Research Area: Microbiology

New research supports finding explaining why some patients may test positive for COVID-19 long after recovery

SARS-CoV-2, the virus that causes COVID-19, seems to have become a permanent presence in our lives. Research from Whitehead Institute Founding Member Rudolf Jaenisch’s lab reveals that this may be true on multiple levels.

Greta Friar | Whitehead Institute
February 28, 2023

SARS-CoV-2, the virus that causes COVID-19, seems to have become a permanent presence in our lives. Research from Whitehead Institute Founding Member Rudolf Jaenisch’s lab reveals that this may be true on multiple levels. Jaenisch, postdoc Liguo Zhang, and colleagues have shown that when the virus infects people, it is capable of integrating parts of its genetic code into the human genome through a process called reverse transcription. This genomic integration is rare, but due to how many hundreds of millions of people have been infected, it has likely occurred many times.

In a paper published in the journal Viruses on February 25, the researchers use and compare multiple methods to show that SARS-CoV-2 can integrate into host cells’ genomes. The paper is a follow up to Jaenisch and Zhang’s 2021 paper in the Proceedings of the National Academy of Sciences, which provided initial evidence of SARS-CoV-2 genomic integration. The original paper intended to solve the puzzle of why some people who had had COVID-19 were still testing positive long after recovering from the disease. The answer the researchers found was that parts of the viral genome were reverse transcribed into the human genome, meaning the viral RNA was transcribed or “read” into DNA (a reverse of the usual process) and then that DNA was stitched into the cell’s DNA. Then, when the cells’ genomes were transcribed into RNA, the portion of the virus’ genome that had been incorporated would be included and could be recognized by a PCR test, leading to a positive result.

In order to further substantiate the findings described in the previous paper, Jaenisch and Zhang have now performed additional experiments and analyses. The new paper explains why some experiments testing for viral genomic integration would come up with a negative result, and how this is consistent with Jaenisch and Zhang’s conclusion. Additionally, Jaenisch and Zhang examine whether viral RNA put into cells, as a model of the COVID-19 mRNA vaccines, can also integrate into the human genome, and find initial evidence that it cannot.

“This paper puts our data on a very firm footing,” Jaenisch says. “Hopefully, it will clarify some of the issues raised in the discussion that followed the first paper, and provide some reassurance to people who were worried about the implications for the vaccine.”

Hunting for a needle in a haystack

The main challenge in finding evidence of SARS-CoV-2 integrating into the human genome is that this event appears to be very rare. In the new paper, Jaenisch and Zhang used digital PCR, an approach that can sensitively detect specific DNA sequences in cells, to see how commonly the sequence that they would find in instances of viral RNA being read into DNA appeared in infected cells. Specifically, they looked for reverse transcribed SARS-CoV-2 complementary DNA (cDNA), DNA that is made from the virus’ original mRNA. Digital PCR revealed that for every one thousand cells, reverse transcribed viral cDNA was only present in around four to twenty cells. This number includes all detected instances of viral cDNA, whether integrated into the genome or not, so genomic integration is likely even rarer—indeed, the new research suggests that only a fraction of the total cDNA identified is from genomic integration.

Because genomic viral integration is so rare, Jaenisch and Zhang needed to use multiple complementary methods to test for it. One approach, called whole genome sequencing (WGS), is able to search cells’ genomes in great detail. When it does come across an instance of viral genomic integration, it can identify not only the reverse transcribed viral sequence, but also two sequences near the viral sequence that are added when it is integrated into the genome by a common reverse transcription complex called LINE1, which is encoded in the host cells. The combination of viral cDNA plus the two nearby cellular host sequences provides very strong evidence that viral cDNA is not only present but has been incorporated into the cell’s genome. However, WGS can only search the equivalent of a few cells’ genomes, and so when searching for a rare event, like SARS-CoV-2 integration, it often comes up empty. People skeptical of the first paper performed this type of experiment and came up with a negative result; Jaenisch and Zhang were not surprised by that, and it is consistent with their own findings when using this approach.

“Because the human cell genome coverage by whole genome sequencing is very limited, you would need to run the sequencing experiment many times in order to have a good chance of detecting one viral genome copy,” Zhang says.

In order to make the most of WGS, Jaenisch and Zhang induced their cells to overexpress LINE1, the cellular machinery that reverse transcribes viral RNA into the human genome. This exponentially increases the amount of viral cDNA that gets made; when the researchers performed digital PCR on their cells with overexpression, it detected fourteen to twenty thousand cDNA copies per thousand cells. Consequently, WGS was able to detect instances of viral cDNA plus the two nearby sequences that are the telltale signature of genomic integration in these cells.

