identify several new binding interactions with various proteins. This leap in understanding confirms and expands upon previous experiments measuring FtsZ’s biological activity. 

This progress is significant in part because it was made without solving the disordered region’s structure, and because it exhibits the potential power of FragFold.

“This is one example of how AlphaFold is fundamentally changing how we can study molecular and cell biology,” Keating says. “Creative applications of AI methods, such as our work on FragFold, open up unexpected capabilities and new research directions.”

Inhibition, and beyond

The researchers accomplished these predictions by computationally fragmenting each protein and then modeling how those fragments would bind to interaction partners they thought were relevant.

They compared the maps of predicted binding across the entire sequence to the effects of those same fragments in living cells, determined using high-throughput experimental measurements in which millions of cells each produce one type of protein fragment. 

AlphaFold uses co-evolutionary information to predict folding, and typically evaluates the evolutionary history of proteins using something called multiple sequence alignments for every single prediction run. The MSAs are critical, but are a bottleneck for large-scale predictions — they can take a prohibitive amount of time and computational power. 

For FragFold, the researchers instead pre-calculated the MSA for a full-length protein once and used that result to guide the predictions for each fragment of that full-length protein. 

Savinov, together with Keating Lab alum Sebastian Swanson, PhD ‘23, predicted inhibitory fragments of a diverse set of proteins in addition to FtsZ. Among the interactions they explored was a complex between lipopolysaccharide transport proteins LptF and LptG. A protein fragment of LptG inhibited this interaction, presumably disrupting the delivery of lipopolysaccharide, which is a crucial component of the E. coli outer cell membrane essential for cellular fitness.

“The big surprise was that we can predict binding with such high accuracy and, in fact, often predict binding that corresponds to inhibition,” Savinov says. “For every protein we’ve looked at, we’ve been able to find inhibitors.”

The researchers initially focused on protein fragments as inhibitors because whether a fragment could block an essential function in cells is a relatively simple outcome to measure systematically. Looking forward, Savinov is also interested in exploring fragment function outside inhibition, such as fragments that can stabilize the protein they bind to, enhance or alter its function, or trigger protein degradation. 

Design, in principle 

This research is a starting point for developing a systemic understanding of cellular design principles, and what elements deep-learning models may be drawing on to make accurate predictions. 

“There’s a broader, further-reaching goal that we’re building towards,” Savinov says. “Now that we can predict them, can we use the data we have from predictions and experiments to pull out the salient features to figure out what AlphaFold has actually learned about what makes a good inhibitor?” 

Savinov and collaborators also delved further into how protein fragments bind, exploring other protein interactions and mutating specific residues to see how those interactions change how the fragment interacts with its target. 

Experimentally examining the behavior of thousands of mutated fragments within cells, an approach known as deep mutational scanning, revealed key amino acids that are responsible for inhibition. In some cases, the mutated fragments were even more potent inhibitors than their natural, full-length sequences. 

“Unlike previous methods, we are not limited to identifying fragments in experimental structural data,” says Swanson. “The core strength of this work is the interplay between high-throughput experimental inhibition data and the predicted structural models: the experimental data guides us towards the fragments that are particularly interesting, while the structural models predicted by FragFold provide a specific, testable hypothesis for how the fragments function on a molecular level.”

Savinov is excited about the future of this approach and its myriad applications.

“By creating compact, genetically encodable binders, FragFold opens a wide range of possibilities to manipulate protein function,” Li agrees. “We can imagine delivering functionalized fragments that can modify native proteins, change their subcellular localization, and even reprogram them to create new tools for studying cell biology and treating diseases.” 

Kingdoms collide as bacteria and cells form captivating connections

Studying the pathogen R. parkeri, researchers discovered the first evidence of extensive and stable interkingdom contacts between a pathogen and a eukaryotic organelle.

Lillian Eden | Department of Biology
January 24, 2025

In biology textbooks, the endoplasmic reticulum is often portrayed as a distinct, compact organelle near the nucleus, and is commonly known to be responsible for protein trafficking and secretion. In reality, the ER is vast and dynamic, spread throughout the cell and able to establish contact and communication with and between other organelles. These membrane contacts regulate processes as diverse as fat metabolism, sugar metabolism, and immune responses.

Exploring how pathogens manipulate and hijack essential processes to promote their own life cycles can reveal much about fundamental cellular functions and provide insight into viable treatment options for understudied pathogens.

New research from the Lamason Lab in the Department of Biology at MIT recently published in the Journal of Cell Biology has shown that Rickettsia parkeri, a bacterial pathogen that lives freely in the cytosol, can interact in an extensive and stable way with the rough endoplasmic reticulum, forming previously unseen contacts with the organelle.

It’s the first known example of a direct interkingdom contact site between an intracellular bacterial pathogen and a eukaryotic membrane.

