Tag Archives: Enzyme

Experimental decoy provides long-term protection from SARS-Cov-2 infection

31 May, 2023 Admin Leave a comment

Reviewed by Danielle Ellis, B.Sc.May 30 2023

An experimental “decoy” provided long-term protection from infection by the pandemic virus in mice, a new study finds.

Led by researchers at NYU Grossman School of Medicine, the work is based on how the virus that causes COVID-19, SARS-CoV-2, uses its spike protein to attach to a protein on the surface of the cells that line human lungs. Once attached to this cell surface protein, called angiotensin converting enzyme 2 (ACE2), the virus spike pulls the cell close, enabling the virus to enter the cell and hijack its machinery to make viral copies.

Earlier in the pandemic, pharmaceutical companies designed monoclonal antibodies to glom onto the spike and neutralize the virus. Treatment of patients soon after infection was successful in preventing hospitalization and death. However the virus rapidly evolved through random genetic changes (mutations) that altered the spike’s shape enough to evade even combinations of therapeutic monoclonal antibodies. Thus, such antibodies, which neutralized early variants, became about 300 times less effective against more recent delta and omicron variants.

Published online this week in the Proceedings of the National Academy of Sciences, the study describes an alternative approach from which the virus cannot escape. It employs a version of ACE2, the surface protein to which the virus attaches, which, unlike the natural, cell-bound version, is untethered from the cell surface. The free-floating “decoy” binds to the virus by its spikes so that it can no longer attach to ACE2 on cells in airways. Unlike the monoclonal antibodies, which are shaped to interfere with a certain spike shape, the decoy mimics the spike’s main target, and the virus cannot easily evolve away from binding to ACE2 and still invade cells.

Treatment with the decoy, either by injection or droplets in the nose, protected 100 percent of the study mice when they were infected in the lab with an otherwise lethal dose of SARS-CoV-2. The decoy lowered the virus load in the mice by 100,000-fold, while mice exposed to a non-active control treatment died. Decoy treatment of mice that were already infected with SARS-CoV-2 caused a rapid drop in viral levels and return to health. This suggests that the decoy could be effective as a therapy post-infection, similar to monoclonal antibodies, the researchers say.

What is remarkable about our study is that we delivered the decoy using a harmless, adeno-associated virus or AAV vector, a type of gene therapy that has been found in previous studies to be safe for use in humans. The viral vector instructs cells in the body to produce the decoy so that the mouse or person is protected long-term, without the need for continual treatment.”

Nathanial Landau, PhD, senior study author, professor, Department of Microbiology at NYU Langone Health

Administered with the vector, says Landau, the treatment caused cells, not only to make the decoy, but to continue making it for several months, and potentially for years.

Importantly, vaccines traditionally include harmless parts of a virus they are meant to protect against, which trigger a protective immune response should a person later be exposed. Vaccines are less effective, however, if a person’s immune system has been compromised, by diseases like cancer or in transplant patients treated with drugs that suppress the immune response to vaccination. Decoy approaches could be very valuable for immunocompromised patients globally, adds Landau.

Future pandemics

For the new study, the research team made key changes to a free ACE2 receptor molecule, and then fused the spike-binding part of it to the tail end of an antibody with the goal of strengthening its antiviral effect. Attaching ACE2 to the antibody fragment to form what the team calls an “ACE2 microbody” increases the time that the molecule persists in tissues (its half-life). The combination also causes the molecules to form dimers, mirror-image molecular pairs that increase the strength with which the decoy attaches to the viral spike.

Whether administered via injection into muscle, or through droplets in the nasal cavity, the study’s AAV vectors provided mice with long-lasting protection COVID infection, including the current Omicron variants.

The approach promises to be effective even if another coronavirus, a type of virus common in birds and bats or apes, were to be transferred to humans in the future, an event termed “zoonosis.” As long as the future virus also uses ACE2 to target cells, the decoy would be ready for “off-the-shelf” soon after an outbreak. If the virus were to somehow switch its receptor a different protein on the surface of lung cells, the decoy could be modified to target the new virus, says Landau.

Along with Landau, the study authors were Takuya Tada and Julia Minnee in the Department of Microbiology at NYU Grossman School of Medicine. The study was supported by a grant from the National Institutes of Health.

Source:

NYU Langone Health / NYU Grossman School of Medicine

Journal reference:

Tada, T., et al. (2023) Vectored immunoprophylaxis and treatment of SARS-CoV-2 infection in a preclinical model. PNAS. doi.org/10.1073/pnas.2303509120.

Microbiology

Novel antibodies target human receptors to neutralize SARS-CoV-2 variants and future sarbecoviruses

17 May, 2023 Admin Leave a comment

By Neha MathurMay 17 2023Reviewed by Lily Ramsey, LLM

In a recent study published in the Nature Microbiology Journal, researchers generated six human monoclonal antibodies (mAbs) that prevented infection by all human angiotensin-converting enzyme 2 (ACE2) binding sarbecoviruses tested, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, Delta and Omicron.

They targeted the hACE2 epitope that binds to the SARS-CoV-2 spike (S) glycoprotein rather than targeting the S protein, which all previous therapeutic mAbs for SARS-CoV-2 targeted.

Study: Pan-sarbecovirus prophylaxis with human anti-ACE2 monoclonal antibodies. Image Credit: paulista/Shutterstock.com

Background

The emergence of new variants of SARS-CoV-2, especially Omicron sublineages, made all therapeutic mAbs targeting SARS-CoV-2 S obsolete.

Any new S-targeting mAb therapy will also probably have limited utility because SARS-CoV-2 will continue to adapt to human antibodies. Ideally, mAbs developed in anticipation of future pandemics caused by sarbecoviruses should be resilient to mutations that arise in them.