“This is unambiguous proof of viral genomic integration,” Zhang says.

This type of experiment is called a positive control. Researchers use it to prove that, in ideal circumstances, the biological phenomenon they are curious about can occur. The question then becomes: does the phenomenon happen in normal circumstances? This was a criticism raised by some researchers in response to the first paper: they were not convinced that viral genomic integration happens in the cells of an infected person, which do not have the same levels of LINE1.

The search gets shallower but wider

Jaenisch and Zhang used another approach to hunt for evidence of viral genomic integration in cells without LINE1 overexpression. The approach, called an enrichment method and performed with the tool TagMap, can analyze thousands of cells—enough cells to reliably find evidence of a rare event. However, it cannot get the same detail as whole genome sequencing; TagMap enriches and captures shorter sequences of DNA, so it can only capture one of the two nearby sequences that act as a signature alongside viral cDNA. However, the smaller stretch of DNA that the researchers focused on still has features that can be used as evidence of integration. With this approach, Jaenisch and Zhang detected many instances of viral cDNA linked to the nearby cellular sequence.

Jaenisch and Zhang argue that the combined results of these experiments show strong proof of viral integration. Whole genome sequencing provides very strong proof that viral genomic integration can occur in the right conditions. Enrichment with TagMap provides reasonably strong proof that viral genomic integration occurs in normal cells.

“Each of these methods has advantages and disadvantages. You have to combine them to get the complete picture,” Jaenisch says.

Turning to the vaccine

After reaffirming their results that genomic integration of SARS-CoV-2 happens following viral infection, the researchers wanted to know whether the same thing happens with mRNA from the COVID-19 vaccines—which had been a concern expressed by many in the wake of the first paper. Jaenisch and Zhang could not get access to the actual vaccine RNA, packaged into a lipid coat, which is used for vaccination. Instead, they created a model of vaccine injection, inserting a bit of SARS-CoV-2 genetic material (mRNA) into cells through transfection, or non-infection “delivery” of genetic content into cells.

The researchers found that transfection of SARS-CoV-2 mRNA did not lead to genomic integration in the same way that infection did. Infection naturally produces a large amount of viral RNA and causes an inflammatory response in cells. Such cellular stresses increase the level of the reverse transcription machinery. Transfection does not do this, and correspondingly, the researchers found no evidence with TagMap that it led to viral genomic integration by LINE1 in normal cells.

The researchers’ model of vaccine injection is missing several key features of the actual vaccine. In the future, Jaenisch hopes to follow up on this research using the actual vaccine RNA sequence, and testing in an animal model to more closely match what happens during vaccine injection. In the meantime, the researchers hope that these initial results are reassuring.

“We need to do further testing, but our results are consistent with vaccine RNA not integrating,” Jaenisch says.

Notes

Zhang, Liguo, Punam Bisht, Anthony Flamier, M. Inmaculada Barrasa, Max Friesen, Alexsia Richards, Stephen H. Hughes, and Rudolf Jaenisch. 2023. “LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected Cells” Viruses 15, no. 3: 629. https://doi.org/10.3390/v15030629

Daniel Lew

Education

  • Graduate: PhD, 1990, Rockefeller University
  • Undergraduate: BA, 1984, Genetics, Cambridge University

Research Summary

Different cells take on an astonishing variety of shapes, which are often critical to be able to perform specialized cell functions like absorbing nutrients or contracting muscles. We study how different cell shapes arise and how cells control the spatial distribution of their internal constituents. We take advantage of the tractability of fungal model systems, and address these questions using approaches from cell biology, genetics, and computational biology to understand molecular mechanisms. 

Honors and Awards

  • Fellow, American Academy of Microbiology, 2008
  • Fellow, American Association for the Advancement of Science, 2010
  • Duke Equity, Diversity, and Inclusion Award, 2019
Scientists discover a new way of sharing genetic information in a common ocean microbe

Prochlorococcus, the world’s most abundant photosynthetic organism, reveals a gene-transfer mechanism that may be key to its abundance and diversity.

David L. Chandler | MIT News Office
January 5, 2023

From the tropics to the poles, from the sea surface to hundreds of feet below, the world’s oceans are teeming with one of the tiniest of organisms: a type of bacteria called Prochlorococcus, which despite their minute size are collectively responsible for a sizable portion of the oceans’ oxygen production. But the remarkable ability of these diminutive organisms to diversify and adapt to such profoundly different environments has remained something of a mystery.