The Lamason Lab studies R. parkeri as a model for infection of the more virulent Rickettsia rickettsii. R. rickettsii, carried and transmitted by ticks, causes Rocky Mountain Spotted Fever. Left untreated, the infection can cause symptoms as severe as organ failure and death.

Rickettsia is difficult to study because it is an obligate pathogen, meaning it can only live and reproduce inside living cells, much like a virus. Researchers must get creative to parse out fundamental questions and molecular players in the R. parkeri life cycle, and much remains unclear about how R. parkeri spreads.

Detour to the junction

First author Yamilex Acevedo-Sánchez, a BSG-MSRP-Bio program alum and a graduate student at the time, stumbled across the ER and R. parkeri interactions while trying to observe Rickettsia reaching a cell junction.

The current model for Rickettsia infection involves R. parkeri spreading cell to cell by traveling to the specialized contact sites between cells and being engulfed by the neighboring cell in order to spread. Listeria monocytogenes, which the Lamason Lab also studies, uses actin tails to forcefully propel itself into a neighboring cell. By contrast, R. parkeri can form an actin tail, but loses it before reaching the cell junction. Somehow, R. parkeri is still able to spread to neighboring cells.

After an MIT seminar about the ER’s lesser-known functions, Acevedo-Sánchez developed a cell line to observe whether Rickettsia might be spreading to neighboring cells by hitching a ride on the ER to reach the cell junction.

Instead, she saw an unexpectedly high percentage of R. parkeri surrounded and enveloped by the ER, at a distance of about 55 nanometers. This distance is significant because membrane contacts for interorganelle communication in eukaryotic cells form connections from 10-80 nanometers wide. The researchers ruled out that what they saw was not an immune response, and the sections of the ER interacting with the R. parkeri were still connected to the wider network of the ER.

“I’m of the mind that if you want to learn new biology, just look at cells,” Acevedo-Sánchez says. “Manipulating the organelle that establishes contact with other organelles could be a great way for a pathogen to gain control during infection.”

The stable connections were unexpected because the ER is constantly breaking and reforming connections, lasting seconds or minutes. It was surprising to see the ER stably associating around the bacteria. As a cytosolic pathogen that exists freely in the cytosol of the cells it infects, it was also unexpected to see R. parkeri surrounded by a membrane at all.

Small margins

Acevedo-Sánchez collaborated with the Center for Nanoscale Systems at Harvard University to view her initial observations at higher resolution using focused ion beam scanning electron microscopy. FIB-SEM involves taking a sample of cells and blasting them with a focused ion beam in order to shave off a section of the block of cells. With each layer, a high-resolution image is taken. The result of this process is a stack of images.

From there, Acevedo-Sánchez marked what different areas of the images were — such as the mitochondria, Rickettsia, or the ER — and a program called ORS Dragonfly, a machine learning program, sorted through the thousand or so images to identify those categories. That information was then used to create 3D models of the samples.

Acevedo-Sánchez noted that less than 5 percent of R. parkeri formed connections with the ER — but small quantities of certain characteristics are known to be critical for R. parkeri infection. R. parkeri can exist in two states: motile, with an actin tail, and nonmotile, without it. In mutants unable to form actin tails, R. parkeri are unable to progress to adjacent cells — but in nonmutants, the percentage of R. parkeri that have tails starts at about 2 percent in early infection and never exceeds 15 percent at the height of it.

The ER only interacts with nonmotile R. parkeri, and those interactions increased 25-fold in mutants that couldn’t form tails.

Creating connections

Co-authors Acevedo-Sánchez, Patrick Woida, and Caroline Anderson also investigated possible ways the connections with the ER are mediated. VAP proteins, which mediate ER interactions with other organelles, are known to be co-opted by other pathogens during infection.

During infection by R. parkeri, VAP proteins were recruited to the bacteria; when VAP proteins were knocked out, the frequency of interactions between R. parkeri and the ER decreased, indicating R. parkeri may be taking advantage of these cellular mechanisms for its own purposes during infection.

Although Acevedo-Sánchez now works as a senior scientist at AbbVie, the Lamason Lab is continuing the work of exploring the molecular players that may be involved, how these interactions are mediated, and whether the contacts affect the host or bacteria’s life cycle.

Senior author and associate professor of biology Rebecca Lamason noted that these potential interactions are particularly interesting because bacteria and mitochondria are thought to have evolved from a common ancestor. The Lamason Lab has been exploring whether R. parkeri could form the same membrane contacts that mitochondria do, although they haven’t proven that yet. So far, R. parkeri is the only cytosolic pathogen that has been observed behaving this way.

“It’s not just bacteria accidentally bumping into the ER. These interactions are extremely stable. The ER is clearly extensively wrapping around the bacterium, and is still connected to the ER network,” Lamason says. “It seems like it has a purpose — what that purpose is remains a mystery.”

An abundant phytoplankton feeds a global network of marine microbes

New findings illuminate how Prochlorococcus’ nightly “cross-feeding” plays a role in regulating the ocean’s capacity to cycle and store carbon.

