Tag Archives: Microscopy

Microbiology

Transforming antibiotic resistance testing: a novel, rapid and affordable technique

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Lily Ramsey, LLMInterview conducted by Lily Ramsey, LLMMay 19 2023
Thought LeadersDr. Sandor KasasResearch LeadEcole Polytechnique Fédérale de Lausanne

News Medical speaks with Dr. Sandor Kasas, a lead researcher at Ecole Polytechnique Fédérale de Lausanne in Switzerland. Here we discuss his recent development of a novel and highly efficient method for rapid antibiotic susceptibility testing using optical microscopy.

The new technique, known as Optical Nanomotion Detection (ONMD), is an extremely rapid, label-free, and single-cell sensitive method to test for antibiotic sensitivity. ONMD requires only a traditional optical microscope equipped with a camera or mobile phone. The simplicity and efficiency of the technique could prove to be a game changer in the field of antibiotic resistance.

Please can you introduce yourself, tell us about your career background, and what inspired your career in biology and medicine?

I graduated in medicine but never practiced in hospitals or medical centers. After my studies, I started working as an assistant in histology at the University of Fribourg in Switzerland. My first research projects included image processing, scanning tunneling, and atomic force microscopy.

Later, and for most of the rest of my scientific carrier, I focused primarily on the biological applications of AFM. For the past ten years, my research interest is about nanomotion, i.e., the study of oscillations at a nanometric scale of living organisms.

Image Credit: dominikazara/Shutterstock.comImage Credit: dominikazara/Shutterstock.com

You started working on biological applications of the atomic force microscope (AFM) in 1992. From your perspective, how has the antibiotic resistance landscape changed over the last two decades? What role has the advancement in technology played in furthering our understanding?

In the early ’90s, the AFM was mainly used for imaging. Later, AFM microscopists noticed that the instrument could also be used to explore the mechanical properties of living organisms. More recently, many “exotic” applications of the AFM have emerged, such as its use to weigh single cells or study their oscillations at the nanometric scale. In the 1990s, antibiotic resistance was not as serious a problem as today, but several teams were already using AFM to assess the effects of antibiotics on bacterial morphology.

The first investigations were limited to structural changes, but later, as the fields of application of AFM expanded, the instrument made it possible to monitor the mechanical properties of the bacterial cell wall upon exposure to antibiotics. In the 2010s, with G. Longo and G. Dietler, we demonstrated that AFM could also track nanoscale oscillations of living organisms. The very first application we had in mind was using the instrument to perform rapid antibiotic susceptibility testing.

We have therefore developed devices based on dedicated AFM technology to perform fast AST (i.e., in 2-4h). AFM-based nanomotion detection instruments are already implemented in medical centers in Switzerland, Spain, and Austria. However, this type of device has some drawbacks, including the need to fix the organism of interest on a cantilever. To overcome this limitation, we have developed with R. Willaert a nanomotion detector based on an optical microscope.

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Your most recent research has led to the development of a novel and highly efficient technique for rapid antibiotic susceptibility testing using optical microscopy. Please could you tell us why the development of rapid, affordable, and efficient testing methods is so important in the world of antimicrobial resistance?

Rapid antibiotic susceptibility testing could reduce the use of broad-spectrum antibiotics. Traditional ASTs based on replication rate require 24 hours (but up to 1 month in the case of tuberculosis) to identify the most effective antibiotic. Doctors prescribe broad-spectrum antibiotics between the patient’s admission to a medical center and the results of the AST.

These drugs quickly improve patients’ conditions but, unfortunately, promote resistance. A rapid AST that could identify the most suitable antibiotic within 2-4 hours would eliminate broad-spectrum antibiotics and increase treatment efficiency and reduce the development of resistant bacterial strains. Since bacterial resistance is a global problem, rapid ASTs should also be implemented in developing countries. Therefore, affordable and simple-to-use tests are needed.

Image Credit: Fahroni/Shutterstock.comImage Credit: Fahroni/Shutterstock.com

Were there any limitations and obstacles you faced in the research process? If so, how did you overcome them?

Antibiotic sensitivity detection with ONMD is very similar to the AFM-based technique. As long as the bacterium is alive, it oscillates; if the antibiotic is effective, it kills the micro-organism, and its oscillations stop. The first limitation we faced when developing the ONMD was our microscopes’ depth of field of view. To prevent the bacteria from leaving the focal plane of the optical microscope during the measurement, we had to constrain the microbes into microfluidic channels a few micrometers high.

Microfabrication of such devices is relatively straightforward in an academic environment, but we were looking for simpler solutions. One option for constructing such a device is to use 10-micron double-sided rubber tape. It allows you to “build” a microfluidic chamber in 5 minutes with two glass coverslips and a puncher.

Another challenge was nanoscale motion detection. For this purpose, we used freely available cross-correlation algorithms that achieve sub-pixel resolution. (i.e., a few nanometers). We first developed the ONMD for larger organisms, such as yeast cells, and expanded the method to bacteria. This further development took us around two years.

You worked alongside Dr. Ronnie Willaert, a professor of structural biology at Vrije Universiteit Brussel, on developing this new rapid AST technique. How did your areas of expertise and research backgrounds complement each other in developing ONMD?