Now, new research reveals that these tiny bacteria exchange genetic information with one another, even when widely separated, by a previously undocumented mechanism. This enables them to transmit whole blocks of genes, such as those conferring the ability to metabolize a particular kind of nutrient or to defend themselves from viruses, even in regions where their population in the water is relatively sparse.

The findings describe a new class of genetic agents involved in horizontal gene transfer, in which genetic information is passed directly between organisms — whether of the same or different species — through means other than lineal descent. The researchers have dubbed the agents that carry out this transfer “tycheposons,” which are sequences of DNA that can include several entire genes as well as surrounding sequences, and can spontaneously separate out from the surrounding DNA. Then, they can be transported to other organisms by one or another possible carrier system including tiny bubbles known as vesicles that cells can produce from their own membranes.

The research, which included studying hundreds of Prochlorococcus genomes from different ecosystems around the world, as well as lab-grown samples of different variants, and even evolutionary processes carried out and observed in the lab, is reported today in the journal Cell, in a paper by former MIT postdocs Thomas Hackl and Raphaël Laurenceau, visiting postdoc Markus Ankenbrand, Institute Professor Sallie “Penny” Chisholm, and 16 others at MIT and other institutions.

Chisholm, who played a role in the discovery of these ubiquitous organisms in 1988, says of the new findings, “We’re very excited about it because it’s a new horizontal gene-transfer agent for bacteria, and it explains a lot of the patterns that we see in Prochlorococcus in the wild, the incredible diversity.” Now thought to be the world’s most abundant photosynthetic organism, the tiny variants of what are known as cyanobacteria are also the smallest of all photosynthesizers.

Hackl, who is now at the University of Groningen in the Netherlands, says the work began by studying the 623 reported genome sequences of different species of Prochlorococcus from different regions, trying to figure out how they were able to so readily lose or gain particular functions despite their apparent lack of any of the known systems that promote/boost horizontal gene transfer, such as plasmids or viruses known as prophages.

What Hackl, Laurenceau, and Ankenbrand investigated were “islands” of genetic material that seemed to be hotspots of variability and often contained genes that were associated with known key survival processes such as the ability to    assimilate essential, and often limiting, nutrients such as iron, or nitrogen, or phosphates. These islands contained genes that varied enormously between different species, but they always occurred in the same parts of the genome and sometimes were nearly identical even in widely different species — a strong indicator of horizontal transfer.

But the genomes showed none of the usual features associated with what are known as mobile genetic elements, so initially this remained a puzzle. It gradually became apparent that this system of gene transfer and diversification was different from any of the several other mechanisms that have been observed in other organisms, including in humans.

Hackl describes what they found as being something like a genetic LEGO set, with chunks of DNA bundled together in ways that could almost instantly confer the ability to adapt to a particular environment. For example, a species limited by the availability of particular nutrients could acquire genes necessary to enhance the uptake of that nutrient.

The microbes appear to use a variety of mechanisms to transport these tycheposons (a name derived from the name of the Greek goddess Tyche, daughter of Oceanus). One is the use of membrane vesicles, little bubbles pouched off from the surface of a bacterial cell and released with tycheposons inside it. Another is by “hijacking” virus or phage infections and allowing them to carry the tycheposons along with their own infectious particles, called capsids. These are efficient solutions, Hackl says, “because in the open ocean, these cells rarely have cell-to-cell contacts, so it’s difficult for them to exchange genetic information without a vehicle.”

And sure enough, when capsids or vesicles collected from the open ocean were studied, “they’re actually quite enriched” in these genetic elements, Hackl says. The packets of useful genetic coding are “actually swimming around in these extracellular particles and potentially being able to be taken up by other cells.”

Chisholm says that “in the world of genomics, there’s a lot of different types of these elements” — sequences of DNA that are capable of being transferred from one genome to another. However, “this is a new type,” she says. Hackl adds that “it’s a distinct family of mobile genetic elements. It has similarities to others, but no really tight connections to any of them.”

While this study was specific to Prochlorococcus, Hackl says the team believes the phenomenon may be more generalized. They have already found similar genetic elements in other, unrelated marine bacteria, but have not yet analyzed these samples in detail. “Analogous elements have been described in other bacteria, and we now think that they may function similarly,” he says.

