Structure of viral membrane-penetrating machines by electron cryo-microscopy and tomography

Abstract

My PhD thesis aims to obtain the structure of several bacteriophages, including so-called “jumbo” phages, using the electron cryo-microscopy (cryo-EM) and to study the structural transformation of these viruses as they attach and infect the host cell. We aim to understand the process of host cell membrane penetration at the molecular level and to achieve the level of knowledge necessary to understand how the genome and proteins, which are originally packaged into the capsid, are delivered into the host cell. This structural information can be of great importance since bacteriophage therapy is a potentially promising alternative to antibiotics, which are urgently needed. Bacteriophages, or phages are the most abundant and diverse form of life on Earth, and have developed various strategies through which to infect a susceptible bacterial host. A vast majority of phages have evolved to use a special organelle, called a “tail”, for host recognition, attachment and genome delivery into the cell. The host cell envelope is penetrated with the help of the tail. Unlike most eukaryotic viruses, infection of a host by tailed bacteriophages usually requires only one virion per bacterium, indicating that tailed bacteriophages have evolved an extremely efficient infection mechanism. Bacteriophages with contractile tails are complex viruses that infect Bacteria and Archaea. They share a common evolutionary origin and some functional aspect with other large macromolecular machines, such as the Serratia entomophila antifeeding prophage, the Photorhabdus virulence cassette, and the type VI secretion system (T6SS), which function to attack other cells by translocating toxic effector proteins into the target cell’s cytoplasm. The T6SS and the so-called “jumbo” phages are the most complex of these contractile injection systems. Jumbo phages have genomes exceeding 250kb and virus particle sizes of 300 nm and greater. Hundreds of different proteins are involved in the morphogenesis and assembly of a jumbo phage particle. A number of bacteriophages infecting important pathogens were isolated and characterized by means of cryo-EM and bioinformatics tools. Bacteriophage ɸ92 is a virus infecting a wide variety of acapsular Escherichia coli laboratory strains, Salmonella strains, and pathogenic E.Coli strains carrying a polysialic acid, which cause severe invasive infections including meningitis, pneumonia, septicaemia, osteomyelitis, septic arthritis and pyelonephritis. A cryo-EM reconstructions of the ɸ92 capsid, baseplate and long contractile tail were calculated, and complete three-dimensional model of the phage virion was built. Two bacteriophages, ɸEco32 infecting mastitis-causing strains of E.Coli and 7-11 infecting Salmonella strains, were isolated. They are a members of the Podoviridae family with the rare C3 morphotype, which occurs in <1% of phage virions. Two functional states of mature ɸEco32 virion, before and after DNA ejection, and mature 7-11 tail were characterized by cryo-EM and cryo-ET. Bacteriophage AR9 is a flagella-specific Bacillus subtilis jumbo phage with a tail of remarkable complexity. The AR9 adsorption apparatus could be used for attachment and “riding on” the motile B. subtilis flagellum. A three-dimensional model of the jumbo virion was built and flagella-specific adsorption apparatus was characterized. Bacterial phage CBA120 belongs to the Myoviridae family distinct from T4 in morphology and behavior and specifically infects E.Coli strain O157, an important food-borne pathogen. CBA120 represents a newly described group of phages. Extended and contracted conformations of CBA120 tail were characterized by cryo-EM. Mutant bacteriophage T4 lacking the tip protein of the cell-puncturing device responsible for the first interaction with the host cell membrane was isolated. Three-dimensional cryo-EM reconstruction of the baseplate and a tail fragment was calculated and compared with that of the wild type T4 phage and the distant member of T4-superfamily phage RB43.