Genetics Updates vol.62
October 31st 2024
Check out latest research updates in the field
TL;DR
- Cellular superhero that protects us against RNA viruses
- Gut bacteria transfer genes to disable weapons of their competitors
- Plastic chemical causes DNA breakage and chromosome defects in sex cells
- New tool enables more complete and rapid decoding of the language of algal gene expression
- Structural biology analysis of a pseudomonas bacterial virus reveals a genome ejection motor
Latest Research
by Lucía Álvarez, Kevin Haubrich, Louisa Iselin, Laurent Gillioz, et al in Nature Communications
Every second of every day, our body is under attack. The invading agents are viruses, bacteria, parasites, toxins -- living and non-living entities that might negatively impact our body's functioning. What keeps us safe is a squad of patrolling superheroes -- proteins that form an essential part of our innate immune system, the body's first line of defence against invaders.
A new study from EMBL Heidelberg researchers has brought us one step closer to understanding how one such superhero -- a protein called TRIM25 -- exercises its superpowers to fight viruses.
"We were inspired to study TRIM25 because of its critical role in the body's innate immune response to RNA viruses, such as influenza or Zika viruses," said Lucía Álvarez, the study's first author and EIPOD4 postdoctoral fellow in EMBL's Hennig Group. "We wanted to understand the role of TRIM25's RNA binding in antiviral defence."
NMR and ITC analysis of RNA binding by TRIM25 PRY/SPRY domain.
TRIM25 belongs to a large family of enzymes that can tag other proteins in the cell with a small protein called ubiquitin, altering their function. Its superpower is the ability to trigger a series of signaling events that eventually leads to the foreign agent being identified and neutralised. While scientists had previously shown that TRIM25 can bind RNA, it wasn't clear why this action is important for its immune activity.
TRIM25 also faces the proverbial needle in a haystack problem -- after all, our cells are swimming in RNA, much of it essential for our biology and functioning. So, does TRIM25 have a way to distinguish friend from foe and selectively bind to RNA that comes from viruses?
The scientists used a combination of biophysical and cell biological techniques to investigate this question in more detail. "We found that TRIM25 doesn't just randomly bind to any RNA," said Álvarez. "It has specific preferences, which may explain how it efficiently targets regions of viral RNA."
The scientists also found that this binding to viral RNA was critical for TRIM25's antiviral activity, as well as its ability to find its way to 'factories' inside the cell where the virus makes copies of itself. To test this, the researchers created a mutant version of TRIM25, which could not bind RNA. Cells that had this 'defective' version of TRIM25 were less effective in fighting an infection by the Sindbis virus -- an RNA virus that can be transferred from mosquitoes to vertebrates.
The study, recently published in the journal Nature Communications, was carried out in collaboration with Alfredo Castello's group at the Centre for Virus Research (CVR) in Glasgow. The researchers also worked closely with Fred Allain's group at ETH Zurich.
"This project was made possible by the EIPOD4 grant and the infection biology transversal theme (IBTT) synergy grant, which allowed me to travel from EMBL to CVR to benefit from the synergies between the groups," said Álvarez.
As a next step, the researchers are investigating whether TRIM25's RNA binding is important not just for the Sindvis virus but also for defence against other RNA viruses. The team is also collaborating with Julia Mahamid's group at EMBL Heidelberg to use cryo-electron tomography to get a closer look at the viral replication organelles inside cells where TRIM25 localises. A recent German Research Foundation grant, jointly submitted by the two groups, will enable this part of the work.
"TRIM25 plays a key role in how our bodies respond to viruses, such as influenza, dengue, and coronaviruses," said Janosch Hennig, EMBL Visiting Group Leader and the study's senior author. "By better understanding how TRIM25 works, we could potentially develop strategies to enhance this immune response, making it a potential target for antiviral therapies. In addition, the study could be applied to wider research into RNA-binding proteins and innate immunity, helping to uncover similar mechanisms in other proteins or immune pathways."
A ubiquitous mobile genetic element changes the antagonistic weaponry of a human gut symbiont
by Madeline L. Sheahan, Katia Flores, Michael J. Coyne, Leonor García-Bayona, Maria Chatzidaki-Livanis, Andrea Q. Holst, Rita C. Smith, Anitha Sundararajan, Blanca Barquera, Laurie E. Comstock in Science
Bacteria evolve rapidly in the human gut by sharing genetic elements with each other. Bacteriodales is a prolific order of gut bacteria that trade hundreds of genetic elements. Little is known, however, about the effects of these DNA transfers, either to the fitness of the bacteria or the host.
New research from the University of Chicago shows that a large, ubiquitous mobile genetic element changes the antagonistic weaponry of Bacteroides fragilis, a common bacterium of the human gut. Acquisition of this element shuts down a potent weapon of B. fragilis, yet arms it with a new weapon to which the strain that donated the DNA is protected. These weapons help the bacteria carve out niches in the tightly packed recesses of the gut.