“It’s kind of a plug-and-play mechanism, where you can have pieces that you can play around with and make all these different combinations,” he says. “And with the enormous population size of Prochlorococcus, it can play around a lot, and try a lot of different combinations.”

Nathan Ahlgren, an assistant professor of biology at Clark University who was not associated with this research, says “The discovery of tycheposons is important and exciting because it provides a new mechanistic understanding of how Prochlorococcus are able to swap in and out new genes, and thus ecologically important traits. Tycheposons provide a new mechanistic explanation for how it’s done.” He says “they took a creative way to fish out and characterize these new genetic elements ‘hiding’ in the genomes of Prochlorococcus.”

He adds that genomic islands, the portions of the genome where these tycheposons were found, “are found in many bacteria, not just marine bacteria, so future work on tycheposons has wider implications for our understanding of the evolution of bacterial genomes.”

The team included researchers at MIT’s Department of Civil and Environmental Engineering, the University of Wuerzburg in Germany, the University of Hawaii at Manoa, Ohio State University, Oxford Nanopore Technologies in California, Bigelow Laboratory for Ocean Sciences in Maine, and Wellesley College. The work was supported by the Simons Foundation, the Gordon and Betty Moore Foundation, the U.S. Department of Energy, and the U.S. National Science Foundation.

Uncovering how cells control their protein output

Gene-Wei Li investigates the rules that cells use to maintain the correct ratio of the proteins they need to survive.

Anne Trafton | MIT News Office
January 4, 2023

A typical bacterial genome contains more than 4,000 genes, which encode all of the proteins that the cells need to survive. How do cells know just how much of each protein they need for their everyday functions?

Gene-Wei Li, an MIT associate professor of biology, is trying to answer that question. A physicist by training, he uses genome-wide measurements and biophysical modeling to quantify cells’ protein production and discover how cells achieve such precise control of those quantities.

Using those techniques, Li has found that cells appear to strictly control the ratios of proteins that they produce, and that these ratios are consistent across cell types and across species.

“Coming from a physics background, it’s surprising to me that these cells have evolved to be really precise in making the right amount of their proteins,” Li says. “That observation was enabled by the fact that we are able to design measurements with a precision that matches what is actually happening in biology.”

From physics to biology

Li’s parents — his father, a marine biologist who teaches at a university in Taiwan, and his mother, a plant biologist who now runs a science camp for high school students — passed their affinity for science on to Li, who was born in San Diego while his parents were graduate students there.

The family returned to Taiwan when Li was 2 years old, and Li soon became interested in math and physics. In Taiwan, students choose their college major while still in high school, so he decided to study physics at National Tsinghua University.

While in college, Li was drawn to optical physics and spectroscopy. He went to Harvard University for graduate school, where after his first year, he started working in a lab that works on single-molecule imaging of biological systems.

“I realized there are a lot of really exciting fields at the boundary between disciplines. It’s something that we didn’t have in Taiwan, where the departments are very strict that physics is physics, and biology is biology,” Li says. “Biology is a lot messier than physics, and I had some hesitancy, but I was happy to see that biology does have rules that you can observe.”

For his PhD research, Li used single-molecule imaging to study proteins called transcription factors — specifically, how quickly they can bind to DNA and initiate the copying of DNA into RNA. Though he had never taken a class in biology, he began to learn more about it and decided to do a postdoc at the University of California at San Francisco, where he worked in the lab of Jonathan Weissman, a professor of cellular and molecular pharmacology.

Weissman, who is now a professor of biology at MIT, also trained as a physicist before turning to biology. In Weissman’s lab, Li developed techniques for studying gene expression in bacterial cells, using high-throughput DNA sequencing. In 2015, Li joined the faculty at MIT, where his lab began to work on tools that could be used to measure gene expression in cells.

When genes are expressed in cells, the DNA is first copied into RNA, which carries the genetic instructions to ribosomes, where proteins are assembled. Li’s lab has developed ways to measure protein synthesis rates in cells, along with the amount of RNA that is transcribed from different genes. Together, these tools can yield precise measurements of how much a particular gene is expressed in a given cell.

“We had the qualitative tools before, but now we can really have quantitative information and learn how much protein is made and how important those protein levels are to the cell,” Li says.

Precise control

Using these tools, Li and his students have discovered that different species of bacteria can have different strategies for making proteins. In E. coli, transcription of DNA and translation of RNA into proteins had long been known to be a coupled process, meaning that after RNA is produced, ribosomes immediately translate it into protein.