Jennifer Chu | MIT News
January 3, 2025

One of the hardest-working organisms in the ocean is the tiny, emerald-tinged Prochlorococcus marinus. These single-celled “picoplankton,” which are smaller than a human red blood cell, can be found in staggering numbers throughout the ocean’s surface waters, making Prochlorococcus the most abundant photosynthesizing organism on the planet. (Collectively, Prochlorococcus fix as much carbon as all the crops on land.) Scientists continue to find new ways that the little green microbe is involved in the ocean’s cycling and storage of carbon.

Now, MIT scientists have discovered a new ocean-regulating ability in the small but mighty microbes: cross-feeding of DNA building blocks. In a study appearing today in Science Advances, the team reports that Prochlorococcus shed these extra compounds into their surroundings, where they are then “cross-fed,” or taken up by other ocean organisms, either as nutrients, energy, or for regulating metabolism. Prochlorococcus’ rejects, then, are other microbes’ resources.

What’s more, this cross-feeding occurs on a regular cycle: Prochlorococcus tend to shed their molecular baggage at night, when enterprising microbes quickly consume the cast-offs. For a microbe called SAR11, the most abundant bacteria in the ocean, the researchers found that the nighttime snack acts as a relaxant of sorts, forcing the bacteria to slow down their metabolism and effectively recharge for the next day.

Through this cross-feeding interaction, Prochlorococcus could be helping many microbial communities to grow sustainably, simply by giving away what it doesn’t need. And they’re doing so in a way that could set the daily rhythms of microbes around the world.

“The relationship between the two most abundant groups of microbes in ocean ecosystems has intrigued oceanographers for years,” says co-author and MIT Institute Professor Sallie “Penny” Chisholm, who played a role in the discovery of Prochlorococcus in 1986. “Now we have a glimpse of the finely tuned choreography that contributes to their growth and stability across vast regions of the oceans.”

Given that Prochlorococcus and SAR11 suffuse the surface oceans, the team suspects that the exchange of molecules from one to the other could amount to one of the major cross-feeding relationships in the ocean, making it an important regulator of the ocean carbon cycle.

“By looking at the details and diversity of cross-feeding processes, we can start to unearth important forces that are shaping the carbon cycle,” says the study’s lead author, Rogier Braakman, a research scientist in MIT’s Department of Earth, Atmospheric and Planetary Sciences (EAPS).

Other MIT co-authors include Brandon Satinsky, Tyler O’Keefe, Shane Hogle, Jamie Becker, Robert Li, Keven Dooley, and Aldo Arellano, along with Krista Longnecker, Melissa Soule, and Elizabeth Kujawinski of Woods Hole Oceanographic Institution (WHOI).

Spotting castaways

Cross-feeding occurs throughout the microbial world, though the process has mainly been studied in close-knit communities. In the human gut, for instance, microbes are in close proximity and can easily exchange and benefit from shared resources.

By comparison, Prochlorococcus are free-floating microbes that are regularly tossed and mixed through the ocean’s surface layers. While scientists assume that the plankton are involved in some amount of cross-feeding, exactly how this occurs, and who would benefit, have historically been challenging to probe; any stuff that Prochlorococcus cast away would have vanishingly low concentrations,and be exceedingly difficult to measure.

But in work published in 2023, Braakman teamed up with scientists at WHOI, who pioneered ways to measure small organic compounds in seawater. In the lab, they grew various strains of Prochlorococcus under different conditions and characterized what the microbes released. They found that among the major “exudants,” or released molecules, were purines and pyridines, which are molecular building blocks of DNA. The molecules also happen to be nitrogen-rich — a fact that puzzled the team. Prochlorococcus are mainly found in ocean regions that are low in nitrogen, so it was assumed they’d want to retain any and all nitrogen-containing compounds they can. Why, then, were they instead throwing such compounds away?

Global symphony

In their new study, the researchers took a deep dive into the details of Prochlorococcus’ cross-feeding and how it influences various types of ocean microbes.

They set out to study how Prochlorococcus use purine and pyridine in the first place, before expelling the compounds into their surroundings. They compared published genomes of the microbes, looking for genes that encode purine and pyridine metabolism. Tracing the genes forward through the genomes, the team found that once the compounds are produced, they are used to make DNA and replicate the microbes’ genome. Any leftover purine and pyridine is recycled and used again, though a fraction of the stuff is ultimately released into the environment. Prochlorococcus appear to make the most of the compounds, then cast off what they can’t.

The team also looked to gene expression data and found that genes involved in recycling purine and pyrimidine peak several hours after the recognized peak in genome replication that occurs at dusk. The question then was: What could be benefiting from this nightly shedding?

For this, the team looked at the genomes of more than 300 heterotrophic microbes — organisms that consume organic carbon rather than making it themselves through photosynthesis. They suspected that such carbon-feeders could be likely consumers of Prochlorococcus’ organic rejects. They found most of the heterotrophs contained genes that take up either purine or pyridine, or in some cases, both, suggesting microbes have evolved along different paths in terms of how they cross-feed.