R. Willaert is an expert in yeast microbiology and microfluidics, while our team in Lausanne is primarily involved in AFM-based nanomotion detection and applying AFM to clinically relevant problems. The two teams were supported by a joint grant from the Swiss National Science Foundation and the Research Foundation Flanders (FWO) which enabled the development of the method.

The field of antimicrobial resistance requires a high level of international collaboration, with everyone working together to achieve a common goal. With antimicrobial resistance rising to dangerously high levels in all parts of the world, how important is collaboration in this field?

Our project required expertise in various fields, such as microbiology, microscopy, microfluidics, programming, and data processing. In the development of rapid AST instruments and many others, only a multidisciplinary approach and close collaboration between teams with complementary expertise is today the only path to success.

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You and Dr. Willaert have said, ‘The simplicity and efficiency of the method make it a game-changer in the field of AST.’ Can you please expand on what makes ONMD a game changer in the AST field and what implications this research could have in clinical and research settings?

As mentioned earlier, bacterial resistance is a global health problem. Rapid AST should also be easily implemented in developing countries to limit the spread of resistant strains. The cheaper and simpler the technique, the more likely it is to be used on a large scale. We are convinced that the ONMD approach can meet these requirements. ONMD could also be used for drug discovery or basic research.

While we recognize the importance of rapid AST, what next steps must be taken before this technique can be used globally in research and clinical landscapes?

For fundamental research, there are no other important developments to be made. Any reasonably equipped research center can implement the technique and use it. Regarding implementing the technique in developing countries or extreme environments, stand-alone devices have to be used, which have yet to be manufactured.

There is a rapidly expanding need for efficient AST globally; however, the need for affordable, accessible, and simple techniques are of grave importance in developing countries disproportionately affected by antibiotic resistance due to existing global health disparities. Could this rapid AST technique be utilized in low-middle-income countries to slow the growing spread of multi-resistant bacteria? What would this mean for global health?

We are confident that ONMD-based AST testing can soon be implemented in research centers in both developed and developing countries. However, accreditation by the health authorities is necessary to use it as a standard diagnostic tool; this process can take several years, depending on the government health policy.

What’s next for you and your research? Are you involved in any exciting upcoming projects?

We want to develop a self-contained device for extreme environments. It would consist of a small microscope equipped with a camera and a data processing unit. The microfluidic part of the device could contain different antibiotics ready to be tested.

The ONMD technique could also monitor contamination levels in enclosed environments such as submarines, spacecraft, and space stations. One of our recent projects is funded by the European Space Agency (ESA) to develop a rapid antifungal susceptibility test that could work in microgravity. Additionally, ONMD could be used for even more exciting projects, such as chemistry-independent life detection in the search for extraterrestrial life.

Where can readers find more information?

  • Villalba MI, Rossetti E, Bonvallat A, Yvanoff C, Radonicic V, Willaert RG*, Kasas S.*.Simple optical nanomotion method for single-bacterium viability and antibiotic response testing. PNAS 2023, May 2;120(18):e2221284120. doi: 10.1073/pnas.2221284120. Epub 2023 Apr 24. PMID: 37094120. * Contributed equally. https://doi.org/10.1073/pnas.2221284120
  • Radonicic, V.; Yvanoff, C.; Villalba, M.I.; Devreese, B.; Kasas, S.; Willaert, R.G. Single-Cell Optical Nanomotion of Candida albicans in Microwells for Rapid Antifungal Susceptibility Testing. Fermentation 2023, 9:365. https://doi.org/10.3390/fermentation9040365
  • Parmar P, Villalba MI, Horii Huber AS, Kalauzi A, Bartolić D, Radotić K, Willaert RG, MacFabe DF and Kasas S. Mitochondrial nanomotion measured by optical microscopy. Front. Microbiol. 2023, 14:1133773. https://doi.org/10.3389/fmicb.2023.1133773
  • Starodubtseva MN, Irina A. Chelnokova IA, Shkliarava NM, Villalba MI, Tapalski DV, Kasas S, Willaert RG. Modulation of the nanoscale motion rate of Candida albicans by X-rays. Front. Microbiol. 2023, 14:1133027. https://doi.org/10.3389/fmicb.2023.1133027
  • Radonicic V, Yvanoff C, Villalba MI, Kasas S, Willaert RG. The Dynamics of Single-Cell Nanomotion Behaviour of Saccharomyces cerevisiae in a Microfluidic Chip for Rapid Antifungal Susceptibility Testing. Fermentation. 2022; 8(5):195. https://doi.org/10.3390/fermentation8050195

About Dr. Sandor Kasas

Nanomotion is a fascinating and novel approach to observing living organisms.

Our team focuses almost exclusively on recording the nanomotion of bacterial mitochondria and mammalian cells with optical and AFM-based devices.

Recently, we demonstrated that the technique could be used not only for fast antimicrobial sensitivity testing but also to explore the metabolism of unicellular organisms. We hope our efforts will permit us to expand the application domains of ONMD.

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Microbiology

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

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Neha MathurBy 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.comStudy: 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.

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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 VH and VL 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.

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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.