Laurie Comstock, PhD, Professor of Microbiology and member of the Duchossois Family Institute at UChicago, and senior author of the new study, has been studying different antagonistic mechanisms of Bacteroidales and the way they transfer DNA for more than 10 years. "These organisms evolve rapidly by DNA transfers. It's quite amazing," she said. “We knew that some strains of B. fragilis couldn't fire their weapons, but when we saw it was due to the acquisition of a large mobile genetic element, that's when we knew we found something interesting.”
Many Bacteroidales species can kill neighboring bacteria by producing toxins. Some of these toxins simply diffuse from the bacterial cell into the surrounding environment, killing nearby sensitive strains. Another weapon is the type VI secretion system (T6SS), which is a nanomachine containing a pointed, spring-loaded tube loaded with toxins. When it fires, it injects toxins directly into neighboring cells like a poison-tipped spear.
The Bacteroidales T6SS comes in three different types, or genetic architectures. One, genetic architecture 3 (GA3), is exclusive to B. fragilis and is very effective at killing other Bacteroidales species. The other two types, GA1 and GA2, are encoded by genes contained on large mobile genetic elements called integrative and conjugative elements (ICEs). These GA1 and GA2 ICEs are rapidly transferring between Bacteroidales species in the human gut throughout the world. However, scientists have yet to observe the same, lethal potency in GA1 and GA2 T6SSs as they have for the GA3 T6SS.
"The ICE containing the GA1 T6SS ICE is racing through human populations, and rapidly transferring to numerous Bacteroidales species in a person's gut," Comstock said.
Comstock's team started studying natural B. fragilis isolates that had a GA3 T6SS or had both a GA3 and GA1 ICE. Those with both ICEs no longer fired the GA3 weapon and could no longer kill other Bacteriodales species. To show this was due to the addition of the GA1 ICE to these strains, they transferred the GA1 ICE into B. fragilis strains with only the GA3 T6SS and showed that the resulting new strains, or "transconjugants," were similarly unable to antagonize other strains with their GA3 T6SS.
The researchers then deleted portions of the GA1 ICE to see which region of the 116 kilobase ICE was shutting off the GA3 weapon. They found that a portion of the GA1 T6SS region encoding the membrane complex of the GA1 nanomachine prevented GA3 T6SS firing.
Next, the team wanted to see how the strains would compete in the mammalian gut. They orally inoculated gnotobiotic (germ-free) mice with equal numbers of isogenic, wild-type B. fragilis (GA3 T6SS only) and the GA3/GA1 ICE transconjugant. The transconjugant quickly outcompeted the wild-type strain in the mice. The investigators went on to show that that this competition was due to antagonism using the GA1 T6SS, the first demonstration of potent antagonism by the GA1 T6SS.
"We didn't know if the GA1 containing strain was going to be antagonistic, so we thought the progenitor GA3 strain would win that battle in the gut," Comstock said. “But that was not what happened.”
The most unexpected finding from this experiment was that in the mouse gut, the GA3 T6SS was not being made at all. They later showed that a gene carried on the GA1 ICE encodes a transcriptional repressor that shuts down transcription of the entire GA3 T6SS, allowing even better production of the GA1 T6SS.
The overall effect of the transfer of this DNA element has consequences for the gut microbial community. The Bacteroidales strains containing the GA1 ICE are killed by the B. fragilis GA3 T6SS, but if one of these strains can transfer their GA1 ICE into the attacking B. fragilis strain, they create a strain that outcompetes the progenitor B. fragilis strain. This new strain no longer targets the donor strain and can also use the GA1 T6SS to communally defend the ecosystem from invasion by other Bacteroidales strains.
Comstock plans to continue studying this diverse family of transcriptional repressors that are frequently carried on mobile genetic elements of the Bacteroidales and their effects in recipient strains.
"This family of transcriptional repressors can be inactivated when they bind specific ligands. We would love to identify the ligands in the gut that derepress their activity," she said.
The study also showed that in the mouse gut, the GA1 ICE transfer occurred rapidly, helping the transconjugant become a large component of the of the population. This suggests that researchers creating synthetic consortia of bacteria for therapeutics need to account for the effects of genetic transfer.
"As bacteria are being selected for inclusion in consortia as biotherapeutics, it is important to safeguard against introducing anything that could be transferred into or out of these strains that might have deleterious effects," Comstock said.
by Ayana L. Henderson, Rajendiran Karthikraj, Emma L. Berdan, Shannan Ho Sui, Kurunthachalam Kannan, Monica P. Colaiácovo in PLOS Genetics
A new study conducted in roundworms finds that a common plastic ingredient causes breaks in DNA strands, resulting in egg cells with the wrong number of chromosomes. Monica Colaiácovo of Harvard Medical School led the study.
