Researchers at the University of Alabama at Birmingham have used cryo-electron microscopy to reveal the structures of bacterial viruses in unprecedented detail. This is the first structure of a virus capable of infecting Staphylococcus epidermidis, and high-resolution structural knowledge is a key link between virus biology and the potential therapeutic use of this virus to inhibit bacterial infections.
Bacteriophage, or “bacteriophage,” is the term used for viruses that infect bacteria. UAB researchers led by Dr. Terje Dokland, in collaboration with Dr. Asma Hatoum-Aslan of the University of Illinois at Urbana-Champaign, described the atomic model bacteriophage Andra of all or part of 11 different structural proteins. The study was published in Science Advances.
Andhra is a member of the parvivirus group. Its host range is limited to Staphylococcus epidermidis. This skin bacteria is mostly benign but is also a leading cause of infection with indwelling medical devices. “Picoviruses are rarely found in phage collections and remain understudied and underutilized for therapeutic applications,” said Hatoum-Aslan, a phage biologist at the University of Illinois.
With the emergence of antibiotic resistance in Staphylococcus epidermidis and the related pathogen Staphylococcus aureus, researchers have renewed interest in the possible use of phages to treat bacterial infections. Microviruses always kill the cells they infect after binding to the bacterial cell wall, breaking through the cell wall enzymatically, penetrating the cell membrane and injecting viral DNA into the cell. They also have other properties that make them attractive candidates for therapeutic use, including small genomes and the inability to transfer bacterial genes between bacteria.
Understanding protein structures in Andhra Pradesh and understanding how these structures allow viruses to infect bacteria will make it possible to use genetic manipulation to produce phages that are customized for specific purposes.
“The structural basis of host specificity between phages that infect S. aureus and S. epidermidis remains poorly understood,” said Dokland, UAB professor of microbiology and director of the UAB Cryo-electron Microscopy Core. “Through the current study, we gain a better understanding of the structure and function of the Andhra gene product and the determinants of host specificity, paving the way for more rational design of custom phages for therapeutic applications. Our findings shed light on Key features virion assembly, host recognition and penetration.”
Staphylococcal phages can usually infect a narrow range of bacteria, depending on the variable polymers of teichoic acid in the surface wall of different bacterial strains. “This narrow host range is a double-edged sword: On the one hand, it allows the phage to target only the specific pathogen that causes the disease; on the other hand, it means that the phage may need to be tailored to each patient’s specific situation,” said Doc. Lan said.
The overall structure of Andhra is a 20-sided circular icosahedral capsid head containing the viral genome. The capsid is attached to a short tail. The tail is primarily responsible for binding to S. epidermidis and enzymatically disrupting the cell wall. Viral DNA is injected into bacteria through the tail. The parts of the tail include the entrance from the shell to the tail, as well as the stem, appendages, nodes and tail tip.
The 11 different proteins that make up each virus particle exist in multiple copies assembled together. For example, the capsid consists of 235 copies of each of the two proteins, and the other nine virion proteins range in copy number from 2 to 72. The virion consists of a total of 645 protein fragments, including two copies of the 12th protein, whose structure was predicted using the protein structure prediction program AlphaFold.
Atomic models described by Dokland, Hatoum-Aslan, and co-first authors N’Toia C. Hawkins, Ph.D., and James L. Kizziah, Ph.D., of the UAB Department of Microbiology show the structure of each protein — such as alpha-helix, beta-helix, beta -strand, beta-barrel or beta-prism as described in molecular languages. The researchers describe how each protein binds to other copies of the same protein type, such as the hexameric and pentameric faces that make up the capsid, and how each protein interacts with neighboring different protein types.
Electron microscopes use a beam of accelerated electrons to illuminate objects, providing much higher resolution than light microscopes. The added element of cryo-electron microscopy makes it particularly useful for near-atomic structural resolution of complexes of larger proteins, membrane proteins, or lipid-containing samples (such as membrane-bound receptors) and several biomolecules.
In the past eight years, new electron detectors have enabled cryo-electron microscopes to achieve a huge leap in resolution over ordinary electron microscopes. The key elements of this so-called “resolution revolution” in cryo-EM are:
- Rapid freezing of aqueous samples in liquid ethane, chilled to below -256°F. Instead of ice crystals that destroy samples and scatter electron beams, the water freezes into “glass ice” that forms windows.
- Samples are kept at ultra-cold temperatures in the microscope, and low-dose electrons are used to avoid damage to proteins.
- Extremely fast direct electron detectors are able to count individual atoms at hundreds of frames per second, allowing instant correction for sample motion.
- Advanced computing combines thousands of images to generate high-resolution 3D structures. Graphics processing units are used to process terabytes of data.
- The microscope stage on which the sample is placed can also be tilted while the image is taken, creating a three-dimensional tomographic image, similar to a CT scan in a hospital.
The UAB researchers’ analysis of the Andhra virion structure began with 230,714 images of the particle. Molecular reconstructions of the capsid, tail, tail distal end, and tail tip start from 186,542, 159,489, 159,489, and 159,489 images, respectively. Resolutions range from 3.50 to 4.90 Angstroms.