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Journal reference:
Microbiology

“Simpler and Much Cheaper” – A New Way To Test Antibiotic Resistance

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A new method for quick antibiotic susceptibility testing using optical microscopy has been created by scientists from EPFL and Vrije Universiteit Brussel. The approach, called Optical Nanomotion Detection, is fast, accurate at the single-cell level, label-free, and can be carried out using a basic optical microscope, equipped with a camera or a mobile phone.

“We have developed a technique in our laboratories that allows us to obtain an antibiogram within 2-4 hours – instead of the current 24 hours for the most common germs and one month for tuberculosis,” says Dr. Sandor Kasas at EPFL. Professor Ronnie Willaert at Vrije Universiteit Brussel adds: “Our technique is not only faster but also simpler and much cheaper than all those existing now.”

Antibiotic resistance occurs when bacteria gain the capability to overcome the medicines created to eradicate them. It has now become a worldwide concern for public health. In 2019, it was the cause of at least 1.27 million fatalities globally and was implicated in nearly five million deaths. In the United States, there are nearly three million cases of antimicrobial-resistant infections annually, and the cost of treating the top six of these infections exceeds $4.6 billion. In the European Union, there are nearly 700,000 cases each year, with an estimated cost of €1.5 billion.

Antibiotic sensitivity testing (AST) uses culture methods that expose bacteria to antibiotics, or genetic methods to determine if bacteria possess genes that confer resistance. Typical ASTs last up to 24 hours or even longer for slow-growing bacteria – a timeframe that can mean life or death in a clinical setting. There have been some faster ASTs developed in recent years, but they tend to be complex, needing sophisticated and expensive equipment.

Now, researchers led by Kasas and Willaert have developed a fast, cheap, and widely accessible method based on optical microscopy that can perform an AST with single-cell sensitivity without needing to attach or label bacteria. The technique uses a basic, conventional optical microscope, a camera or mobile phone, and dedicated software. The joint research project was published in the journal PNAS.

The new technique is called optical nanomotion detection (ONMD), and involves the monitoring of nanoscale vibrations of single bacterial before and while being exposed to antibiotics. The monitoring is performed with a basic optical microscope, a video camera, or a mobile phone.

The ONMD technique monitors the microscopic oscillations of bacterial cells (nanomotion) that characterize living organisms and can be considered as a “signature of life”. Indeed, nanomotion lasts as long as the organism is alive but stops immediately when it is dead. In the ONMD technique, bacterial nanomotion is recorded in a movie in which all individual cell displacements are monitored with sub-pixel resolution.

The researchers used ONMD to successfully detect the sensitivity of numerous bacteria to antibiotics. Escherichia coli, Staphylococcus aureus, Lactobacillus rhamnosus, and Mycobacterium smegmatis (a non-pathogenic bacterial model for tuberculosis) sensitivities to the antibiotics ampicillin, streptomycin, doxycycline, and vancomycin was determined in less than two hours.

The ONMD not only monitors the bacteria’s life-death transitions upon exposure to different antibiotics but also highlights changes in the bacteria’s metabolism caused by the availability of nutrients. The tests showed that ONMD can assess the sensitivity or resistance of bacterial cells to antibiotics in a simple and rapid way by monitoring cellular oscillations.

The authors state: “The simplicity and efficiency of the method make it a game-changer in the field of AST” as it can be applied to a wide range of bacteria, which has significant implications for clinical and research applications.

Reference: “Simple optical nanomotion method for single-bacterium viability and antibiotic response testing” by Maria I. Villalba, Eugenia Rossetti, Allan Bonvallat, Charlotte Yvanoff, Vjera Radonicic, Ronnie G. Willaert and Sandor Kasas, 24 April 2023, Proceedings of the National Academy of Sciences.
DOI: 10.1073/pnas.2221284120

The study was funded by the Swiss National Science Foundation, Research Foundation – Flanders (FWO), the Belgian Federal Science Policy Office (Belspo), and the European Space Agency.

Ecole Polytechnique Fédérale de Lausanne

Microbiology

Inhibition of cell wall formation arrests staphylococcal cell division

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Reviewed by Emily Henderson, B.Sc.Mar 23 2023

We still do not understand exactly how antibiotics kill bacteria. However, this understanding is necessary if we want to develop new antibiotics. And that is precisely what is urgently needed, because bacteria are currently showing more and more resistance to existing antibiotics. Therefore, researchers from the University Hospital Bonn (UKB) and the University of Bonn used high-performance microscopes to observe the effect of different antibiotics on the cell division of Staphylococcus aureus. They found that the biosynthesis of peptidoglycan, core component of the bacterial cell wall, is the driving force during the entire process of cell division. In addition, they clarified how exactly different antibiotics block cell division within a few minutes. The results have now been published in the journal Science Advances.

The bacterial cell wall maintains the shape and integrity of unicellular organisms. Cell wall synthesis plays a key role in bacterial growth: the cell division protein FtsZ forms the so-called Z-ring in the center of the cell, thus initiating the division process. A new cell wall is formed there, for which peptidoglycan is produced as the core component. This constriction thus gives rise to two identical daughter cells.

Fluorescent proteins in Staphylococcus aureus under the microscope

The UKB research team led by Fabian Grein and Tanja Schneider, together with the team led by Ulrich Kubitscheck, Professor of Biophysical Chemistry at the University of Bonn, selected the bacterium Staphylococcus aureus, one of the most dangerous human pathogenic bacteria, as the model organism for their study. The focus was on the influence of antibiotics that inhibit peptidoglycan synthesis on cell division.