About the study

In the present study, researchers developed hACE2-binding mAbs that blocked infection by pseudotypes of all tested sarbecoviruses at potencies matching SARS-CoV-2 S targeting therapeutic mAbs. The binding affinity of these mAbs to hACE2 was in the nanomolar to picomolar range.

To develop these mAbs, researchers used the KP and Av AlivaMab mouse strains that generate a human Kappa (κ) light chain and Kappa (κ) and Lambda (λ) light chains carrying antibodies, respectively.

They immunized these mice with monomeric and dimeric recombinant hACE2 extracellular domains. Fusion to the fraction, crystallizable (Fc) portion of human immunoglobulin G1 (IgG1) rendered them dimeric.

Further, the team generated hybridomas from mice using sera that inhibited SARS-CoV-2 pseudotyped viruses. They used enzyme-linked immunosorbent assay (ELISA) to screen hybridoma supernatants for hACE2-binding mAbs.

Furthermore, the researchers tested the ability of the six most potent mAbs to inhibit Wuhan-hu-1 S pseudotyped infection in Huh-7.5 target cells.

The team purified chimeric mAbs from the hybridoma culture supernatants and used a SARS-CoV-2 pseudotype assay to reconfirm their antiviral activity. They also sequenced the human Fab variable regions, VH and VL.

The team cloned V_H and V_L domains from the six most potent chimeric human-mouse mAbs into a human IgG1 expression vector to generate fully human anti-hACE2 mAbs.

They used single-particle cryo-electron microscopy (cryo-EM) to delineate the structural basis for broad neutralization of anti-hACE2 mAbs.

Specifically, they determined the structure of soluble hACE2 bound to the antigen-binding fragment (Fab) of 05B04, one of the most potent mAbs unaffected by naturally occurring human ACE2 variations.

Finally, the researchers tested these hACE2 mAbs in an animal model and determined their pharmacokinetic behavior.

Results

The researchers identified 82 hybridomas expressing hACE2-binding mAbs, of which they selected ten based on their potency in inhibiting pseudotyped virus infection of Huh-7.5 cells.

These ten mAbs were 1C9H1, 4A12A4, 05B04, 2C12H3, 2F6A6, 2G7A1, 05D06, 05E10, 05G01 and 05H02. Four of the five mAbs from the KP AlivaMab mice, viz., 05B04, 05E10, 05G01, and 05D06, shared identical complementarity-determining regions (CDRs). Conversely, AV AlivaMab mice-derived mAbs were diverse.

While allosteric inhibition of hACE2 activity by the mAbs was theoretically feasible, such inhibition did not occur.

Also, the anti-hACE2 mAbs did not affect hACE2 internalization or recycling, suggesting that the anti-hACE2 mAbs would unlikely undergo accelerated target-dependent clearance from the circulation during in vivo use.

These two findings confirmed that these mAbs would not have harmful side effects based on their target specificity.

In addition, the anti-hACE2 mAbs showed favorable pharmacokinetics and no ill effects on the hACE2 knock-in mice. When used prophylactically in hACE2 knock-in mice, these mAbs conferred near-sterilizing protection against lung SARS-CoV-2 infection.

Moreover, they presented a high genetic barrier to the acquisition of resistance by SARS-CoV-2.

The six anti-hACE2 mAbs also inhibited infection by pseudotyped SARS-CoV-2 variants, Delta, and Omicron, with similar potency, i.e., half maximal inhibitory concentration (IC₅₀) values ranging between 8.2 ng ml⁻¹ and 197 ng ml⁻¹.

A cryo-EM structure of the 05B04-hACE2 complex at 3.3 Å resolution revealed a 05B04 Fab bound to the N-terminal helices of hACE2.

05B04-mediated inhibition of ACE2-binding sarbecoviruses through molecular mimicry of SARS-CoV-2 receptor-binding domain (RBD) interactions, providing high binding affinity to hACE2 despite the smaller binding footprint on hACE2.

None of the four most potent mAbs affected hACE2 enzymatic activity or induced the internalization of hACE2 localized on the host cell surface. Thus, based on their target specificity, these mAbs shall not have deleterious side effects.

Though these anti-ACE2 antibodies could effectively inhibit sarbecovirus infection in humans, the fact that the antibodies target a host receptor molecule rather than the SARS-CoV-2 S protein will necessitate their testing in terms of safety, efficacy, and pharmacological behavior in primate models before human clinical trials.

Conclusions

SARS-CoV-2 might evolve and start using receptors other than ACE2, creating another genetic hurdle to overcome for researchers working on the development of SARS-CoV-2 therapeutics.

However, the human anti-hACE2 mAbs engineered in this study showed exceptional breadth and potency in inhibiting infection by hACE2-utilizing sarbecoviruses.

Thus, they represent a long-term, ‘resistance-proof’ prophylaxis and treatment for SARS-CoV-2, even for future outbreaks of SARS-like coronaviruses.

In addition, these mAbs might prove particularly useful for susceptible patients like those with immunodeficiency and in which vaccine-induced protective immunity is unattainable or difficult to attain.

Journal reference:

Zhang, F. et al. (2023) “Pan-sarbecovirus prophylaxis with human anti-ACE2 monoclonal antibodies”, Nature Microbiology. doi: 10.1038/s41564-023-01389-9. https://www.nature.com/articles/s41564-023-01389-9

Microbiology

Elucidating the mysteries of enzyme evolution at the macromolecular level

8 May, 2023 Admin Leave a comment

Professor Nicolas Doucet and his team at Institut national de la recherche scientifique (INRS) made a major breakthrough earlier this year in the field of evolutionary conservation of molecular dynamics in enzymes. Their work, published in the journal Structure, points to potential applications in health, including the development of new drugs to treat serious diseases such as cancer or to counter antibiotic resistance.

