Bacteriophages or “phages” is the terms used for viruses that infect bacteria. The UAB researchers, led by Terje Dokland, Ph.D., in collaboration with Asma Hatoum-Aslan, Ph.D., at the University of Illinois Urbana-Champaign, have described atomic models for all or part of 11 different structural proteins in phage Andhra. The study is published in Science Advances.

Andhra is a member of the picovirus group. Its host range is limited to S. epidermidis. This skin bacterium is mostly benign but also is a leading cause of infections of indwelling medical devices. “Picoviruses are rarely found in phage collections and remain understudied and underused for therapeutic applications,” said Hatoum-Aslan, a phage biologist at the University of Illinois.

With emergence of antibiotic resistance in S. epidermidis and the related pathogen Staphylococcus aureus, researchers have renewed interest in potentially using bacteriophages to treat bacterial infections. Picoviruses always kill the cells they infect, after binding to the bacterial cell wall, enzymatically breaking through that wall, penetrating the cell membrane and injecting viral DNA into the cell. They also have other traits that make them attractive candidates for therapeutic use, including a small genome and an inability to transfer bacterial genes between bacteria.

Knowledge of protein structure in Andhra and understanding of how those structures allow the virus to infect a bacterium will make it possible to produce custom-made phages tailored to a specific purpose, using genetic manipulation.

“The structural basis for host specificity between phages that infect S. aureus and S. epidermidis is still poorly understood,” said Dokland, a professor of microbiology at UAB and director of the UAB Cryo-Electron Microscopy Core. “With the present study, we have gained a better understanding of the structures and functions of the Andhra gene products and the determinants of host specificity, paving the way for a more rational design of custom phages for therapeutic applications. Our findings elucidate critical features for virion assembly, host recognition and penetration.”

Staphylococcal phages typically have a narrow range of bacteria they can infect, depending on the variable polymers of wall teichoic acid on the surface of different bacterial strains. “This narrow host range is a double-edged sword: On one hand, it allows the phages to target only the specific pathogen causing the disease; on the other hand, it means that the phage may need to be tailored to the patient in each specific case,” Dokland said.

The general structure of Andhra is a 20-faced, roundish icosahedral capsid head that contains the viral genome. The capsid is attached to a short tail. The tail is largely responsible for binding to S. epidermidis and enzymatically breaking the cell wall. The viral DNA is injected into the bacterium through the tail. Segments of the tail include the portal from the capsid to the tail, and the stem, appendages, knob and tail tip.

The 11 different proteins that make up each virus particle are found in multiple copies that assemble together. For instance, the capsid is made of 235 copies each of two proteins, and the other nine virion proteins have copy numbers from two to 72. In total, the virion is made up of 645 protein pieces that include two copies of a 12th protein, whose structure was predicted using the protein structure prediction program AlphaFold.

The atomic models described by Dokland, Hatoum-Aslan, and co-first authors N’Toia C. Hawkins, Ph.D., and James L. Kizziah, Ph.D., UAB Department of Microbiology, show the structures for each protein — as described in molecular language like alpha-helix, beta-helix, beta-strand, beta-barrel or beta-prism. The researchers have described how each protein binds to other copies of that same protein type, such as to make up the hexameric and pentameric faces of the capsid, as well as how each protein interacts with adjacent different protein types.

Electron microscopes use a beam of accelerated electrons to illuminate an object, providing much higher resolution than a light microscope. Cryo-electron microscopy adds the element of super-cold temperatures, making it particularly useful for near-atomic structure resolution of larger proteins, membrane proteins or lipid-containing samples like membrane-bound receptors, and complexes of several biomolecules together.

In the past eight years, new electron detectors have created a tremendous jump in resolution for cryo-electron microscopy over normal electron microscopy. Key elements of this so-called “resolution revolution” for cryo-electron microscopy are:

• Flash-freezing aqueous samples in liquid ethane cooled to below -256 degrees F. Instead of ice crystals that disrupt samples and scatter the electron beam, the water freezes to a window-like “vitreous ice.”
• The sample is kept at super-cold temperatures in the microscope, and a low dose of electrons is used to avoid damage to the proteins.
• Extremely fast direct electron detectors are able to count individual atoms at hundreds of frames per second, allowing sample movement to be corrected on the fly.
• Advanced computing merges thousands of images to generate three-dimensional structures at high resolution. Graphics processing units are used to churn through terabytes of data.
• The microscope stage that holds the sample can also be tilted as images are taken, allowing construction of a three-dimensional tomographic image, similar to a CT scan at the hospital.

The analysis of Andhra virion structure by the UAB researchers started with 230,714 particle images. Molecular reconstruction of the capsid, tail, distal tail and tail tip started with 186,542, 159,489, 159,489 and 159,489 images, respectively. Resolution ranged from 3.50 to 4.90 angstroms.

It’s almost impossible to study this phenomenon in a lab, though, because water crystallizes into ice so quickly at such low temperatures. Now, new research from the Georgia Institute of Technology uses machine learning models to better understand water’s phase changes, opening more avenues for a better theoretical understanding of various substances. With this technique, the researchers found strong computational evidence in support of water’s liquid-liquid transition that can be applied to real-world systems that use water to operate.