We found a rapid and strong effect of oxacillin and the glycopeptide antibiotics vancomycin and telavacin on cell division. The cell division protein FtsZ served as a marker here and we monitored it.”

Jan-Samuel Puls, a PhD student at the Institute of Pharmaceutical Microbiology at UKB

For this purpose, FtsZ was fluorescently labeled alongside other proteins. Then the researchers analyzed the effects on individual living bacterial cells over time and also used super-resolution microscopy. They established an automated image analysis for microscopy images that allowed them to quickly analyze all cells in the sample under study. “Staphylococcus aureus is only about one micrometer, which is one-thousandth of a millimeter. This makes microscopy particularly challenging,” says Dr. Fabian Grein, junior research group leader at the UKB’s Institute of Pharmaceutical Microbiology and a scientist at the German Center for Infection Research (DZIF).

Antibiotic effect on cell wall biosynthesis machinery inhibits cell division immediately

The Bonn research team found that the formation of peptidoglycan is the driving force during the entire process of cell division. Previously, peptidoglycan synthesis was thought to be essential only during a specific part of this process. The team showed that inhibition of cell wall assembly by glycopeptide antibiotics in Staphylococcus aureus occurs rapidly and with a dramatic effect on cell division. In addition, they clarified in detail the specific role of essential penicillin-binding protein 2 (PBP2), which links cell wall components, in cell division. The β-lactam antibiotic oxacillin prevents the proper localization of this protein. “This means that PBP2 does not get to the place where it is needed. As a result, the cell can’t divide,” Grein says. “Importantly, this all happens immediately after the antibiotics are added. So the first cellular effects, which have not been studied very intensively so far, are crucial.” Therefore, in view of the alarming increase in antibiotic resistance worldwide, he hopes the study results will provide a better understanding of how exactly these agents work at the cellular level, and thus a key to the development of new antibiotics. Understanding cellular mechanisms of antibiotic action and production is the goal of the DFG Collaborative Research Center TRR 261 “Antibiotic CellMAP”, which conducted these studies.

Source:

University of Bonn

Journal reference:

Puls, J.-S., et al. (2023). Inhibition of peptidoglycan synthesis is sufficient for total arrest of staphylococcal cell division. Science Advances. doi.org/10.1126/sciadv.ade9023

Microbiology

Multiplex PCR panels associated with reduced administration of antibiotics to hospitalized GI patients

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Reviewed by Emily Henderson, B.Sc.Mar 16 2023

Acute gastroenteritis afflicts adults of all ages, causing significant suffering and inflicting significant costs on the American healthcare system. A new study encompassing nearly 40,000 hospital visits from a geographically diverse healthcare database shows that sampling a single stool, using multiple polymerase chain reaction (PCR) panels, can identify more pathogens, notably diarrhea-causing E. coli and enteric viruses, and do so more rapidly than a conventional workup. The research is published in Journal of Clinical Microbiology, a publication of the American Society for Microbiology.

Using multiple PCR panels, “Fewer patients received antibiotics, required additional visits or diagnostic tests, or were hospitalized for gastroenteritis within 30 days [of index visit],” said Rena C. Moon, M.D., M.P.H., Principal Research Scientist, PINC AI Applied Sciences, Charlotte, NC. Additionally, healthcare costs were lower than with a conventional workup. Conventional workups may include testing a stool culture for a single suspect species of pathogen, use of a single pathogen PCR test, or identifying a pathogen using microscopy, immunology or an ova and parasites test.

Earlier studies showed that large multiplex PCR panels improve the speed and accuracy of diagnostic testing in patients with acute gastroenteritis, but their impact on costs and clinical outcomes had been uncertain. Our study shows that the benefits of multiplex panels can be achieved without increasing overall healthcare costs, and also facilitates more appropriate use of antibiotics.”

Ferric C. Fang, M.D., Professor of Laboratory Medicine, Pathology, and Microbiology at the University of Washington School of Medicine, Seattle

“This study illustrates the power of big data to analyze the healthcare impacts of diagnostic testing, and help laboratories select testing approaches that improve meaningful clinical outcomes,” Fang said.

Using multiplex PCR, more patients could be discharged and did not require hospitalization during the following month. That resulted in similar healthcare costs to patients undergoing the traditional stool work-up plus follow-up visits over the following month. Furthermore, multiplex PCR (using 12 or more) panels were associated with reduced administration of antibiotics to hospitalized patients.

The overall result: improved care with lower costs.

Source:

American Society for Microbiology

Microbiology

Using the origami technique to design RNA nanostructures

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Reviewed by Emily Henderson, B.Sc.Feb 28 2023

Researchers from Aarhus University and Berkeley Laboratory have designed RNA molecules, that folds into nanoscale rectangles, cylinders, and satellites, and have studied their 3D structure and dynamics with advanced nanotechnological methods. In an article in the journal Nature Nanotechnology, the researchers describe their work and how it has led to the discovery of rules and mechanisms for RNA folding that will make it possible to build more ideal and functional RNA particles for use in RNA-based medicine.