Benzyl butyl phthalate (BBP) is a chemical that makes plastic more flexible and durable, and is found in many consumer products, including food packaging, personal care products and children's toys. Previous studies have shown that BBP interferes with the body's hormones and affects human reproduction and development, but the details of how it impacts reproduction has been unclear.
BP dose-response curve reveals non-monotonic effects of BBP on rate of X-chromosome nondisjunction, apoptosis, and chromosome organization defects in the germline.
In the new study, researchers tested a range of doses of BBP on the nematode Caenorhabditis elegans and looked for abnormal changes in egg cells. They saw that at levels similar to those detected in humans, BBP interferes with how newly copyied chromosomes are distributed into the sex cells. Specifically, BBP causes oxidative stress and breaks in the DNA strands, which lead to cell death and egg cells with the wrong number of chromosomes.
Based on these findings, the researchers propose that BBP exposure alters gene expression in ways that cause significant damage to the DNA, ultimately leading to lower quality egg cells with abnormal chromosomes. The study also showed that C. elegans metabolizes BBP in the same way as mammals, and is impacted at similar BBP levels that occur in humans, suggesting that C. elegans is an effective model for studying the impacts in people. Overall, the study underscores the toxic nature of this very common plastic ingredient and the damage it causes to animal reproduction.
The authors summarize: "Here, examining the female germline in the nematode C. elegans, this study found that a level of exposure within the range detected in human serum and urine, alters gene expression linking increased germline oxidative stress with compromised genomic integrity and errors in meiotic chromosome segregation."
by James J. Pesavento, Megan S. Bindra, Udayan Das, Sarah R. Rommelfanger, Mowei Zhou, Ljiljana Paša-Tolić, James G. Umen in Analytical Chemistry
A new method that research teams can use to measure and compare different forms of proteins and protein complexes helped reveal a previously unseen molecular signature of how algal genomes are controlled during the cell cycle.
The research collaboration included James Umen, PhD, member and principal investigator, Danforth Plant Science Center, James (Jim) Pesavento, PhD, associate professor at Saint Mary's College of California (SMC), Mowei Zhou, PhD, Qiushi scholar for experiments, Zhejiang University and Ljiljana Paša-Tolić, PhD, lead scientist for Visual Proteomics, Department of Energy Environmental and Molecular Sciences Laboratory in Richland, WA.
The Umen lab is well known for longstanding research to understand how algal cells multiply and differentiate into sexually distinct types. Pesavento is an expert in using mass spectrometry to identify biological molecules with extremely high precision and to quantify their abundance. His special expertise is on a very important group of proteins called histones that are used to package DNA in all organisms whose cells have a nucleus including plants, algae and humans. Histones are not only essential for packaging and DNA into a compact form called chromatin, but also are decorated with chemical modifications that serve as signals or signposts marking locations of genes and whether they should be expressed. These markings are sometimes referred to as an epigenome as they add an additional layer of information to the DNA with which they associate.
A major challenge in this field has been figuring out which histones have which chemical modifications, where on the histone protein those modifications occur, and whether they are dynamic (added and removed under specific conditions). The combinatorial possibilities make this task especially difficult even with the most advanced mass spectrometry instrumentation and software available. Moreover, each group of organisms such as plants and green algae, seem to have their own variant histone code language, and it is important to learn this language as a tool for helping make improved varieties with beneficial traits.
Although commercial software exists to help with this task, there was no software that was robust enough to tackle histone modifications on whole histone proteins. Pesavento realized that the repetitive and time-consuming tasks to identify histone modifications could be partially automated, and he set out to create an open-source tool called pyMS-Vis to help solve this problem for algal histones and other histone researchers.
"This work started as a collaboration with SMC professor Udayan Das, PhD, as we co-mentored an undergraduate computer science student Megan Bindra during the SMC undergraduate Summer Research Program in 2022," said Pesavento. "We were able to make significant progress, and Bindra presented this work at a professional conference the following year (2023). The combination of NSF funding support for my small lab at SMC and invested collaborators across diverse scientific disciplines, were essential to this work's publication."
To test this method, he used a set of histone samples the Umen lab prepared from cells that were in different stages of their cell division cycle to answer questions about what happens to marks on histones in cells when they are replicating their DNA versus when they are growing but not replicating DNA or dividing. pyMS-Vis made it possible to rapidly analyze the data and discover a new and unexpectedly large population of a specific histone sub-type that was missing a mark which had always been assumed to be present on nearly every histone of this sub-type.
"pyMS-Vis has allowed us to see histone dynamics that we could not easily see before and opened the door to a more complete understanding of the language of algal gene expression," said Umen. "This deeper understanding will be a critical part of developing algae as productive crop species that stably express beneficial traits such as increased yields of oil or high value products."