Many researchers assumed that this would be true for all bacteria, but in a 2020 study, Li found that Bacillus subtilis and hundreds of other bacterial species use a different strategy.

“A lot of other species have what we call runaway transcription, where the transcription happens really fast and the proteins don’t get made at the same time. And because of this uncoupling, these species have very different mechanisms of regulating their gene expression,” Li says.

Li’s lab has also found that across species, cells make the same proportions of certain proteins that work together. Many cellular processes, such as breaking down sugar and storing its energy as ATP, are coordinated by enzymes that perform a series of reactions in a specified sequence.

“Evolution, it turns out, gives us the same proportion of those enzymes, whether in E. coli or other bacteria or in eukaryotic cells,” Li says. “There are apparently rules and principles for designing these pathways that we didn’t know of before.”

Mutations that cause too much or too little of a protein to be produced can cause a variety of human diseases. Li now plans to investigate how the genome encodes the rules governing the correct quantities of each protein, by measuring how changes to genetic and regulatory sequences affect gene expression at each step of the process — from initiation of transcription to protein assembly.

“The next level that we’re trying to focus on is: How is that information stored in the genome?” he says. “You can easily read off protein sequences from a genome, but it’s still impossible to tell how much protein is going to be made. That’s the next chapter.”

Hot off the press: parasite researchers melt down proteins to understand their roles in infection
Eva Frederick | Whitehead Institute
August 31, 2022

Much like humans, plants, and bacteria, the single-celled parasite Toxoplasma gondii (T. gondii) uses calcium as a messenger to coordinate important cellular processes. But while the messenger is the same, the communication pathways that form around calcium differ significantly between organisms.

“Since Toxoplasma parasites are so divergent from us, they have evolved their own sets of proteins that are involved in calcium signaling pathways,”  said Alice Herneisen, a graduate student in the lab of Whitehead Institute Member Sebastian Lourido.

Lourido and his lab study the molecular mechanisms that allow the single-celled parasite T. gondii and related pathogens to be so widespread and potentially deadly — and calcium signaling is an important part of the parasite’s process of invading its hosts. “Calcium governs this very important transition from the parasites replicating inside of host cells to parasites leaving those cells and seeking out new ones to infect,” said Lourido. “We’ve been really interested in how calcium plays into the regulation of proteins inside the parasite.”

A paper published August 17 in eLife provides some insight. In the paper, Herneisen, Lourido and collaborators used an approach called thermal profiling to broadly survey which parasite proteins are involved in calcium signaling in T. gondii. The new work reveals that an unexpected protein plays a role in parasite calcium pathways, and provides new targets that scientists could potentially use to stop the spread of the parasite. The data will also serve as a resource that other Toxoplasma researchers can use to find out whether their own proteins of interest interact with calcium pathways in parasite cells.

The heat is on

When studying calcium pathways in humans, researchers can often draw parallels from work in mice. “But parasites are very different from us,” said Lourido. “All of the principles that we’ve learned about calcium signaling in humans or mice can’t be readily translated over to parasites.”

So to study these mechanisms in Toxoplasma, the researchers had to start from scratch to determine which proteins were involved. That’s where the thermal profiling method came in. The method is based on the observation that proteins are designed to work well at specific temperatures, and when it becomes too hot for them, they melt. Consider eggs: when the proteins in egg whites and egg yolks are heated in a frying pan, the proteins begin to melt and congeal. “When we think about a protein melting, what we mean is the proteins unraveling,” said Lourido. “When proteins unravel, they expose side chains that bind to each other. They stop being individual proteins that are well-folded, and become a mesh.”

Small changes to the chemical structure of a protein — such as the changes resulting from binding a small molecule such as calcium — can alter the melting point of a protein. Researchers can then trace these alterations using proteomic methods. “Proteins that are binding calcium are changing in response to calcium, and are ultimately changing their thermal stability,” Herneisen said. “That’s kind of the language of proteins, alterations in their thermal stability.”

The thermal profiling method works by applying heat to parasite cells and graphing how each of the parasite’s proteins responds to changes in temperature under different conditions (for example, the presence or absence of calcium). In a 2020 paper, the researchers used the thermal profiling method to investigate the role of a protein called ENH1 in calcium signaling.

In their new paper, Lourido and Herneisen investigated the effect of calcium on all proteins in the parasite using two approaches. The researchers combined parasites with specific amounts of calcium, applied heat, and then performed proteomics techniques to track how the calcium affected the melting behavior of each protein. If a protein’s melting point was higher or lower than usual, the researchers could deduce that that protein was changed either by calcium itself or by another player in a calcium signaling pathway.