The RNA molecule is commonly recognized as messenger between DNA and protein, but it can also be folded into intricate molecular machines. An example of a naturally occurring RNA machine is the ribosome, that functions as a protein factory in all cells. Inspired by natural RNA machines, researchers at the Interdisciplinary Nanoscience Center (iNANO) have developed a method called “RNA origami”, which makes it possible to design artificial RNA nanostructures that fold from a single stand of RNA. The method is inspired by the Japanese paper folding art, origami, where a single piece of paper can be folded into a given shape, such as a paper bird.

Frozen folds provide new insight

The research paper in Nature Nanotechnology describes how the RNA origami technique was used to design RNA nanostructures, that were characterized by cryo-electron microscopy (cryo-EM) at the Danish National cryo-EM Facility EMBION. Cryo-EM is a method for determining the 3D structure of biomolecules, which works by freezing the sample so quickly that water does not have time to form ice crystals, which means that frozen biomolecules can be observed more clearly with the electron microscope. Images of many thousands of molecules can be converted on the computer into a 3D map, that is used to build an atomic model of the molecule. The cryo-EM investigations provided valuable insight into the detailed structure of the RNA origamis, which allowed optimization of the design process and resulted in more ideal shapes.

With precise feedback from cryo-EM, we now have the opportunity to fine-tune our molecular designs and construct increasingly intricate nanostructures.”

Ebbe Sloth Andersen, Associate Professor at iNANO, Aarhus University

Discovery of a slow folding trap

Cryo-EM images of an RNA cylinder sample turned out to contain two very different shapes, and by freezing the sample at different times it was evident that a transition between the two shapes was taking place. Using the technique of small-angle X-ray scattering (SAXS), where the samples are not frozen, the researchers were able to observe this transition in real time and found that the folding transition occurred after approx. 10 hours. The researchers had discovered a so-called “folding trap” where the RNA gets trapped during transcription and only later gets released (see video).

“It was quite a surprise to discover an RNA molecule that refolds this slow since folding typically takes place in less than a second” tells Jan Skov Pedersen, Professor at Department of Chemistry and iNANO, Aarhus University.

“We hope to be able to exploit similar mechanisms to activate RNA therapeutics at the right time and place in the patient”, explains Ewan McRae, the first author of the study, who is now starting his own research group at the “Centre for RNA Therapeutics” at the Houston Methodist Research Institute in Texas, USA.

Construction of a nanosatellite from RNA

To demonstrate the formation of complex shapes, the researchers combined RNA rectangles and cylinders to create a multi-domain “nanosatellite” shape, inspired by the Hubble Space Telescope.

“I designed the nanosatellite as a symbol of how RNA design allows us to explore folding space (possibility space of folding) and intracellular space, since the nanosatellite can be expressed in cells”, says Cody Geary, assistant professor at iNANO, who originally developed the RNA-origami method.

However, the satellite proved difficult to characterize by cryo-EM due to its flexible properties, so the sample was sent to a laboratory in the USA, where they specialize in determining the 3D structure of individual particles by electron tomography, the so-called IPET-method.

“The RNA satellite was a big challenge! But by using our IPET method, we were able to characterize the 3D shape of individual particles and thus determine the positions of the dynamic solar panels on the nanosatellite”, says Gary Ren from the Molecular Foundry at Lawrence Berkeley National Laboratory, California, USA.

The future of RNA medicine

The investigation of the RNA origamis contributes to improving the rational design of RNA molecules for use in medicine and synthetic biology. A new interdisciplinary consortium, COFOLD, supported by the Novo Nordisk Foundation, will continue the investigations of RNA folding processes by involving researchers from computer science, chemistry, molecular biology, and microbiology to design, simulate and measure folding at higher time resolution.

“With the RNA design problem partially solved, the road is now open to creating functional RNA nanostructures that can be used for RNA-based medicine, or act as RNA regulatory elements to reprogram cells”, predicts Ebbe Sloth Andersen.

Source:

Aarhus University

Journal reference:

McRae, E.K.S., et al. (2023) Structure, folding and flexibility of co-transcriptional RNA origami. Nature Nanotechnology. doi.org/10.1038/s41565-023-01321-6.

Microbiology

Simple blood tests for telomeric protein could provide a valuable screen for certain cancers

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Reviewed by Emily Henderson, B.Sc.Feb 21 2023

Once thought incapable of encoding proteins due to their simple monotonous repetitions of DNA, tiny telomeres at the tips of our chromosomes seem to hold a potent biological function that’s potentially relevant to our understanding of cancer and aging.

Reporting in the Proceedings of the National Academy of Science, UNC School of Medicine researchers Taghreed Al-Turki, PhD, and Jack Griffith, PhD, made the stunning discovery that telomeres contain genetic information to produce two small proteins, one of which they found is elevated in some human cancer cells, as well as cells from patients suffering from telomere-related defects.

Based on our research, we think simple blood tests for these proteins could provide a valuable screen for certain cancers and other human diseases. These tests also could provide a measure of ‘telomere health,’ because we know telomeres shorten with age.”

Jack Griffith, PhD, the Kenan Distinguished Professor of Microbiology and Immunology and Member of the UNC Lineberger Comprehensive Cancer Center

Telomeres contain a unique DNA sequence consisting of endless repeats of TTAGGG bases that somehow inhibit chromosomes from sticking to each other. Two decades ago, the Griffith laboratory showed that the end of a telomere’s DNA loops back on itself to form a tiny circle, thus hiding the end and blocking chromosome-to-chromosome fusions. When cells divide, telomeres shorten, eventually becoming so short that the cell can no longer divide properly, leading to cell death.

