Atomic structure of a staphylococcal bacteriophage using cryo-electron microscopy

Cryo-electron microscopy by researchers at the University of Alabama at Birmingham has exposed the structure of a bacterial virus in unprecedented detail. This is the first structure of a virus capable of infecting Staphylococcus epidermidis, and high-resolution knowledge of the structure is a key link between viral biology and the potential therapeutic use of the virus to suppress bacterial infections.

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

Andhra is part of the picovirus group. Its host range is limited to S. epidermidis. This skin bacterium is mostly benign, but it is also a leading cause of indwelling medical device infections. “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 S. epidermidis and the related pathogen Staphylococcus aureus, researchers have renewed interest in the potential use of bacteriophages to treat bacterial infections. Picoviruses always kill the cells they infect, following binding to the bacterial cell wall, enzymatically crossing this 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.

Knowing the structure of proteins in Andhra and understanding how these structures allow the virus to infect a bacterium will allow the production of tailor-made phages suitable for 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, professor of microbiology at UAB and director of UAB Cryo-Electron Microscopy. Core. “With the present study, we have gained a better understanding of the structures and functions of Andhra gene products and the determinants of host specificity, paving the way for more rational design of custom phages for therapeutic applications. Our results elucidate essential features for virion assembly, host recognition and penetration. »

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

The general structure of Andhra is a rounded 20-sided icosahedral capsid head which contains the viral genome. The capsid is attached to a short tail. The tail is largely responsible for binding to S. epidermidis and enzymatic disruption of the cell wall. The viral DNA is injected into the bacterium through the tail. The tail segments include the capsid portal to the tail, as well as the stem, appendages, knob, and tip of the tail.

The 11 different proteins that make up each virus particle are found in multiple copies that fit together. For example, the capsid is made up of 235 copies of each of two proteins, and the other nine proteins in the virion have a copy number of two to 72. In total, the virion is made up of 645 pieces of protein that include two copies of a 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., UAB Department of Microbiology, show the structures of each protein – as described in molecular language as alpha-helix, beta-helix, beta-strand, beta-barrel or beta-prism. The researchers described how each protein binds to other copies of the same type of protein, for example to form the hexameric and pentameric faces of the capsid, as well as how each protein interacts with different types of adjacent proteins.

Electron microscopes use a beam of accelerated electrons to illuminate an object, providing much higher resolution than an optical microscope. Cryo-electron microscopy adds the element of super cold temperatures, which makes it particularly useful for resolving the quasi-atomic structure of larger proteins, membrane proteins, or samples containing lipids such as membrane-bound receptors and complexes of several biomolecules together.

Over the past eight years, new electron detectors have created a huge leap in resolution for cryo-electron microscopy compared to normal electron microscopy. The key elements of this so-called “resolution revolution” for cryo-electron microscopy are:

• Instant freezing of aqueous samples in liquid ethane cooled to less than -256 degrees F. Instead of ice crystals disturbing the samples and scattering the electron beam, the water freezes into a “glassy ice” shaped of window.
• The sample is kept at super cold temperatures in the microscope and a low dose of electrons is used to prevent protein damage.
• Extremely fast direct electron detectors are capable of counting individual atoms at hundreds of frames per second, allowing sample motion to be corrected on the fly.
• Advanced computing merges thousands of images to generate high-resolution three-dimensional structures. Graphics processing units are used to process terabytes of data.
• The microscope stage that holds the sample can also be tilted as images are taken, allowing the construction of a three-dimensional tomographic image, similar to a CT scan in the hospital.

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