They then treated the parasites with a chemical that caused them to release stored calcium in a controlled manner and measured how a protein modification called phosphorylation changed over time. Together, these methods allowed them to infer how proteins might sense and respond to calcium within the signaling network.

Their approach provided data on nearly every expressed protein in the parasite cells, but the researchers zeroed in on one particular protein called Protein Phosphatase 1 (or PP1). The protein is ubiquitous across many species, but has never previously been implicated in calcium signaling pathways. They found that the protein was concentrated at the front end of the parasite. This region of the parasite cell is involved in motility and host invasion.

The protein’s role in the parasites — and in the other organisms in which it appears — is to remove the small molecules called phosphates from phosphorylated proteins. “This is a modification that can often change the activity of individual proteins, because it’s this big charge that’s been covalently stuck onto the surface of the protein,” Lourido said. “This ends up being a principle through which many, many different biological processes are regulated.”

How exactly PP1 interacts with calcium remains to be seen. When the researchers depleted PP1 in parasite cells, they found that the protein is somehow involved in helping the parasite take in calcium necessary for movement. It’s unclear whether or not it actually binds calcium or is involved in the pathway through another mechanism.

Because parasites use calcium signaling to coordinate life cycle changes such as entering or leaving  host cells, insights into the key players in calcium pathways could be a boon to public health. “These are kind of the pressure points or the hubs that would be ideal to target in order to prevent the spread and pathogenesis of these parasites,” Herneisen said.

Herneisen and collaborators focused primarily on PP1, but there are many other proteins to investigate using the data from this project. “I think part of the reason why I wanted to release this paper is so that the field could take the next steps,” she said. “I’m just one person — it would be great if 20 other people find that the protein that they were studying is calcium responsive, and they can chase down the exact reason for that or how it is involved in this greater calcium signaling network. This was exciting for us with regards to PP1, and I’m sure other researchers will make their own connections.”

Notes

Alice L. Herneisen,  Zhu-Hong Li, Alex W. Chan, Silvia NJ Moreno, and Sebastian Lourido. “Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca2+-responsive pathways”. eLife, August 17, 2022. DOI: https://doi.org/10.7554/eLife.80336

Scientists identify a plant molecule that sops up iron-rich heme

The peptide is used by legumes to control nitrogen-fixing bacteria; it may also offer leads for treating patients with too much heme in their blood.

Anne Trafton | MIT News Office
August 11, 2022

Symbiotic relationships between legumes and the bacteria that grow in their roots are critical for plant survival. Without those bacteria, the plants would have no source of nitrogen, an element that is essential for building proteins and other biomolecules, and they would be dependent on nitrogen fertilizer in the soil.

To establish that symbiosis, some legume plants produce hundreds of peptides that help bacteria live within structures known as nodules within their roots. A new study from MIT reveals that one of these peptides has an unexpected function: It sops up all available heme, an iron-containing molecule. This sends the bacteria into an iron-starvation mode that ramps up their production of ammonia, the form of nitrogen that is usable for plants.

“This is the first of the 700 peptides in this system for which a really detailed molecular mechanism has been worked out,” says Graham Walker, the American Cancer Society Research Professor of Biology at MIT, a Howard Hughes Medical Institute Professor, and the senior author of the study.

This heme-sequestering peptide could have beneficial uses in treating a variety of human diseases, the researchers say. Removing free heme from the blood could help to treat diseases caused by bacteria or parasites that need heme to survive, such as P. gingivalis (periodontal disease) or toxoplasmosis, or diseases such as sickle cell disease or sepsis that release too much heme into the bloodstream.

“This study demonstrates that basic research in plant-microbe interactions also has potential to be translated to therapeutic applications,” says Siva Sankari, an MIT research scientist and the lead author of the study, which appears today in Nature Microbiology.

Other authors of the paper include Vignesh Babu, an MIT research scientist; Kevin Bian and Mary Andorfer, both MIT postdocs; Areej Alhhazmi, a former KACST-MIT Ibn Khaldun Fellowship for Saudi Arabian Women scholar; Kwan Yoon and Dante Avalos, MIT graduate students; Tyler Smith, an MIT instructor in biology; Catherine Drennan, an MIT professor of chemistry and biology and a Howard Hughes Medical Institute investigator; Michael Yaffe, a David H. Koch Professor of Science and a member of MIT’s Koch Institute for Integrative Cancer Research; and Sebastian Lourido, the Latham Family Career Development Professor of Biology at MIT and a member of the Whitehead Institute for Biomedical Research.