Scientist first identified telomeres about 80 years ago, and because of their monotonous sequence, the established dogma in the field held that telomeres could not encode for any proteins, let alone ones with potent biological function.

In 2011 a group in Florida working on an inherited form of ALS reported that the culprit was an RNA molecule containing a six-base repeat which by a novel mechanism could generate a series of toxic proteins consisting of two amino acids repeating one after the other. Al-Turki and Griffith note in their paper a striking similarity of this RNA to the RNA generated from human telomeres, and they hypothesized that the same novel mechanism might be in play.

They conducted experiments – as described in the PNAS paper – to show how telomeric DNA can instruct the cell to produce signaling proteins they termed VR (valine-arginine) and GL (glycine-leucine). Signaling proteins are essentially chemicals that trigger a chain reaction of other proteins inside cells that then lead to a biological function important for health or disease.

Al-Turki and Griffith then chemically synthesized VR and GL to examine their properties using powerful electron and confocal microscopes along with state-of-the-art biological methods, revealing that the VR protein is present in elevated amounts in some human cancer cells, as well as cells from patients suffering from diseases resulting from defective telomeres.

“We think it’s possible that as we age, the amount of VR and GL in our blood will steadily rise, potentially providing a new biomarker for biological age as contrasted to chronological age,” said Al-Turki, a postdoctoral researcher in the Griffith lab. “We think inflammation may also trigger the production of these proteins.”

Griffith noted, “When you go against current thinking, you are usually wrong because you are bucking many people who’ve worked so diligently in their fields. But occasionally scientists have failed to put observations from two very distant fields together and that’s what we did. Discovering that telomeres encode two novel signaling proteins will change our understanding of cancer, aging, and how cells communicate with other cells.

“Many questions remain to be answered, but our biggest priority now is developing a simple blood test for these proteins. This could inform us of our biological age and also provide warnings of issues, such as cancer or inflammation.”

Source:

University of North Carolina Health Care

Journal reference:

Al-Turki, T., et al. (2023) Mammalian Telomeric RNA (TERRA) can be translated to produce valine-arginine and glycine-leucine dipeptide repeat proteins. PNAS. doi.org/10.1073/pnas.2221529120.

Microbiology

Penn scientists discover a cellular pathway that keeps Ebola virus from exiting human cells

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Reviewed by Emily Henderson, B.Sc.Feb 3 2023

In their evolutionary battle for survival, viruses have developed strategies to spark and perpetuate infection. Once inside a host cell, the Ebola virus, for example, hijacks molecular pathways to replicate itself and eventually make its way back out of the cell into the bloodstream, where it can spread further.

But our own cells, in the case of Ebola and many other viruses, aren’t without defenses. In a study published in the Proceedings of the National Academy of Sciences, a team led by University of Pennsylvania School of Veterinary Medicine scientists discovered a way human cells hamper the Ebola virus’ ability to exit.

An interaction between viral and host proteins prompts host cells to ramp up activity of a pathway responsible for digesting and recycling proteins, the team found. This activity, known as autophagy “self-eating,” allows fewer viral particles to reach the surface of a host cell, thus reducing the number that can exit into the bloodstream and further propagate infection.

This interaction seems to be part of an innate defense mechanism. Human cells appear to specifically target a key Ebola virus protein and direct it into the autophagy pathway, which is how cells process and recycle waste.”

Ronald N. Harty, professor at Penn Vet and senior author on the study

The investigation emerged from a longtime area of focus for Harty’s lab: the interaction between the viral protein VP40, found in both Ebola and Marburg viruses, and various human proteins. In the group’s previous work, they’ve found that one area of VP40, known as a PPXY motif, binds corresponding motifs known as WW domains on specific host proteins.

In many instances, this PPXY-WW interaction causes more viral particles to exit the cell in a process called “budding.” But in screening various host proteins thought to play a role in the process, Harty and postdoc Jingjing Liang, the study’s lead author, uncovered some that did the opposite upon binding VP40, causing budding to decrease. One of these was a protein called Bag3, on which they reported in a PLOS Pathogens paper in 2017.

Though Ebola is a potentially deadly virus, Harty and colleagues can safely study its workings in a Biosafety Level 2 laboratory, substituting virus-like particles (VLPs) that express VP40 for the virus itself. These VP40 VLPs are not infectious but can bud out from host cells like the real thing.

In the new work, the Penn Vet researchers and colleagues from the Texas Biomedical Research Institute dug deeper to learn about the mechanism by which Bag3 reduced budding. Bag3 is known as a “co-chaperone” protein, involved in forming a complex with other proteins and chaperoning them on their trip to be digested, ultimately in organelles called autolysosomes, part of the process of autophagy. Using VP40 VLPs, Harty’s group confirmed that VP40 bound to Bag3 and formed the protein complex. When the researchers added a compound that is known to block formation of this complex, they saw VP40 being released; VLP budding activity subsequently increased.