The group zeroed in on one purine-preferring microbe, SAR11, as it is the most abundant heterotrophic microbe in the ocean. When they then compared the genes across different strains of SAR11, they found that various types use purines for different purposes, from simply taking them up and using them intact to breaking them down for their energy, carbon, or nitrogen. What could explain the diversity in how the microbes were using Prochlorococcus’ cast-offs?

It turns out the local environment plays a big role. Braakman and his collaborators performed a metagenome analysis in which they compared the collectively sequenced genomes of all microbes in over 600 seawater samples from around the world, focusing on SAR11 bacteria. Metagenome sequences were collected alongside measurements of various environmental conditions and geographic locations in which they are found. This analysis showed that the bacteria gobble up purine for its nitrogen when the nitrogen in seawater is low, and for its carbon or energy when nitrogen is in surplus — revealing the selective pressures shaping these communities in different ocean regimes.

“The work here suggests that microbes in the ocean have developed relationships that advance their growth potential in ways we don’t expect,” says co-author Kujawinski.

Finally, the team carried out a simple experiment in the lab, to see if they could directly observe a mechanism by which purine acts on SAR11. They grew the bacteria in cultures, exposed them to various concentrations of purine, and unexpectedly found it causes them to slow down their normal metabolic activities and even growth. However, when the researchers put these same cells under environmentally stressful conditions, they continued growing strong and healthy cells, as if the metabolic pausing by purines helped prime them for growth, thereby avoiding the effects of the stress.

“When you think about the ocean, where you see this daily pulse of purines being released by Prochlorococcus, this provides a daily inhibition signal that could be causing a pause in SAR11 metabolism, so that the next day when the sun comes out, they are primed and ready,” Braakman says. “So we think Prochlorococcus is acting as a conductor in the daily symphony of ocean metabolism, and cross-feeding is creating a global synchronization among all these microbial cells.”

This work was supported, in part, by the Simons Foundation and the National Science Foundation.

Imperiali Lab News Brief: combining bioinformatics and biochemistry

Parsing endless possibilities

Lillian Eden | Department of Biology
December 11, 2024

New research from the Imperiali Lab in the Department of Biology at MIT combines bioinformatics and biochemistry to reveal critical players in assembling glycans, the large sugar molecules on bacterial cell surfaces responsible for behaviors such as evading immune responses and causing infections.

In most cases, single-celled organisms such as bacteria interact with their environment through complex chains of sugars known as glycans bound to lipids on their outer membranes. Glycans orchestrate biological responses and interactions, such as evading immune responses and causing infections. 

The first step in assembling most bacterial glycans is the addition of a sugar-phosphate group onto a lipid, which is catalyzed by phosphoglycosyl transferases (PGTs) on the inner membrane. This first sugar is then further built upon by other enzymes in subsequent steps in an assembly-line-like pathway. These critical biochemical processes are challenging to explore because the proteins involved in these processes are embedded in membranes, which makes them difficult to isolate and study. 

Although glycans are found in all living organisms, the sugar molecules that compose glycans are especially diverse in bacteria. There are over 30,000 known bacterial PGTs, and hundreds of sugars for them to act upon. 

Research recently published in PNAS from the Imperiali Lab in the Department of Biology at MIT uses a combination of bioinformatics and biochemistry to predict clusters of “like-minded” PGTs and verify which sugars they will use in the first step of glycan assembly. 

Defining the biochemical machinery for these assembly pathways could reveal new strategies for tackling antibiotic-resistant strains of bacteria. This comprehensive approach could also be used to develop and test inhibitors, halting the assembly pathway at this critical first step. 

Exploring Sequence Similarity

First author Theo Durand, an undergraduate student from Imperial College London who studied at MIT for a year, worked in the Imperiali Lab as part of a research placement. Durand was first tasked with determining which sugars some PGTs would use in the first step of glycan assembly, known as the sugar substrates of the PGTs. When initially those substrate-testing experiments didn’t work, Durand turned to the power of bioinformatics to develop predictive tools. 

Strategically exploring the sugar substrates for PGTs is challenging due to the sheer number of PGTs and the diversity of bacteria, each with its own assorted set of glycans and glycoconjugates. To tackle this problem, Durand deployed a tool called a Sequence Similarity Network (SSN), part of a computational toolkit developed by the Enzyme Function Initiative. 

According to senior author Barbara Imperiali, Class of 1922 Professor of Biology and Chemistry, an SSN provides a powerful way to analyze protein sequences through comparisons of the sequences of tens of thousands of proteins. In an optimized SSN, similar proteins cluster together, and, in the case of PGTs, proteins in the same cluster are likely to share the same sugar substrate. 

For example, a previously uncharacterized PGT that appears in a cluster of PGTs whose first sugar substrate is FucNAc4N would also be predicted to use FucNAc4N. The researchers could then test that prediction to verify the accuracy of the SSN. 