Iron control

For nearly 40 years, Walker’s lab has been studying the symbiosis between legumes and rhizobia, a type of nitrogen-fixing bacteria. These bacteria convert nitrogen gas to ammonia, a critical step of the Earth’s nitrogen cycle that makes the element available to plants (and to animals that eat the plants).

Most of Walker’s work has focused on a clover-like plant called Medicago truncatula. Nitrogen-fixing bacteria elicit the formation of nodules on the roots of these plants and eventually end up inside the plant cells, where they convert to their symbiotic form called bacteroids.

Several years ago, plant biologists discovered that Medicago truncatula produces about 700 peptides that contribute to the formation of these bacteroids. These peptides are generated in waves that help the bacteria make the transition from living freely to becoming embedded into plant cells where they act as nitrogen-fixing machines.

Walker and his students picked one of these peptides, known as NCR247, to dig into more deeply. Initial studies revealed that when nitrogen-fixing bacteria were exposed to this peptide, 15 percent of their genes were affected. Many of the genes that became more active were involved in importing iron.

The researchers then found that when they fused NCR247 to a larger protein, the hybrid protein was unexpectedly reddish in color. This serendipitous observation led to the discovery that NCR247 binds heme, an organic ring-shaped iron-containing molecule that is an important component of hemoglobin, the protein that red blood cells use to carry oxygen.

Further studies revealed that when NCR247 is released into bacterial cells, it sequesters most of the heme in the cell, sending the cells into an iron-starvation mode that triggers them to begin importing more iron from the external environment.

“Usually bacteria fine-tune their iron metabolism, and they don’t take up more iron when there is already enough,” Sankari says. “What’s cool about this peptide is that it overrides that mechanism and indirectly regulates the iron content of the bacteria.”

Nitrogenase, the main enzyme that bacteria use to fix nitrogen, requires 24 to 32 atoms of iron per enzyme molecule, so the influx of extra iron likely helps those enzymes to become more active, the researchers say. This influx is timed to coincide with nitrogen fixation, they found.

“These peptides are produced in a wave in the nodules, and the production of this particular peptide is higher when the bacteria are preparing to fix nitrogen. If this peptide was secreted throughout the whole process, then the cell would have too much iron all the time, which is bad for the cell,” Sankari says.

Without the NCR247 peptide, Medicago truncatula and rhizobium cannot form an effective nitrogen-fixing symbiosis, the researchers showed.

“Many possible directions”

The peptide that the researchers studied in this work may have potential therapeutic uses. When heme is incorporated into hemoglobin, it performs a critical function in the body, but when it’s loose in the bloodstream, it can kill cells and promote inflammation. Free heme can accumulate in stored blood, so having a way to filter out the heme before the blood is transfused into a patient could be potentially useful.

A variety of human diseases lead to free heme circulating in the bloodstream, including sickle cell anemia, sepsis, and malaria. Additionally, some infectious parasites and bacteria depend on heme for their survival but cannot produce it, so they scavenge it from their environment. Treating such infections with a protein that takes up all available heme could help prevent the parasitic or bacterial cells from being able to grow and reproduce.

In this study, Lourido and members of his lab showed that treating the parasite Toxoplasma gondii with NCR427 prevented the parasite from forming plaques on human cells.

The researchers are now pursuing collaborations with other labs at MIT to explore some of these potential applications, with funding from a Professor Amar G. Bose Research Grant.

“There are many possible directions, but they’re all at a very early stage,” Walker says. “The number of potential clinical applications is very broad. You can place more than one bet in this game, which is an intriguing thing.”

Currently, the human protein hemopexin, which also binds to heme, is being explored as a possible treatment for sickle cell anemia. The NCR247 peptide could provide an easier to deploy alternative, the researchers say, because it is much smaller and could be easier to manufacture and deliver into the body.

The research was funded in part by the MIT Center for Environmental Health Sciences, the National Science Foundation, and the National Institutes of Health.

Yiyin Erin Chen and Sam Chunte Peng named as core members of Broad Institute and MIT
Broad Communications
July 12, 2022

Broad Institute of MIT and Harvard has named Erin Chen, a dermatologist and microbiologist, and Sam Peng, a biophysicist and physical chemist with expertise in single-molecule imaging, as core institute members.