To follow the activity of VP40 in real time, the team used powerful confocal microscopy, labeling each actor of interest with a different fluorescent tag. They observed that Bag3 was involved in sequestering VP40 in vesicles in the cell that would go on to undergo autophagy. Stuck in these vesicles and destined for the cellular “recycling center,” VP40 was unable to move to the cell membrane and bud.

“I think one of the most interesting things that we showed is the selectivity of the cargo,” Liang says. “We show that autophagy doesn’t just happen passively. Bag3 acts through the PPXY-WW interaction to specifically target VP40 to undergo autophagy.”

When the researchers added the drug rapamycin, which enhances autophagy, VP40 sequestration went up and VLP budding went down. Rapamycin works by inhibiting the activity of a pathway governed by a protein complex called mTORC1, a cellular sensor that turns on protein synthesis when a cell needs raw material to grow. The researchers found this pathway appeared to be important in regulating Ebola infection; in experiments with live virus conducted in a Biosafety Level 4 laboratory, they observed that the virus could activate mTORC1 signaling, causing the cellular “factory” to produce materials the virus would need to expand and spread. In contrast, inhibiting mTORC1 with rapamycin directed the virus toward the autophagy pathway, where it would be digested by the cell’s autolysosomes.

“The virus wants the cell growing so it activates mTORC1,” says Harty. “Autophagy does the opposite, keeping the cellular materials in balance.”

Autophagy is important for normal cellular processes, ensuring that the cell doesn’t become cluttered with unnecessary or misfolded proteins and other materials floating around. But this work also suggests autophagy can be harnessed by the body to defend against harmful infection.

“Our conception is that this is part of the arms race between our bodies and the virus,” Liang says. “The virus wants to shape its environment to benefit itself and its own survival, so it evolved to manipulate mTORC1. But the cell can also use this pathway to defend against viral infection.”

With these insights into the human body’s innate defenses against Ebola, the researchers hope to see if autophagy may be a factor in other hemorrhagic viral infections, such as those that cause Marburg and Lassa fever. And while the current experiments were primarily conducted using human liver cell lines, the team would also like to test whether autophagy and the mTORC1 pathway are involved in viral defense in other cell types, such as the immune system’s macrophages, the primary cells involved in propagating infection.

Ultimately, Harty, Liang, and colleagues hope to find as many viral vulnerabilities as possible, helping inform drugs that could be one component of a therapeutic cocktail, each targeting different stages of infection, from viral entry to exit.

“This all ties together in our overall goal of understanding viral-host interactions and, by understanding them, working to intervene to slow or stop infection,” Harty says.

Source:

University of Pennsylvania

Journal reference:

Liang, J., et al. (2023) Chaperone-assisted selective autophagy targets filovirus VP40 as a client and restricts egress of virus particles. PNAS. doi.org/10.1073/pnas.2210690120.

Microbiology

Structure and function of the first FDA-approved treatment for Ebola virus discovered

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Reviewed by Emily Henderson, B.Sc.Jan 30 2023

Scientists at La Jolla Institute for Immunology (LJI) have uncovered the structure and function of the first FDA-approved treatment for Zaire ebolavirus (Ebola virus).

Inmazeb (REGN-EB3), developed by Regeneron, is a three-antibody cocktail designed to target the Ebola virus glycoprotein. The drug was first approved for clinical use in October 2020, but its exact mechanism of action has remained unclear.

In the cover story of the latest issue of Cell Host & Microbe, LJI researchers present a high-resolution, 3D structure of the three antibodies as they bind to the Ebola virus glycoprotein (the viral protein that launches Ebola virus infection). The model reveals new information about both the drug and the virus, and how their interaction fights infection and protects against future viral mutations.

Before this, we had a general idea of what the drug was doing, but we didn’t know exactly how. We now know which specific amino acids the antibodies are latching onto and how their binding affects the viral glycoprotein.”

Ollmann Saphire, Ph.D., LJI President and CEO, Study’s Senior Author

The new research also shows the potential for Inmazeb in treating additional species of Ebolavirus.

The new study shows how three antibodies (light blue, dark blue, and yellow) used in Inmazeb (REGN-EB3) bind to different regions of the Ebola virus glycoprotein (grey) to combat infection. Image credit: Ethan MacKenzie (Phospho Biomedical Animation)

How the antibody cocktail works

At 3.1 angstroms, the 3D structure is the highest-resolution image of the Ebola virus surface protein ever assembled using asymmetric reconstruction. The researchers achieved this detailed view through an imaging technique called cryogenic electron microscopy (cryo-EM).

“It’s like getting mugshots of a protein,” said first author Vamseedhar Rayaprolu, Ph.D., who spearheaded the project as a postdoctoral associate at LJI and now serves at The Pacific Northwest Center for Cryo-EM. “We take photos of the complex that is frozen in all different angles and then stitch them together to get a 3D model.”

Thanks to these images, the LJI team immediately made a discovery not just about the drug, but also about Ebola virus itself. While the overall structure of the Ebola glycoprotein has been known for some time, one region had yet to be modeled effectively-;the β17-β18 loop on the protein’s glycan cap.

“This piece is normally too floppy to be imaged,” said Rayaprolu, “but when the antibodies were bound to the virus, they locked the loop into place and we were able to finally capture its location and structure.”