As a researcher specializing in protein dynamics, Professor Doucet is captivated by things that are invisible to the naked eye, yet full of mysteries and essential to all forms of life. He studies proteins and enzymes, and the poorly understood links between their structure, function, and motion at the atomic scale.

To better envision unexplored avenues of enquiry, the enzyme engineering specialist starts by examining problems from a conceptual standpoint.

“A bit of imagination may be all it takes to envision multiple paths of enquiry in this tiny world we still know relatively little about, but the scientific process is very meticulous,” said Professor Doucet, a researcher at the Armand-Frappier Santé Biotechnologie Research Centre and scientific co-head of the Nuclear Magnetic Resonance Spectroscopy Lab at INRS.

Towards a better understanding of macromolecular function

As part of this study, Professor Doucet’s team investigated an issue considered fundamental by experts in the field: if a particular protein or enzyme relies on the conformational change of its three-dimensional structure to perform its biological function in humans, do homologous enzymes in other vertebrates or other living organisms also depend on these same conformational changes? In other words, if certain motions are essential to the biological function of proteins and enzymes, are these conformation changes selected and conserved as a molecular evolutionary mechanism in all forms of life?

Despite our very limited understanding of how these macromolecules essential to life on Earth actually work, the team attempted to answer this question.

Developments in biochemical and biophysical technology in recent decades have made it easier to observe the molecular structures of proteins and enzymes.

“We studied different enzymes of the same family to analyze several proteins exhibiting the same biological function. We compared their atomic-scale motions to uncover whether they are preserved throughout evolution. Despite overall similarities between species, we were surprised to find that, on the contrary, movements are divergent,” explained the lead author of the study, David Bernard, an INRS graduate who was a PhD student in Professor Doucet’s lab at the time. He now works as a researcher at NMX.

Molecular motions of great importance

The molecular function of a protein or enzyme depends on its amino acid sequence, but also on its three-dimensional (3D) structure. In recent years, scientists have discovered that protein dynamics are closely linked to the biological activity of certain enzymes and proteins.

If this is the case for a given enzyme, what about the conservation of these motions from an evolutionary standpoint? In other words, are specific atomic motions in an enzyme family always present and similarly conserved to preserve biological function?

This would imply that the atomic-scale motions within proteins are an important determinant of the selective pressure experienced to preserve biological function, similar to the preservation of an amino acid sequence or a protein structure.

In the article, Professor Doucet’s team and their U.S. collaborators present a molecular and dynamic analysis of several ribonucleases, enzymes known as RNases that catalyze the degradation of RNA into smaller elements. RNases from a handful of vertebrate species, including primates and humans, were selected based on their structural and functional homology.

This study, which builds on *previously published research by the team, convincingly demonstrates that RNases that retain specific biological functions in various species also maintain a very similar dynamic profile among themselves. In contrast, structurally similar RNases with distinct biological function demonstrate a unique dynamic profile, strongly suggesting that the preservation of dynamics is related to biological function in these biocatalysts.

Elucidating the motions essential to the function of a protein or enzyme therefore holds promise for exploiting its therapeutic potential. This could provide a potential target for controlling protein and enzyme functions in the cell, a field known as allosteric modulation or inhibition.

For example, successfully inhibiting an enzyme by binding a drug to its active (or orthosteric) site while also targeting an allosteric site on the surface of a protein could kill two birds with one stone. The idea here is to inhibit the active site of the enzyme while at the same time disrupting its molecular dynamics by targeting an allosteric site. This inhibitory action would also significantly reduce the development of antibiotic resistance.

Drug resistance is a global health issue. In recent years, one of the most compelling and widely publicized examples of this has been antibiotic resistance in the fight against bacteria that infect humans and farm animals.

In conclusion, since specific molecular motions are uniquely observable in some enzyme families, this would allow researchers to achieve a remarkable degree of selectivity in developing unique allosteric inhibitors — all without affecting structurally or functionally homologous enzymes.

David N. Bernard, Chitra Narayanan, Tim Hempel, Khushboo Bafna, Purva Prashant Bhojane, Myriam Létourneau, Elizabeth E. Howell, Pratul K. Agarwal, Nicolas Doucet. Conformational exchange divergence along the evolutionary pathway of eosinophil-associated ribonucleases. Structure, 2023; 31 (3): 329 DOI: 10.1016/j.str.2022.12.011

Institut national de la recherche scientifique – INRS

Microbiology

Discovery offers a potential target for TB therapies

19 April, 2023 Admin Leave a comment

Reviewed by Emily Henderson, B.Sc.Apr 19 2023

In ongoing research aimed at developing more effective treatments for tuberculosis (TB), University of Massachusetts Amherst microbiologists have identified a long-sought gene that plays a critical role in the growth and survival of the TB pathogen.

The discovery offers a potential target for drug therapies for a deadly disease that has few effective treatments and in 2021 alone sickened 10.6 million worldwide and caused 1.6 million deaths, according to the World Health Organization.

Published in the journal mBio, the research showed that the putative gene cfa encodes an essential enzyme directly involved in the first step of forming tuberculostearic acid (TBSA), a unique fatty acid in the cell membranes of mycobacteria. TBSA was first isolated from mycobacteria nearly 100 years ago but exactly how it’s synthesized had remained elusive.

“There is a long history associated with this very fascinating fatty acid,” says senior author Yasu Morita, associate professor of microbiology, in whose lab lead authors Malavika Prithviraj and Takehiro Kado carried out the research.