“We are doing this with very detailed quantum chemistry calculations that are trying to be as close as possible to the real physics and physical chemistry of water,” said Thomas Gartner, an assistant professor in the School of Chemical and Biomolecular Engineering at Georgia Tech. “This is the first time anyone has been able to study this transition with this level of accuracy.”

The research was presented in the paper, “Liquid-Liquid Transition in Water From First Principles,” in the journal Physical Review Letters, with co-authors from Princeton University.

Simulating Water

To better understand how water interacts, the researchers ran molecular simulations on supercomputers, which Gartner compared to a virtual microscope.

“If you had an infinitely powerful microscope, you could zoom in all the way down to the level of the individual molecules and watch them move and interact in real time,” he said. “This is what we’re doing by creating almost a computational movie.”

The researchers analyzed how the molecules move and characterized the liquid structure at different water temperatures and pressures, mimicking the phase separation between the high and low-density liquids. They collected extensive data — running some simulations for up to a year — and continued to fine-tune their algorithms for more accurate results.

Even a decade ago, running such long and detailed simulations wouldn’t have been possible, but machine learning today offered a shortcut. The researchers used a machine learning algorithm that calculated the energy of how water molecules interact with each other. This model performed the calculation significantly faster than traditional techniques, allowing the simulations to progress much more efficiently.

Machine learning isn’t perfect, so these long simulations also improved the accuracy of the predictions. The researchers were careful to test their predictions with different types of simulation algorithms. If multiple simulations gave similar results, then it validated their accuracy.

“One of the challenges with this work is that there’s not a lot of data that we can compare to because it’s a problem that’s almost impossible to study experimentally,” Gartner said. “We’re really pushing the boundaries here, so that’s another reason why it’s so important that we try to do this using multiple different computational techniques.”

Beyond Water

Some of the conditions the researchers tested were extremes that probably don’t exist on Earth directly, but potentially could be present in various water environments of the solar system, from the oceans of Europa to water in the center of comets. Yet these findings could also help researchers better explain and predict water’s strange and complex physical chemistry, informing water’s use in industrial processes, developing better climate models, and more.

The work is even more generalizable, according to Gartner. Water is a well-studied research area, but this methodology could be expanded to other difficult-to-simulate materials like polymers, or complex phenomena like chemical reactions.

“Water is so central to life and industry, so this particular question of whether water can undergo this phase transition has been a longstanding problem, and if we can move toward an answer, that’s important,” he said. “But now we have this really powerful new computational technique, but we don’t yet know what the boundaries are and there’s a lot of room to move the field forward.”

The first-of-its-kind study, published in PLOS ONE, examined both the exterior surface and interior microbiome of mosquitoes found in homes in Africa’s Cote d’Ivoire — the Ivory Coast.

“When you’re exposed to mosquitoes, you worry about blood feeding,” said R. Michael Roe, William Neal Reynolds Distinguished Professor of Entomology at NC State and co-corresponding author of the study. “Our hypothesis is that mosquitoes can physically transfer bacteria by landing on you or by defecating on household surfaces, like flies do.

“They may not, but no one has studied it before.”

Research collaborators at the Centre Suisse de Recherches Scientifiques collected 79 adult female Anopheles coluzzii mosquitoes from homes in a rice-producing province in Cote d’Ivoire. The mosquitoes were sent to NC State for analysis of the microbiome inside and on external body surfaces.

Some of the findings were surprising.

“We found greater bacterial diversity internally than externally, which didn’t match what has been found with blow flies, for example,” said Loganathan Ponnusamy, an NC State principal research scholar in entomology and co-corresponding author of the paper.

“At the same time, we found lots of external bacterial differences between homes, but not much difference internally between homes, which makes sense. Much of what is found internally relates to nectar or honey consumed as mosquitoes forage outdoors.”

The researchers also found — for the first time in the academic literature — fructobacillus, which is generally found in nectar sources like flowers and beehives, pointing to mosquitoes visiting those plants or nectar sources, said Kaiying Chen, an NC State postdoctoral researcher and first author of the paper.

Perhaps more ominously, the researchers also found large amounts of Staphylococcus and two variants of Rickettsia. The genus of these bacteria are associated with human and animal diseases.

“This is another risk,” Roe said. “Mosquitoes carry bacteria externally and internally and come into your home, possibly transferring pathogenic bacteria.”

The researchers hope to continue the work by exposing mosquitoes to a bacteria that would never be found on human skin and seeing whether the bacteria transfers to an artificial membrane. They then could perform the same test on human arms.

NC State Ph.D. researchers Chouaïbou S. Mouhamadou and Jean M. Deguenon co-authored the paper, as did Behi Kouadio Fodjo, Gba Christabelle Sadia and France Paraudie Kouadio Affoue from the Centre Suisse de Recherches Scientifiques, Abidjan, Cote d’Ivoire, Africa. Funding was provided by a grant from the Department of the Army under a Deployed Warfighter Protection (DWFP) Program Grant W911QY1910003.

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Materials provided by North Carolina State University. Original written by Mick Kulikowski. Note: Content may be edited for style and length.