FucNAc4N is the sugar substrate for the PGT of Fusobacterium nucleatum (F. nucleatum), a bacterium that is normally only present in the oral cavity but is correlated with certain cancers and endometriosis, and Streptococcus pneumoniae, a bacterium that causes pneumonia. 

Adjusting the assay

The critical biochemical process of assembling glycans has historically been challenging to define, mainly because assembly is anchored to the interior side of the inner membrane of the bacterium. The purification process itself can be difficult, and the purified proteins don’t necessarily behave in the same manner once outside their native membrane environment.

To address this, the researchers modified a commercially available test to work with proteins still embedded in the membrane of the bacterium, thus saving them weeks of work to purify the proteins. They could then determine the substrate for the PGT by measuring whether there was activity. This first step in glycan assembly is chemically unique, and the test measures one of the reaction products. 

For PGTs whose substrate was unknown, Durand did a deep dive into the literature to find new substrates to test. FucNAc4N, the first sugar substrate for F. nucleatum, was, in fact, Durand’s favorite sugar – he found it in the literature and reached out to a former Imperiali Lab postdoc for the instructions and materials to make it. 

“I ended up down a rabbit hole where I was excited every time I found a new, weird sugar,” Durand recalls with a laugh. “These bacteria are doing a bunch of really complicated things and any tools to help us understand what is actually happening is useful.” 

Exploring inhibitors

Imperiali noted that this research both represents a huge step forward in our understanding of bacterial PGTs and their substrates and presents a pipeline for further exploration. She’s hoping to create a searchable database where other researchers can seed their own sequences into the SSN for their organisms of interest. 

This pipeline could also reveal antibiotic targets in bacteria. For example, she says, the team is using this approach to explore inhibitor development. 

The Imperiali lab worked with Karen Allen, a professor of Chemistry at Boston University, and graduate student Roxanne Siuda to test inhibitors, including ones for F. nucleatum, the bacterium correlated with certain cancers and endometriosis whose first sugar substrate is FucNAc4N. They are also hoping to obtain structures of inhibitors bound to the PGT to enable structure-guided optimization.

“We were able to, using the network, discover the substrate for a PGT, verify the substrate, use it in a screen, and test an inhibitor,” Imperiali says. “This is bioinformatics, biochemistry, and probe development all bundled together, and represents the best of functional genomics.”

Sauer & Davis Lab News Brief: structures of molecular woodchippers reveal mechanism for versatility

Rest in pieces: deconstructing polypeptide degradation machinery

Lillian Eden | Department of Biology
November 12, 2024

Research from the Sauer and Davis Labs in the Department of Biology at MIT shows that conformational changes contribute to the specificity of “molecular woodchippers” 

Degradation is a crucial process for maintaining protein homeostasis by culling excess or damaged proteins whose components can then be recycled. It is also a highly regulated process—for good reason. A cell could potentially waste many resources if the degradation machinery destroys proteins it shouldn’t. 

One of the major pathways for protein degradation in bacteria and eukaryotic mitochondria involves a molecular machine called ClpXP. ClpXP is made up of two components: a star-shaped structure made up of six subunits called ClpX that engages and unfolds proteins tagged for degradation, and an associated barrel-shaped enzyme, called ClpP, that chemically breaks up proteins into small pieces called peptides. 

ClpXP is incredibly adaptable and is often compared to a woodchipper — able to take in materials and spit out their broken-down components. Thanks to biochemical experiments, this molecular degradation machine is known to be able to break down hundreds of different proteins in the cell regardless of physical or chemical properties such as size, shape, or charge. ClpX uses energy from ATP hydrolysis to unfold proteins before they are threaded through its central channel, referred to as the axial channel, and into the degradation chamber of ClpP.

In three papers, one in PNAS and two in Nature Communications, researchers from the Department of Biology at MIT have expanded our understanding of how this molecular machinery engages with, unfolds, and degrades proteins — and how that machinery refrains, by design, from unfolding proteins not tagged for degradation. 

Alireza Ghanbarpour, until recently a postdoc in the Sauer Lab and Davis Lab and first author on all three papers, began with a simple question: given the vast repertoire of potential substrates — that is, proteins to be degraded — how is ClpXP so specific?

Ghanbarpour — now an assistant professor in the Department of Biochemistry and Molecular Biology at Washington University School of Medicine in St. Louis — found that the answer to this question lies in conformational changes in the molecular machine as it engages with an ill-fated protein. 

Reverse Engineering using Structural Insights

Ghanbarpour approached the question of ClpXP’s versatility by characterizing conformational changes of the molecular machine using a technique called cryogenic electron microscopy. In cryo-EM, sample particles are frozen in solution, and images are collected; algorithms then create 3D renderings from the 2D images.