The experiments revealed how TBSA controls the functions of the mycobacterial plasma membrane, which acts as a protective barrier for the TB pathogen to survive in human hosts for decades.

Cfa is directly involved in the formation of tuberculostearic acid and is also involved in the organization of the plasma membrane, and that all fell in place with our hypothesis.”

Malavika Prithviraj, Lead Author

The focus of research in Morita’s lab is to identify ways to interrupt homeostasis of the thick and waxy cell envelope, which includes the plasma membrane, so the mycobacteria are unable to grow or vulnerable to attack. Prithviraj, a Ph.D. student, and colleagues performed cellular lipidomics to confirm what researchers have suspected for some 60 years.

“People have been very, very interested in understanding how this lipid is made and what it is doing in the cell,” Morita says. “Malavika figured out that Cfa is the enzyme that makes this lipid, which is such a unique lipid that researchers have been pursuing this lipid as a diagnostic marker for TB.”

In previous experiments, the Morita lab had noted that plasma membrane domains found at polar regions of the cell were important for the growth of the mycobacteria.

“We were interested in understanding how this particular membrane domain is compartmentalized and organized in the bacteria,” Prithviraj says. “We worked with a deletion strain of cfa and also a complement strain wherein we could add it back into the bacteria and check what exactly was its function.”

The TB pathogen usually stays alive but dormant in the body for years or decades, thanks to its protective surface structure. Morita and his team work on a nonpathogenic model organism primarily to figure out what features of bacteria are needed for them to survive and grow.

The researchers found that TBSA also prevents “tight packing” inside the membrane. “If the membrane is too rigid, it cannot function properly, and so the membrane dynamics, or maintaining membrane fluidity, is very important,” Morita says. “What we showed in this paper is that tuberculostearic acid is likely a very important molecular key for maintaining this proper fluidity.”

The findings will help researchers take the next step toward developing new TB treatments.

“We would be interested in understanding the effects of the gene in TB infection and how Cfa might be helping the bacteria to survive in the human host” Prithviraj says. “If we find a way to disrupt the membrane fluidity maintenance, the cells cannot grow efficiently and would eventually die.”

Morita adds, “There are many drugs used for treating TB, but there has been no previous demonstration that this particular aspect of mycobacteria physiology can be used as a direct target,” Morita says. “This study is showing it could be.”

Source:

University of Massachusetts Amherst

Journal reference:

Prithviraj, M., et al. (2023) Tuberculostearic Acid Controls Mycobacterial Membrane Compartmentalization. mBio. doi.org/10.1128/mbio.03396-22.

Microbiology

Novel assay based on hybrid DNA-RNA probe for detecting food contaminated with salmonella

18 April, 2023 Admin Leave a comment

Apr 18 2023Reviewed by Lily Ramsey, LLM

A team of researchers have developed an easy-to-use colorimetric assay for the detection of food contaminated with salmonella. The assay is based on a novel nucleic acid probe that is cleaved by an RNase enzyme specific to the salmonella species. As the team report in the journal Angewandte Chemie, this specific enzymatic cleavage principle made it possible to build a sensitive but simple and portable test system using colloidal gold.

Image Credit: Angewandte Chemie

Consumption of food contaminated with Salmonella typhimurium, whether eggs, ground meat, or chicken, can lead to severe food poisoning. However, suspected cases of salmonella are usually only confirmed several days later, when the bacteria are detected in microbiology laboratories by growing them in culture. A team of researchers led by Yingfu Li, Tohid Didar, and Carlos Filipe of McMaster University in Hamilton, Canada, have now developed a novel test system based on a hybrid DNA-RNA probe that specifically and rapidly detects salmonella, without the need for microbiological diagnostics or expensive analytical equipment.

Using a multi-round selection process, the McMaster team uncovered an artificial DNA-RNA hybrid probe that is a substrate for a salmonella-specific form of an RNase H enzyme. Based on this highly specific enzymatic recognition, the team first developed a fluorescence-based assay on salmonella RNase H, and then extended the principle to a simple, portable salmonella assay based on a colloidal gold colorimetry.

Colloidal gold is a common color reagent familiar to many of us from its use in SARS-CoV-2 antigen test strips. In a slight departure from this methodology, however, the team did not use a paper strip as the basis for their assay, but instead turned to plastic pipette tips, which are commonly used in the laboratory to measure specific amounts of liquids.

For the preparation of the colorimetric assay, the inner wall of a pipette tip was first coated with DNA-functionalized nanogold. A mixture of reagents composed of nanogold-DNA and the DNA-RNA probe were then sucked up into the pipette tip, causing a double layer of nanogold to form on the walls, because the DNA-RNA hybrid probe links both layers.

However, when the sample mixture contains salmonella, the upper layer is released thanks to the salmonella RNase H specifically cleaving the DNA-RNA linker probe. When the gold-containing solution is then drained onto an absorbent pad with a nylon membrane, a clear red spot indicates the presence of salmonella in the sample being tested. The team also tested the specificity of their system, finding it did not falsely detect the presence of other bacteria containing RNAse H.

The authors highlight that the test is not only much less complex than other methods for detecting salmonella, but also much faster. In contrast to other methods, only one hour of incubation in a pipette tip is required for highly sensitive detection of salmonella, for example, in ground beef. In the future, the team envision developing more nucleic acid probes which can specifically detect other infectious pathogens, for example coliform bacteria such as E. coli.

Source:

Wiley

Journal reference:

Li, J., et al. (2023). A Simple Colorimetric Au‐on‐Au Tip Sensor with a New Functional Nucleic Acid Probe for Food‐borne Pathogen Salmonella typhimurium. Angewandte Chemie International Edition. doi.org/10.1002/anie.202300828.