The team then confirmed that the drug’s three antibodies bind the glycoprotein at distinct, non-overlapping locations, maximizing their effectiveness by minimizing their redundancy.

Atoltivimab (REGN3470) is the specific antibody that binds the β17-β18 loop. When bound, this antibody can serve as a signal to attract the immune system, flagging infected cells to be killed via effector functions.

A second antibody, called odesivimab (REGN3471), binds to amino acids on the glycoprotein’s receptor-binding site, preventing the virus from attaching itself to human cells.

The third antibody, called maftivimab (REGN3479), binds and warps the glycoprotein’s internal fusion loop, which the virus requires to drive itself into a cell. The researchers also found evidence that maftivimab may be valuable in future therapies against other types of Ebolaviruses.

Fighting more than one virus

“Like with SARS-CoV-2, Ebola virus has changed over time and become different than the original virus,” says study collaborator Robert Davey, Ph.D., Professor in the National Emerging Infectious Diseases Laboratories (NEIDL), of the Boston University Chobanian & Avedisian School of Medicine. As Davey points out, Ebola viruses aren’t the only dangerous members of the larger Filovirus family. This family includes closely related Ebolavirus species, such as Sudan ebolavirus (a 2022 outbreak of Sudan ebolavirus killed at least 55 people in Uganda) and the more distantly related Marburg virus.

Through a series of escape studies led by study collaborators in Davey’s lab and at Regeneron, the team found that Inmazeb could potentially protect against several viruses in the Ebolavirus genus of Filoviruses, including Sudan ebolavirus.

The key appears to be the maftivimab antibody. Maftivimab’s target, the viral glycoprotein’s internal fusion loop, is conserved across these Ebolaviruses. This means the loop structure has not changed significantly, even as other parts of the virus have mutated over time.

“We found that, in general, the antibodies in Inmazeb could be effective against the more closely related viruses,” says Davey. “But for the more distantly related species, such as Marburg, more work needs to be done to devise a new antibody cocktail.”

Could Inmazeb also combat new Ebola virus variants? The researchers found that-;in the presence of all three antibodies-;Ebola virus has to undergo ten rounds of replication and multiple mutations to partially escape the effects of the drug. In contrast, using any single antibody alone leads to escape mutations within only one or two passages.

This finding suggests that Inmazeb can provide lasting immunity against variants. The new findings may also guide the development of novel antibody drugs that target the glycoprotein more broadly or effectively.

“We now understand how subtle shifts in the landing site of different antibodies impact function,” says Rayaprolu. “This tells us the differences between more or less effective immune responses.”

“Knowing exactly where a drug contacts the virus helps us predict whether it is likely to still work on a new viral variant,” adds Saphire. “These methods and the insights from our research collaborators will be integral to the development of next-generation vaccines.”

Source:

La Jolla Institute for Immunology

Journal reference:

Rayaprolu, V., et al. (2023) Structure of the Inmazeb cocktail and resistance to Ebola virus escape. Cell Host & Microbe. doi.org/10.1016/j.chom.2023.01.002.

Microbiology

For decades, humans have relied on antibiotics to eliminate bacterial infections, and for a long time, those antibiotics …

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For decades, humans have relied on antibiotics to eliminate bacterial infections, and for a long time, those antibiotics worked reliably. But microbes, like other forms of life, can evolve to find ways around the things that impede their growth and survival. Pathogenic bacteria are doing that with antibiotics, and while scientists are searching for new antibiotic compounds that can be used therapeutically, creating new medicines presents many challenges and can take a long time. Bacteriophages, which are viruses that only infect bacterial cells, might be a solution to the problem of antibiotic resistance. But if they are going to work against infections in humans, we need to know more about them first.

A digitally colorized SEM image of two carbapenem-resistant Klebsiella pneumoniae (CRKP) bacteria (yellow) interacting with a type of human white blood cell called a neutrophil (green) / Credit: National Institute of Allergy and Infectious Diseases (NIAID)

Scientists have now used a variety of methods including cryo-electron microscopy, machine learning, and simulations to characterize the structure and function of a bacteriophage that normally lives in the human gastrointestinal tract. The phage, called ϕKp24, could be useful against multidrug resistant strains of Klebsiella pneumoniae. The work has been reported in Nature Communications.

The World Health Organization has designated K. pneumoniae a priority 1 pathogen, meaning that it is critical to create new antibiotics that can eliminate these infections. There are over one hundred genetically distinct types of K. pneumoniae, which can cause bacteremia, urinary tract infection, pneumonia, and other diseases. Immunocompromised individuals are at particular risk.

Although bacteriophages can destroy multi-drug resistant K. pneumoniae, most of these phages are very individualized, and will only attack specific strains of the pathogen. This study aimed to reveal more about the ϕKp24 phage, which may be able to attack multiple strains of K. pneumoniae.

The study authors suggested that ϕKp24 may be a good candidate for the development of phage therapy for humans.

“The problem with phage therapy is that bacteriophages don’t work on all bacteria, even from the same species. In contrast to many known bacteriophages, this one is special because it works on many different subtypes. This makes it a good candidate for phage therapy,” explained corresponding study author Professor Ariane Briegel of Institute Biology Leiden. “By learning more about how the bacteriophage works, we can hopefully treat people with it in the future.”

Sources: Leiden University, Nature Communications


Carmen Leitch