Integrative structural analysis of Pseudomonas phage DEV reveals a genome ejection motor
by Ravi K. Lokareddy, Chun-Feng David Hou, Francesca Forti, Stephano M. Iglesias, Fenglin Li, Mikhail Pavlenok, David S. Horner, Michael Niederweis, Federica Briani, Gino Cingolani in Nature Communications
The viruses that infect bacteria are the most abundant biological entities on the planet. For example, a recent simple study of 92 showerheads and 36 toothbrushes from American bathrooms found more than 600 types of bacterial viruses, commonly called bacteriophages or phages. A teaspoon of coastal seawater has about 50 million phages.
While largely unnoticed, phages do not harm humans. On the contrary, these viruses are gaining increasing popularity as biomedicines to eradicate pathogenic bacteria, especially those associated with antibiotic-resistant infections.
In a study, Gino Cingolani, Ph.D., of the University of Alabama at Birmingham, and Federica Briani, Ph.D., of the Università degli Studi di Milano, Milan, Italy, have described the full molecular structure of the phage DEV. DEV infects and lyses Pseudomonas aeruginosa bacteria, an opportunistic pathogen in cystic fibrosis and other diseases. DEV is part of an experimental phage cocktail developed to eradicate P. aeruginosa infection in pre-clinical studies.
Cryo-EM analysis of the Pseudomonas phage DEV.
A peculiar feature of DEV is the presence of a 3,398-amino acid virion-associated RNA polymerase inside the capsid expelled into the bacterium upon infection. Unexpectedly, Cingolani and Briani's study revealed the virion-associated RNA polymerase is part of a genome ejection motor that pulls the DNA of the phage out of its head after the phage has attached to the surface of a Pseudomonas bacteria using its tail fibers and has penetrated the cell's outer and inner membranes using its tail tube.
"We posit that the design principles of the DEV ejection apparatus are conserved in all Schitoviridae phages," Cingolani said. "As of October 2024, over 220 Schitoviridae genomes have been sequenced and are available in the public database. As these genomes are largely unannotated and many open-reading frames have unknown functions, our work paves the way for the facile identification of structural components when a new Schitoviridae phage is discovered."
The Schitoviridae family of phages "represents some of biology's most understudied bacterial viruses, increasingly utilized in phage therapy," Cingolani said. "We are using structural biology to decipher the building blocks and map gene products. This is vital when the amino acid sequence evolves too rapidly for conventional phylogenetic analysis."
The researchers used cryo-electron microscopy localized reconstruction, biochemical methods and genetic knockouts to describe the complete molecular architecture of DEV, whose DNA genome has 91 open-reading frames that include the giant virion-associated RNA polymerase. "This vRNAP is part of a three-gene operon conserved all Schitoviridae genomes we analyzed," Cingolani said. "We propose these three proteins are ejected into the host to form a genome ejection motor spanning the cell envelope."
The structure of DEV and many other phages resembles a minuscule version of Neil Armstrong's 1969 lunar lander, with a large head, or capsid, that contains the genome and leg-like fibers supporting the phage as it lands on the surface of bacteria, preparing to infect the living bacterial cell.
The researchers determined structures of all the protein capsid factors and tail components in DEV involved in host attachment. Through genetic experiments, they showed that the DEV long tail fibers were essential for infection of P. aeruginosa but were not needed to infect P. aeruginosa mutants whose surface lipopolysaccharide lacked the O-antigen. In general, viruses attach to different cell surface molecules as the first step of infection.
While this study provides several still images of the phage structure, the researchers do not completely understand the movie of DEV infection. They envision three steps in that infection process.
In step one, as a single DEV phage drifts in isolation, its flexible long tail fibers fluctuate to improve the chance of touching a Pseudomonas lipopolysaccharide surface molecule. After the first touch, all five fibers attach to tether the phage perpendicularly close to the bacterial outer surface.
In step two, the short tail fiber, which also acts as a tail plug, touches a secondary receptor on the Pseudomonas and a mechanical signal releases the tail plug.
Up to this point, the three proteins called gp73, gp72 and gp71 have been stored inside the phage head near its tail, with shapes that will dramatically change when they exit the phage head. In step three, when the plug is gone, the three proteins are expelled out of the head and into the bacterial cell envelope. The lead protein, gp73, refolds its shape to form an outer membrane pore with a hollow center. Below that, gp72 refolds into a hollow tube that spans the Pseudomonas periplasm, the space between the bacteria's outer membrane and its inner membrane. Finally, gp71 crosses the inner membrane and refolds into a large RNA polymerase motor in the bacterial cytoplasm that pulls the phage DNA through the hollow gp73 and gp72 channels and into the Pseudomonas cell.
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