Microbiology

Genetically-engineered probiotic could be a new way to reduce alcohol-induced health problems

11 April, 2023 Admin Leave a comment

Reviewed by Emily Henderson, B.Sc.Apr 11 2023

Excessive alcohol consumption leads to painful hangovers and accompanying headaches, fatigue, and nausea. Drinking alcohol has also been linked to a raft of health problems in the human body, including heart disease, cirrhosis, and immune deficiency. One way to avoid those consequences would be to drink less, but researchers in China have introduced another way to mitigate hangovers and other adverse outcomes -; a genetically-engineered probiotic.

In a paper published this week in Microbiology Spectrum, the researchers described their approach and reported that in experiments on mice, the treatment reduced alcohol absorption, prolonged alcohol tolerance, and shortened the animals’ recovery time after exposure to alcohol. The probiotic hasn’t yet been tested on humans, but the authors predicted that if it confers the same benefits, it could present a new way to reduce alcohol-induced health problems, and liver problems in general.

Meng Dong, Ph.D, at the Chinese Academy of Science’s Institute of Zoology, who worked on the study, noted that clinical applications may extend beyond alcohol-related conditions. “We believe that genetically engineered probiotics will provide new ideas for the treatment of liver diseases,” she said.

The human body primarily uses forms of an enzyme called alcohol dehydrogenase, or ADH, to metabolize alcohol. But some variants are more effective than others: Some studies have found that a form called ADH1B, found primarily in East Asian and Polynesian populations, is 100 times more active than other variants. Previous studies on mice have shown that viral vectors genetically engineered to express ADH1B can accelerate the breakdown of alcohol, but that approach hasn’t been shown to be safe in humans.

Motivated by those findings, Dong and her colleagues looked for a safer delivery method, focusing on the probiotic Lactococcus lactis, a bacterium often used in fermentation. They used molecular cloning to introduce the gene for human ADH1B into a bacterial plasmid, which was then introduced into a strain of L. lactis. Lab tests confirmed that the probiotic secreted the enzyme. The researchers encapsulated the probiotic to ensure it would survive against stomach acid, then tested it on 3 groups of 5 mice, each exposed to different levels of alcohol.

Untreated mice showed signs of drunkenness 20 minutes after exposure to alcohol. When the mice were placed on their backs, for example, they were unable to get back on their feet. But in the group that received a probiotic that expressed human ADH1B, half the mice were still able to turn themselves over an hour after alcohol exposure. A quarter never lost their ability to turn themselves over.

Further tests showed that 2 hours after exposure, blood alcohol levels in the control group continued to rise, while those in the probiotic-treated mice had begun to fall. In addition, the researchers found that treated mice showed lower levels of lipids and triglycerides in their livers, suggesting that the probiotic could alleviate alcohol-related damage to that organ.

The next step, Dong said, is to investigate whether the potential therapeutic effect of the modified probiotic extends to humans.

We are excited about the improvement of recombinant probiotics in acute alcohol-induced liver and intestinal damage.”

Meng Dong, Ph.D, Chinese Academy of Science’s Institute of Zoology

Source:

American Society for Microbiology

Journal reference:

Jiang, X., et al. (2023) Oral Probiotic Expressing Human Ethanol Dehydrogenase Attenuates Damage Caused by Acute Alcohol Consumption in Mice. Microbiology Spectrum. doi.org/10.1128/spectrum.04294-22.

Microbiology

Researchers report surprising first steps that promote resistance to commonly prescribed antibiotics

2 April, 2023 Admin Leave a comment

Reviewed by Emily Henderson, B.Sc.Mar 31 2023

Antibiotic resistance is a global health threat. In 2019 alone, an estimated 1.3 million deaths were attributed to antibiotic resistant bacterial infections worldwide. Looking to contribute a solution to this growing problem, researchers at Baylor College of Medicine have been studying the process that drives antibiotic resistance at the molecular level.

They report in the journal Molecular Cell crucial and surprising first steps that promote resistance to ciprofloxacin, or cipro for short, one of the most commonly prescribed antibiotics. The findings point at potential strategies that could prevent bacteria from developing resistance, extending the effectiveness of new and old antibiotics.

Previous work in our lab has shown that when bacteria are exposed to a stressful environment, such as the presence of cipro, they initiate a series of responses to attempt to survive the toxic effect of the antibiotic.”

Dr. Susan M. Rosenberg, co-corresponding author, Ben F. Love Chair in Cancer Research and professor of molecular and human genetics, biochemistry and molecular biology and molecular virology and microbiology at Baylor

She also is program leader in Baylor’s Dan L Duncan Comprehensive Cancer Center (DLDCCC). “We discovered that cipro triggers cellular stress responses that promote mutations. This phenomenon, known as stress-induced mutagenesis, generates mutant bacteria, some of which are resistant to cipro. Cipro-resistant mutants keep on growing, sustaining an infection that can no longer be eliminated with cipro.”

Cipro induces breaks in the DNA molecule, which accumulate inside bacteria and consequently trigger a DNA damage response to repair the breaks. The Rosenberg lab’s discoveries of the steps involved in stress-induced mutagenesis revealed that two stress responses are essential: the general stress response and the DNA-damage response.

Some of the downstream steps of the process that leads to increased mutagenesis have been revealed previously by the Rosenberg lab and her colleagues. In this study, the researchers discovered the molecular mechanisms of the first steps between the antibiotic causing DNA breaks and the bacteria turning on the DNA damage response.

“We were surprised to find an unexpected molecule involved in modulating DNA repair,” said first author Dr. Yin Zhai, postdoctoral associate in the Rosenberg lab. “Usually, cells regulate their activities by producing specific proteins that mediate the desired function. But in this case, the first step to turn on the DNA repair response was not about activating certain genes that produce certain proteins.”