“It’s really useful to generate different structures in different conditions and then put them together until you know how a machine works,” he says. “I love structural biology, and these molecular machines make fascinating targets for structural work and biochemistry. Their structural plasticity and precise functions offer exciting opportunities to understand how nature leverages enzyme conformations to generate novel functions and tightly regulate protein degradation within the cell.”

Inside the cell, these proteases do not work alone but instead work together with “adaptor” proteins, which can promote — or inhibit — degradation by ClpXP. One of the adaptor proteins that promotes degradation by ClpXP is SspB. 

In E. coli and most other bacteria, ClpXP and SspB interact with a tag called ssrA that is added to incomplete proteins when their biosynthesis on ribosomes stalls. 

The tagging process frees up the ribosome to make more proteins, but creates a problem: incomplete proteins are prone to aggregation, which could be detrimental to cellular health and can lead to disease. By interacting with the degradation tag, ClpXP and SspB help to ensure the degradation of these incomplete proteins. Understanding this process and how it may go awry may open therapeutic avenues in the future.

“It wasn’t clear how certain adapters were interacting with the substrate and the molecular machines during substrate delivery,” Ghanbarpour notes. “My recent structure reveals that the adapter engages with the enzyme, reaching deep into the axial channel to deliver the substrate.” 

Ghanbarpour and colleagues showed that ClpX engages with both the SspB adaptor and the ssrA degradation tag of an ill-fated protein at the same time. Surprisingly, they also found that this interaction occurs while the upper part of the axial channel through ClpX is closed — in fact, the closed channel allows ClpX to contact both the tag and the adaptor simultaneously.

This result was surprising, according to senior author and Salvador E. Luria Professor of Biology Robert Sauer, whose lab has been working on understanding this molecular machine for more than two decades: it was unclear whether the channel through ClpX closes in response to a substrate interaction, or if the channel is always closed until it opens to pass an unfolded protein down to ClpP to be degraded.

Preventing Rogue Degradation

Throughout this project, Ghanbarpour was co-advised by structural biologist and Associate Professor of Biology Joey Davis and collaborated with members of the Davis Lab to better understand the conformational changes that allow these molecular machines to function. Using a cryo-EM analysis approach developed in the Davis lab called CryoDRGN, the researchers showed that there is an equilibrium between ClpXP in the open and closed states: it’s usually closed but is open in about 10% of the particles in their samples. 

The closed state is almost identical to the conformation ClpXP assumes when it is engaged with an ssrA-tagged substrate and the SspB adaptor. 

To better understand the biological significance of this equilibrium, Ghanbarpour created a mutant of ClpXP that is always in the open position. Compared to normal ClpXP, the mutant degraded some proteins lacking obvious degradation tags faster but degraded ssrA-tagged proteins more slowly. 

According to Ghanbarpour, these results indicate that the closed channel improves ClpXP’s ability to efficiently engage tagged proteins meant to be degraded, whereas the open channel allows more “promiscuous” degradation. 

Pausing the Process

The next question Ghanbarpour wanted to answer was what this molecular machine looks like while engaged with a protein it is attempting to unfold. To do that, he created a substrate with a highly stable protein attached to the degradation tag that is initially pulled into ClpX, but then dramatically slows protein unfolding and degradation.

In the structures where the degradation process stalls, Ghanbarpour found that the degradation tag was pulled far into the molecular machine—through ClpX and into ClpP—and the folded protein part of the substrate was pulled tightly against the axial channel of ClpX. 

The opening of the axial channel, called the axial pore, is made up of looping protein structures called RKH loops. These flexible loops were found to play roles both in recognizing the ssrA degradation tag and in how substrates or the SspB adaptor interact with or are pulled against the channel during degradation. 

The flexibility of these RKH loops allows ClpX to interact with a large number of different proteins and adapters, and these results clarify some previous biochemical and mutational studies of interactions between the substrate and ClpXP. 

Although Ghanbarpour’s recent work focused on just one adaptor and degradation tag, he noted there are many more targets — ClpXP is something akin to a Swiss army knife for breaking down polypeptide chains. 

The way those other substrates interact with ClpXP could differ from the structures solved with the SspB adaptor and ssrA tag. It also stands to reason that the way ClpXP reacts to each substrate may be unique. For example, given that ClpX is occasionally in an open state, some substrates may engage with ClpXP only while it’s in an open conformation. 

In his new position at Washington University, Ghanbarpour intends to continue exploring how ClpXP and other molecular machines locate their target substrates and interact with adaptors, shedding light on how cells regulate protein degradation and maintain protein homeostasis.

The structures Ghanbarpour solved involved free-floating protein degradation machinery, but membrane-bound degradation machinery also exists. The membrane-bound version’s structure and conformational adaptions potentially differ from the structures Ghanbarpour found in his previous three papers. Indeed, in a recent preprint, Ghanbarpour worked on the cryo-EM structure of a nautilus shell-shaped protein assembly that seems to control membrane-bound degradation machinery. This assembly plays a critical role in regulating protein degradation within the bacterial inner membrane.