Chen will join in January 2023 and will also serve as an assistant professor in the Department of Biology at MIT and an attending dermatologist at Massachusetts General Hospital. Peng joined in July 2022 and will serve as an assistant professor in the Department of Chemistry at MIT.

Chen’s lab will study the communication between the immune system and the diverse microbes that colonize every surface of the human body, with a focus on the human body’s largest organ, the skin.

Peng’s lab will develop novel probes and microscopy techniques to visualize the dynamics of individual molecules in living cells, which will improve the understanding of molecular mechanisms underlying human diseases.

“We are delighted to welcome Sam and Erin to the Broad community,” said Todd Golub, director of the Broad. “These creative scientists are each taking inventive approaches to understand the molecular signals and interactions that underlie biological processes in health and disease. These insights will help further the Broad’s mission of advancing the understanding and treatment of human disease.”

Erin Chen.
Erin Chen

Erin Chen earned her BA in biology from the University of Chicago, her PhD from MIT, and her MD from Harvard Medical School. Prior to joining the Broad, Chen was a Howard Hughes Medical Institute Hanna Gray Postdoctoral Fellow at Stanford University, in the lab of Michael Fischbach. She was also an attending dermatologist at the University of California San Francisco and at the San Francisco VA Medical Center. During her postdoctoral research, Chen developed genetic methods to study harmless commensal skin bacteria. She engineered these bacteria to generate anti-tumor immunity, pioneering a novel approach to vaccination and cancer immunotherapy.

At the Broad, members of the Chen lab will continue to employ microbial genetics, immunologic approaches, and mouse models to dissect the molecular signals used by commensal microbes to educate the immune system. Ultimately, Chen aims to harness these microbe-host interactions to engineer novel therapeutics for human disease.

“I’m excited to join the collaborative scientific community at the Broad and MIT, including those who have pioneered novel tools for examining biological mechanisms at higher spatial resolution,” said Chen. “The biology I study is quite basic, but I’m motivated by the potential impact it could have on patients. Figuring out how commensal skin bacteria are captured by the immune system could unlock a whole new therapeutic toolbox.”

Sam Peng
Sam Peng

Sam Peng earned his BS in chemistry from the University of California, Berkeley, and his PhD from MIT in physical chemistry. He completed his postdoctoral research at Stanford University as an NIH K99 Pathway to Independence scholar in the lab of Steve Chu. During his postdoctoral research, he developed long-term single molecule imaging in live cells using a novel class of nanoprobes. He applied this new technique to study axonal transport in neurons and the molecular dynamics of dynein — a motor protein involved in transporting cargo in cells.

At the Broad, the Peng group will aim to elucidate the molecular mechanisms underlying human diseases. Lab members will develop and integrate a diverse toolbox spanning single-molecule microscopy, super-resolution microscopy, spectroscopy, nanomaterial engineering, biophysics, chemical biology, and quantitative modeling to uncover previously unexplored biological processes. With bright and photostable probes, lab members will have unprecedented capability to record ultra-long-term “molecular movies” in living systems with high spatiotemporal resolutions and to reveal molecular interactions that drive biological functions. Peng’s group will focus on studying molecular dynamics, protein-protein interactions, and cellular heterogeneity involved in neurobiology and cancer biology. Their long-term goal is to translate these mechanistic insights into drug discovery.

“Because my research is so multi-disciplinary, joining the Broad and MIT communities allows us to integrate a range of experimental tools and to collaborate with colleagues and students from diverse backgrounds,” said Peng. “I’m excited to see how our techniques can enable discoveries for a variety of cellular processes, including those underlying complex brain functions and dysfunctions. Many problems that previously seemed inaccessible now appear to be within reach in the foreseeable future.”

Yiyin Erin Chen

Education

  • Graduate: PhD, 2011, MIT; MD, 2013, Harvard Medical School
  • Undergraduate: BA, 2006, Biology, University of Chicago

Research Summary

Diverse commensal microbes colonize every surface of our bodies. We study the constant communication between these microbes and our immune system. We focus on our largest organ: the skin. By employing microbial genetics, immunologic approaches, and mouse models, we can dissect (1) the molecular signals used by microbes to educate our immune system and (2) how different microbial communities alter immune responses. Ultimately, we aim to harness these microbe-host interactions to engineer novel vaccines and therapeutics for human disease.

Awards

  • Howard Hughes Medical Institute Hanna H. Gray Fellow, 2018-2026
  • A.P. Giannini Postdoctoral Research Fellowship, 2018
  • Dermatology Foundation Research Fellowship, 2017