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Instead, the first step consisted of disrupting the activity of a protein already present, RNA polymerase. RNA polymerase is key to protein synthesis. This enzyme binds to DNA and transcribes DNA-encoded instructions into an RNA sequence, which is then translated into a protein.

“We discovered that RNA polymerase also plays a major role in regulating DNA repair,” Zhai said. “A small molecule called nucleotide ppGpp, which is present in bacteria exposed to a stressful environment, binds to RNA polymerase through two separate sites that are essential for turning on the repair response and the general stress response. Interfering with one of these sites turns off DNA repair specifically at the DNA sequences occupied by RNA polymerase.”

“ppGpp binds to DNA-bound RNA polymerase, telling it to stop and backtrack along the DNA to repair it,” said co-corresponding author Dr. Christophe Herman, professor of molecular and human genetics, molecular virology and microbiology and member of the DLDCCCC. The Herman lab found the repair-RNA-polymerase connection previously, reported in Nature.

Rosenberg’s lab discovered that DNA repair can be an error-prone process. As repair of the broken DNA strands progresses, errors occur that alter the original DNA sequence producing mutations. Some of these mutations will confer bacteria resistance to cipro. “Interestingly, the mutations also induce resistance to two other antibiotic drugs the bacteria have not seen before,” Zhai said.

“We are excited about these findings,” Rosenberg said. “They open new opportunities to design strategies that would interfere with the development of antibiotic resistance and help turn the tide on this global health threat. Also, cipro breaks bacterial DNA in the same way that the anti-cancer drug etoposide breaks human DNA in tumors. We hope this may additionally lead to new tools to combat cancer chemotherapy resistance.”

Other contributors to this work include P.J. Minnick, John P. Pribis, Libertad Garcia-Villada and P.J. Hastings, all at Baylor College of Medicine.

Source:

Baylor College of Medicine

Journal reference:

Zhai, Y., Minnick, P. J., Pribis, J. P., Garcia-Villada, L., Hastings, P. J., Herman, C., & Rosenberg, S. M. (2023). ppGpp and RNA-polymerase backtracking guide antibiotic-induced mutable gambler cells. Molecular Cell. doi.org/10.1016/j.molcel.2023.03.003.

Microbiology

Study offers a novel therapeutic option to combat antibiotic-resistant pneumonia

29 March, 2023 Admin Leave a comment

Reviewed by Emily Henderson, B.Sc.Mar 29 2023

Increases in multidrug-resistance in the bacteria Streptococcus pneumoniae have made it the fourth-leading cause of death associated with antibiotic resistance.

In a study in PLOS Biology, researchers report a new target to fight against pneumonia due to infections by this opportunistic lung pathogen -; interference with the bacteria’s fermentation metabolism. This may offer a novel therapeutic option in the urgent need to discover new strategies to combat drug-resistant S. pneumoniae.

In a proof of principle, University of Alabama at Birmingham researchers showed that giving an existing drug -; one already approved by the United States Food and Drug Administration to treat methanol poisoning – in combination with the antibiotic erythromycin significantly reduced disease in mice infected with a virulent, multidrug-resistant S. pneumoniae. The combination therapy reduced bacterial burden in the lungs by 95 percent, and bacterial burdens in the spleen and heart by 100- and 700-fold, respectively. The FDA-approved drug alone, or erythromycin alone, had no effect.

Fomepizole, the FDA-approved drug, disrupts activity of the enzyme alcohol dehydrogenase in the bacteria. The mice were infected intratracheally with the multidrug-resistant clinical isolate S. pneumoniae serotype 35B strain 162–5678, which has high resistance to erythromycin. Notably, the S. pneumoniae 35B serotype has been reported as an emerging multidrug-resistant serotype in clinical settings. Eighteen hours after infection, the mice were given a single injection of erythromycin, with or without fomepizole.

Fomepizole, or other drugs that inhibit bacterial metabolism, have potential to dramatically increase the efficacy of erythromycin and other antibiotics, respectively, in vivo.”

Carlos Orihuela, Ph.D., professor and interim chair of the UAB Department of Microbiology

A broad foundation of basic research preceded this proof-of-principle experiment.

S. pneumoniae relies on fermentation and glycolysis to produce energy. During fermentation, pyruvate is converted to lactate, acetate and ethanol, and NADH is oxidized to regenerate NAD+, which is needed for glycolysis. Accordingly, maintenance of an available NAD+ pool, necessary for redox balance, is vital for sustained energy production, bacterial growth and survival.

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Orihuela and UAB colleagues made S. pneumoniae mutants in five enzymes involved in fermentation and NAD+ production, and they found, in general, that the mutants had impaired metabolism. Two of the mutants, one for lactate dehydrogenase and one for alcohol dehydrogenase, had stark decreases in intracellular pool of ATP, the energy molecule of living cells. The other three mutants had significant, but more modest, decreases.

NAD+/NADH redox imbalances in the mutants generally interfered with production of S. pneumoniae virulence factors and colonization in the mouse nasopharynx. Some of the mutations influenced susceptibility to antibiotics, as tested with three antibiotics, including erythromycin, that interfere with protein synthesis, two antibiotics that disrupt cell wall synthesis and one antibiotic that targets DNA transcription.

Researchers found that treating a wildtype S. pneumoniae, which did not have mutations in alcohol dehydrogenase or the other enzymes, with fomepizole alone caused redox imbalances. In vitro tests showed that treatment of S. pneumoniae with fomepizole enhanced the susceptibility to antibiotics, including fourfold decreases in the minimal inhibitory concentrations of the antibiotics erythromycin and gentamicin.