“The function of these proteases goes beyond simply degrading damaged proteins. They also target transcription factors, regulatory proteins, and proteins that don’t exist in normal conditions,” he says. “My new lab is particularly interested in understanding how cells use these proteases and their accessory adaptors, both under normal and stress conditions, to reshape the proteome and support recovery from cellular distress.”

Laub Lab News Brief: anti-viral defense system in bacteria modifies mRNA

Killing the messenger

Lillian Eden | Department of Biology
October 23, 2024

Newly characterized anti-viral defense system in bacteria aborts infection through novel mechanism by chemically modifying mRNA.

Like humans and other complex multicellular organisms, single-celled bacteria can fall ill and fight off viral infections. A bacterial virus is known as a bacteriophage, or, more simply, a phage, which is one of the most ubiquitous life forms on Earth. Phages and bacteria are engaged in a constant battle, the virus attempting to circumvent the bacteria’s defenses, and the bacteria racing to find new ways to protect itself.

These anti-phage defense systems are carefully controlled and prudently managed — dormant but always poised to strike. 

New research recently published in Nature from the Laub Lab in the Department of Biology at MIT has characterized an anti-phage defense system in bacteria known as CmdTAC. CmdTAC prevents viral infection by altering mRNA, the single-stranded genetic code used to produce proteins, of both the host and the virus.  

This defense system detects phage infection at a stage when the viral phage has already commandeered the host’s machinery for its own purposes. In the face of annihilation, the ill-fated bacterium activates a defense system that will halt translation, preventing the creation of new proteins and aborting the infection — but dooming itself in the process. 

“When bacteria are in a group, they’re kind of like a multicellular organism that is not connected to one another. It’s an evolutionarily beneficial strategy for one cell to kill itself to save another identical cell,” says Christopher Vassallo, a postdoc and co-author of the study. “You could say it’s like self-sacrifice: one cell dies to protect the other cells.” 

The enzyme responsible for altering the mRNA is called an ADP-ribosyltransferase.  Researchers have characterized hundreds of these enzymes — although only a few are known to target DNA or other types of RNA, all but a handful target proteins. This is the first time these enzymes have been characterized targeting mRNA within cells.

Expanding understanding of anti-phage defense

Co-first author and graduate student Chris Doering noted that it is only within the last decade or so that researchers have begun to appreciate the breadth of diversity and complexity of anti-phage defense systems. For example, CRISPR gene editing, a technique used in everything from medicine to agriculture, is rooted in research on the bacterial CRISPR-Cas9 anti-phage defense system. 

CmdTAC is a subset of a widespread anti-phage defense mechanism called a toxin-antitoxin system. A TA system is just that: a toxin capable of killing or altering the cell’s processes rendered inert by an associated antitoxin. 

Although these TA systems can be identified — if the toxin is expressed by itself, it kills or inhibits the growth of the cell; if the toxin and antitoxin are expressed together, the toxin is neutralized — characterizing the cascade of circumstances that activates these systems requires extensive effort. In recent years, however, many TA systems have been shown to serve as anti-phage defenses. 

Two general questions need to be answered to understand a viral defense system: how do bacteria detect an infection, and how do they respond?

Detecting infection

CmdTAC is a TA system with an additional element, and the three components generally exist in a stable complex: the toxin CmdT, the antitoxin CmdA, and an additional component that mediates the system, the chaperone CmdC. 

If the phage’s protective capsid protein is present, CmdC disassociates from CmdT and CmdA and interacts with the phage capsid protein instead. In the model outlined in the paper, the chaperone CmdC is, therefore, the sensor of the system, responsible for recognizing when an infection is occurring. Structural proteins, such as the capsid that protects the phage genome, are a common trigger because they’re abundant and essential to the phage.

The uncoupling of CmdC leads to the degradation of the neutralizing antitoxin CmdA, which releases the toxin CmdT to do its lethal work.

Toxicity on the loose

Guided by computational tools, the researchers knew that CmdT was likely an ADP-ribosyltransferase due to its similarities to other such enzymes. As the name suggests, the enzyme transfers an ADP ribose onto its target.

To determine how CmdT was altering mRNA, the researchers tested a mix of short sequences of single-stranded RNA to see if the enzyme was drawn to any sequences or positions in particular. RNA has four bases: A, U, G, and C, and the evidence points to the enzyme recognizing GA sequences. 

The CmdT modification of GA sequences in mRNA blocks its translation. The cessation of creating new proteins aborts the infection, preventing the phage from spreading beyond the host to infect other bacteria. 

“Not only is it a new type of bacterial immune system, but the enzyme involved does something that’s never been seen before: the ADP-ribsolyation of mRNA,” Vassallo says. 

Although the paper outlines the broad strokes of the anti-phage defense system, there’s more to learn: it’s unclear how CmdC interacts with the capsid protein, and how the chemical modification of GA sequences prevents translation. 