“We also evaluated whether fomepizole treatment impacted the antibiotic susceptibility of other anaerobic gram-positive bacteria, including other streptococcal pathogens, including Streptococcus pyogenes, Streptococcus agalactiae and Enterococcus faecium, to erythromycin or gentamicin,” Orihuela said. “We observed from twofold to eightfold decreased minimal inhibitory concentration with fomepizole in most cases, including E. faecium.”

“Our results indicate that the blocking of NAD+ regeneration pathways during infection is a way to increase antibiotic susceptibility in drug-resistant gram-positive anaerobic pathogens,” Orihuela said. “This has clinical potential with regard to microbial eradication and treatment of disseminated infection.”

Globally, more than 3 million individuals are hospitalized due to pneumococcal disease annually, and hundreds of thousands die as a result.

Source:

University of Alabama at Birmingham

Journal reference:

Im, H., et al. (2023). Targeting NAD+ regeneration enhances antibiotic susceptibility of Streptococcus pneumoniae during invasive disease. PLOS Biology. doi.org/10.1371/journal.pbio.3002020.

Microbiology

Lupus, Sepsis, and More: Scientists Uncover Promising New Therapeutic Target for Inflammatory Diseases

29 March, 2023 Admin Leave a comment

What goes wrong in our bodies during the progression of an inflammatory disease? Scientists at the School of Biochemistry and Immunology in the Trinity Biomedical Sciences Institute at Trinity College Dublin have made a significant advancement in comprehending the underlying mechanisms of the progression of inflammatory diseases. The discovery has uncovered a promising new target for therapeutic intervention.

The researchers have discovered that the repression of an enzyme called Fumarate Hydratase occurs in macrophages, which are a type of inflammatory cell that play a role in various diseases such as Lupus, Arthritis, Sepsis, and COVID-19.

Professor Luke O’Neill, Professor of Biochemistry at Trinity is the lead author of the research article that has just been published in the leading international journal, Nature.

He said: “No one has made a link from Fumarate Hydratase to inflammatory macrophages before and we feel that this process might be targetable to treat debilitating diseases like Lupus, which is a nasty autoimmune disease that damages several parts of the body including the skin, kidneys, and joints.”

Joint first-author Christian Peace added: “We have made an important link between Fumarate Hydratase and immune proteins called cytokines that mediate inflammatory diseases. We found that when Fumarate Hydratase is repressed, RNA is released from mitochondria which can bind to key proteins ‘MDA5’ and ‘TLR7’ and trigger the release of cytokines, thereby worsening inflammation. This process could potentially be targeted therapeutically.”

Fumarate Hydratase was shown to be repressed in a model of sepsis, an often-fatal systemic inflammatory condition that can happen during bacterial and viral infections. Similarly, in blood samples from patients with Lupus, Fumarate Hydratase was dramatically decreased.

“Restoring Fumarate Hydratase in these diseases or targeting MDA5 or TLR7, therefore, presents an exciting prospect for badly needed new anti-inflammatory therapies,” said Prof O’Neill.

Excitingly, this newly published work is accompanied by another publication by a group led by Professor Christian Frezza, now at the University of Cologne, and Dr. Julien Prudent at the MRC Mitochondrial Biology Unit (MBU), who have made similar findings in the context of kidney cancer.

“Because the system can go wrong in certain types of cancer, the scope of any potential therapeutic target could be widened beyond inflammation,” added Prof O’Neill.

Reference: “Macrophage fumarate hydratase restrains mtRNA-mediated interferon production” by Alexander Hooftman, Christian G. Peace, Dylan G. Ryan, Emily A. Day, Ming Yang, Anne F. McGettrick, Maureen Yin, Erica N. Montano, Lihong Huo, Juliana E. Toller-Kawahisa, Vincent Zecchini, Tristram A. J. Ryan, Alfonso Bolado-Carrancio, Alva M. Casey, Hiran A. Prag, Ana S. H. Costa, Gabriela De Los Santos, Mariko Ishimori, Daniel J. Wallace, Swamy Venuturupalli, Efterpi Nikitopoulou, Norma Frizzell, Cecilia Johansson, Alexander Von Kriegsheim, Michael P. Murphy, Caroline Jefferies, Christian Frezza and Luke A. J. O’Neill, 8 March 2023, Nature.
DOI: 10.1038/s41586-023-05720-6

The Trinity study is a collaboration between eight universities including the MRC MBU, University of Cambridge where Dr. Dylan Ryan is co-first author along with Dr. Alex Hooftman, who is now based at the Swiss Federal Institute of Technology Lausanne. Cedars Sinai Medical Centre in Los Angeles is another key collaborator helping with the study of Lupus patients.

The study was funded by the European Research Council, Medical Research Council, and Science Foundation Ireland. Work in the Frezza lab is also supported by the ERC, further illustrating the importance of ERC funding for EU science.

Trinity College Dublin

Microbiology

Epigenome reprogramming after SARS-CoV-2 infection

28 March, 2023 Admin Leave a comment

By Neha MathurMar 27 2023Reviewed by Lily Ramsey, LLM

In a recent article in published in the journal Nature Microbiology, researchers in Texas, United States (US) performed a three-dimensional (3D) evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected human cells to show a direct cell-autonomous effect elicited by SARS-CoV-2 on the host chromatin.

The study aimed at improving the understanding of coronavirus disease 2019 (COVID-19)-related perturbations in the genome and epigenome of a host cell.

Study: SARS-CoV-2 restructures host chromatin architecture. Image Credit:FUNFUNPHOTO/Shutterstock.com

Background

The 3D folding of chromatin in mammals, including humans, influences deoxyribonucleic acid (DNA) replication, recombination, DNA damage repair, and transcription. It is a key determinant of how human cells act and function. Viruses, including SARS-CoV-2, antagonize host defense by rewiring their chromatin architecture, which typically has several layers, e.g., A/B compartments, chromatin loops, and topological associating domains (TADs).

The A and B compartments superimpose transcriptionally active euchromatin and relatively inactive heterochromatin, respectively. However, studies have barely investigated these effects.

In addition, epigenetic alterations impact gene expression and resulting phenotypes in the long term. Thus, a sneak peek into the interactions between the virus, host chromatin, and epigenome could help find novel methods to fight SARS-CoV-2 in the acute phase. In addition, it could unravel the molecular basis of post-acute SARS-CoV-2 sequelae or long COVID and subsequently mitigate it.

About the study

At 24 hours post-infection (24 hpi), human A549 cells expressing angiotensin-converting enzyme 2 (ACE2), infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1, had high levels of infection. This was shown by ribonucleic acid-sequencing (RNA-seq). Immunofluorescence of the SARS-CoV-2 spike (S) glycoprotein also substantiated an elevated infection ratio.

So, in the present study, researchers used an improved version of in situ Hi-C high-throughput chromosome conformation capture (Hi-C) 3.0 to study host chromatin changes in these cells at 24 hpi and mock-infected cells (Mock).

In addition, the team evaluated the epigenetic features of the altered chromatin regions to understand the vulnerability to compartmental changes due to infection. To this end, they used chromatin immunoprecipitation (ChIP-seq) methods to generate data on representative histone markers and polymerase II (Pol2) in A549-ACE2 cells. This analysis covered four histone markers, viz., H3K27ac, H3K4me3, H3K9me3, and H3K27me3.

It helped them examine the epigenetic features of these six categories of bins. They ranked E1-score changes for each genomic bin to sort bins. They dubbed bins showing E1-score increase and decrease as ‘A-ing’ and ‘B-ing’ bins, respectively.

Results

The Hi-C analysis showed extensive alterations in the hosts’ 3D genome after SARS-CoV-2 infection. The researchers also plotted a Pearson correlation map of their Hi-C analysis that reaffirmed these changes alongside indicating modified chromatin compartmentalization.

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A focused view of the ~0.7 Mb region showed a weakening of the rectangle-shaped chromatin domains and deregulation of chromatin loops. While SARS-CoV-2 prompted a global decline in near-diagonal short-range chromatin contacts (<560 kilobases), as seen in a P(s) curve, chromatin contacts far-separated from the diagonal (>28 megabases) were often deregulated.

Further, a P(s) curve showed that SARS-CoV-2 elicited modest and enhanced interactions in middle-to-long-distance contacts (~560 kb to 8.9 Mb) and far-positioned regions, respectively.

Fold changes in inter-chromosomal interactions or trans-vs-cis contact ratios also depicted the effect of SARS-CoV-2 infection on inter-chromosomal contacts. The enhancement of inter- and intra-chromosomal interactions indicated changes in chromatin compartmentalization. Consequently, principal component analysis (PCA) of a 100-kb bin on Hi-C background showed noticeable defects of chromatin compartmentalization in virus-infected cells.

The total PCA E1 scores quantifying E1 changes in ~30% of genomic regions showed a widespread diminishing of the A compartment, A-to-B switching, or strengthening of the B compartment post-SARS-CoV-2 infection.

Among all, A to weaker A changes were the most common and occurred in ~18% of the genome, which indicated that SARS-CoV-2 extensively weakened the host euchromatin.

Further analysis showed that the ‘B-ing’ and ‘A-ing’ genomic regions were historically enriched in active chromatin markers (e.g., H3K27ac) and repressive histone markers, especially H3K27me3. Unexpectedly, SARS-CoV-2 infection selectively modified the H3K4me3 marker of phytochrome interacting factors (PIF) gene promoters, suggesting unappreciated mechanisms at these promoters that confer deviating inflammation in COVID-19.

A flawed chromatin compartmentalization likely caused the historically well-partitioned A or B compartments to lose their identity. A saddle plot illustrating inter-compartment chromatin interactions across the genome showed these global changes.

The authors also noted weakened compartmentalization between chromosomes. For instance, in chromosomes 17 & 18, while A–B interactions were amplified, A–A/B–B homotypic interactions appeared to have become compromised.

Moreover, SARS-CoV-2 infection mechanistically depleted the cohesin complex in a pervasive but selective manner from intra-TAD regions. These changes provided a molecular explanation for the weakening of intra-TAD interactions.

It supported the notion that defective cohesin loop extrusion inside TADs releases this chromatin to engage in long-distance associations. Intriguingly, chromatin in SARS-CoV-2-infected cells exhibited a higher frequency of long-distance intra-chromosomal and inter-chromosomal interactions.

Conclusions

SARS-CoV-2 infection markedly restructured 3D host chromatin, featuring widespread compartment A weakening and A–B mixing and global reduction in intra-TAD chromatin contacts.

However, it is still unknown exactly how SARS-COV-2 infection restructures host chromatin. Likely, open reading frame 8 (ORF8) disrupts the host epigenome, suggesting that some viral factors are involved in host chromatin rewiring.

It also altered the host epigenome, including a global reduction in active chromatin mark H3K27ac and a specific increase in H3K4me3 at pro-inflammatory gene promoters. Intriguingly, all these host chromatin alterations were unique to SARS-CoV-2 infection, and other common-cold coronaviruses or immune stimuli did not elicit these changes.

Journal reference:

Wang, R. et al. (2023) “SARS-CoV-2 restructures host chromatin architecture”, Nature Microbiology. doi: 10.1038/s41564-023-01344-8. https://www.nature.com/articles/s41564-023-01344-8

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