Beyond Bacteria

While exploring anti-phage defense aligns with the Laub Lab’s overall goal of understanding how bacteria function and evolve, these results may have broader implications beyond bacteria.

Senior author Michael Laub, Salvador E. Luria Professor and HHMI Investigator, says the ADP-ribosyltransferase has homologs in eukaryotes, including human cells. They are not well studied, and not currently among the Laub Lab’s research topics, but they are known to be up-regulated in response to viral infection. 

“There are so many different — and cool — mechanisms by which organisms defend themselves against viral infection,” Laub says. “The notion that there may be some commonality between how bacteria defend themselves and how humans defend themselves is a tantalizing possibility.” 

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 the latest concepts to the field of parasitology,” Lourido says.

A fascination with microbiology

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.”

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 being 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.”

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.”
The 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.”

News brief: Davis Lab

Exploring the cellular neighborhood

Alison Biester | Department of Biology
March 12, 2024

New software allows scientists to model shapeshifting proteins in native cellular environments

Cells rely on complex molecular machines composed of protein assemblies to perform essential functions such as energy production, gene expression, and protein synthesis. To better understand how these machines work, scientists capture snapshots of them by isolating proteins from cells and using various methods to determine their structures. However, isolating proteins from cells also removes them from the context of their native environment, including protein interaction partners and cellular location.

Recently, cryogenic electron tomography (cryo-ET) has emerged as a way to observe proteins in their native environment by imaging frozen cells at different angles to obtain three-dimensional structural information. This approach is exciting because it allows researchers to directly observe how and where proteins associate with each other, revealing the cellular neighborhood of those interactions within the cell.

With the technology available to image proteins in their native environment, graduate student Barrett Powell wondered if he could take it one step further: what if molecular machines could be observed in action? In a paper published today in Nature Methods, Powell describes the method he developed, called tomoDRGN, for modeling structural differences of proteins in cryo-ET data that arise from protein motions or proteins binding to different interaction partners. These variations are known as structural heterogeneity. 

Although Powell had joined the Davis Lab as an experimental scientist, he recognized the potential impact of computational approaches in understanding structural heterogeneity within a cell. Previously, the Davis Lab developed a related methodology named cryoDRGN to understand structural heterogeneity in purified samples. As Powell and Associate Professor of Biology Joey Davis saw cryo-ET rising in prominence in the field, Powell took on the challenge of reimagining this framework to work in cells. 

When solving structures with purified samples, each particle is imaged only once. By contrast, cryo-ET data is collected by imaging each particle more than 40 times from different angles. That meant tomoDRGN needed to be able to merge the information from more than 40 images, which was where the project hit a roadblock: the amount of data led to an information overload.

To address the information overload, Powell successfully rebuilt the cryoDRGN model to prioritize only the highest-quality data. When imaging the same particle multiple times, radiation damage occurs. The images acquired earlier, therefore, tend to be of higher quality because the particles are less damaged.

“By excluding some of the lower quality data, the results were actually better than using all of the data–and the computational performance was substantially faster,” Powell says.

Just as Powell was beginning work on testing his model, he had a stroke of luck: the authors of a groundbreaking new study that visualized, for the first time, ribosomes inside cells at near-atomic resolution, shared their raw data on the Electric Microscopy Public Image Archive (EMPIAR). This dataset was an exemplary test case for Powell, through which he demonstrated that tomoDRGN could uncover structural heterogeneity within cryo-ET data. 

According to Powell, one exciting result is what tomoDRGN found surrounding a subset of ribosomes in the EMPIAR dataset. Some of the ribosomal particles were associated with a bacterial cell membrane and engaged in a process called cotranslational translocation. This occurs when a protein is being simultaneously synthesized and transported across a membrane. Researchers can use this result to make new hypotheses about how the ribosome functions with other protein machinery integral to transporting proteins outside of the cell, now guided by a structure of the complex in its native environment. 

After seeing that tomoDRGN could resolve structural heterogeneity from a structurally diverse dataset, Powell was curious: how small of a population could tomoDRGN identify? For that test, he chose a protein named apoferritin which is a commonly used benchmark for cryo-ET and is often treated as structurally homogeneous. Ferritin is a protein used for iron storage and is referred to as apoferritin when it lacks iron.

Surprisingly, in addition to the expected particles, tomoDRGN revealed a minor population of ferritin particles–with iron bound–making up just 2% of the dataset that was not previously reported. This result further demonstrated tomoDRGN’s ability to identify structural states that occur so infrequently that they would be averaged out with traditional analysis tools. 

Powell and other members of the Davis Lab are excited to see how tomoDRGN can be applied to further ribosomal studies and to other systems. Davis works on understanding how cells assemble, regulate, and degrade molecular machines, so the next steps include exploring ribosome biogenesis within cells in greater detail using this new tool.

“What are the possible states that we may be losing during purification?” Davis says. “Perhaps more excitingly, we can look at how they localize within the cell and what partners and protein complexes they may be interacting with.”