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

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

A Multivalent Adsorption Apparatus Explains the Broad Host Range of Phage phi92: a Comprehensive Genomic and Structural Analysis

Christopher Browning, Petr Leiman, Sergey Nazarov

Bacteriophage phi92 is a large, lytic myovirus isolated in 1983 from pathogenic Escherichia coli strains that carry a polysialic acid capsule. Here we report the genome organization of phi92, the cryoelectron microscopy reconstruction of its virion, and the re-investigation of its host specificity. The genome consists of a linear, double-stranded 148,612-bp DNA sequence containing 248 potential open reading frames and 11 putative tRNA genes. Orthologs were found for 130 of the predicted proteins. Most of the virion proteins showed significant sequence similarities to proteins of myoviruses rv5 and PVP-SE1, indicating that phi92 is a new member of the novel genus of rv5-like phages. Reinvestigation of phi92 host specificity showed that the host range is not limited to polysialic acid-encapsulated Escherichia coli but includes most laboratory strains of Escherichia coli and many Salmonella strains. Structure analysis of the phi92 virion demonstrated the presence of four different types of tail fibers and/or tail-spikes, which enable the phage to use attachment sites on encapsulated and nonencapsulated bacteria. With this report, we provide the first detailed description of a multivalent, multispecies phage armed with a host cell adsorption apparatus resembling a nanosized Swiss army knife. The genome, structure, and, in particular, the organization of the baseplate of phi92 demonstrate how a bacteriophage can evolve into a multi-pathogen-killing agent.

American Society for Microbiology2012

Assembly Mechanisms and Cellular Effects of Bacterial Pore-Forming Toxins

Mirko Bischofberger

The correct assembly of macromolecular protein complexes such as ribosomes or multisubunit membrane channels is essential for their function. Alterations in the process can lead to disease either by loss of function or by acquisition of toxic function. However, the precise mechanisms by which multimeric protein complexes assemble are generally poorly understood owing to the difficulty in reconstituting the process in-vitro. Thus, systems that undergo dynamic assembly and disassembly in solution, such as cytoskeletal proteins, have been particularly attractive. By contrast, the assembly of multi-subunit membrane protein complexes has been notably difficult to study. An interesting, somewhat in between, situation is provided by pore-forming proteins, the best-characterized subclass of which are bacterial pore-forming toxins (PFTs). These proteins can generally be produced recombinantly and in a highly stable soluble monomeric form, but assemble into homo-oligomeric ring-like structures upon addition to cells expressing the appropriate cell surface receptor. These rings concomitantly, or subsequently depending of on the PFT, undergo a conformational change that leads to exposure of hydrophobic surfaces and spontaneous membrane insertion. The built pores then perforate the membrane of the host cell. PFTs do not only occur in bacteria and have a very broad taxonomic distribution, occurring in organisms such as parasites, plants and mammals. However, for pathogenic organisms the PFTs are major virulence factors contributing to the disease caused by the organisms producing them. For this reason bacterial PFTs have been studied extensively during the last century and a lot of insight into their mode of action has been gained. However, despite the increasing knowledge on PFTs, the mechanisms and the kinetics of self-assembly of these complexes remain largely enigmatic. In particular it is unknown whether oligomerization occurs through the sequential addition of monomers or through interaction of multimeric intermediates, whether oligomerization is the rate-limiting step during the pore-formation process and whether the same rules apply to all toxins. This is mostly due to the fact that intermediates have not been visualized by either biochemical or structural methods. Also, with few exceptions, functional assays on the activity of PFTs have always been performed at the level of a population of cells. The aim of this thesis was to measure pore-formation at the single cell level to extract mechanistic information from the analysis of the stochasticity of the pore-formation process. We chose to study three PFTs produced by human pathogens: aerolysin, PFO and Cytolysin A (ClyA). Aerolysin is produced by Aeromonas hydrophila and forms pores of 1-2 nm in diameter, perfringolysin O (PFO) occurs in Clostridium perfringensis and is a cholesterol-dependent cytolysin (CDC) that builds channels of 25-30 nm. Both of these PFTs build structurally similar pores made of transmembrane β-barrels even though the number of strands in the barrel varies by more than 1 order of magnitude. On the contrary, ClyA is a PFT produced by both E. coli and Salmonella enterica strains and forms rings of 12-13 protomers with the membrane spanning domain composed of a-helices. Hence, the pores formed by these three PFTs vary greatly from one another and are representative for the diversity of PFTs. We measured cell permeabilisation at the single cell level using two independent live-cell imaging methods, in erythrocytes and in nucleated cells. Both assays revealed that the assembly mechanism of PFTs is stochastic in its nature and that lowering the initial monomer concentration increases the variability and average time to successfully build first functional pores. The measured stochasticity reflects the chemical kinetics behind the assembly reaction and therefore carries important information about the underlying mechanism. In fact, analysis of the stochasticity of the processes led to the robust determination for each toxin of the minimal number of independent rate-limiting steps required for pore formation. Furthermore, scaling relationships in our data upon changes in initial toxin concentration constrain the possible underlying mechanisms for assembly and reveal that the steps leading to the aerolysin heptamer are composed of at least seven independent reactions whose rates scale with toxin concentration. Taken together, our findings suggest that that what is limiting for aerolysin is the conversion for each monomer to an assembly competent state, while for PFO a single limiting step is observed, likely corresponding to membrane insertion. A second aim of this thesis was to clarify the cellular consequences that occur upon pore formation by different PFTs and to find out how PFT-affected cells sense this event. This was done in collaboration with other members of the lab. Our results suggest that the cells monitor the breaches in the plasma membrane by assessing the correct homeostasis of the potassium ion inside the cell through a yet unknown mechanism. Interestingly, the cells seem to "monitor" the integrity of the membrane through the most upstream effect possible after pore formation, namely the flux of ions. Incidentally, the assays we developed in the first part of this thesis to "monitor" initial pore formation events are based on the same principle, namely the immediate loss of gradients that occur upon pore formation.

EPFL2011

Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin

Petr Leiman

Protein secretion is a common property of pathogenic microbes. Gram-negative bacterial pathogens use at least 6 distinct extracellular protein secretion systems to export proteins through their multilayered cell envelope and in some cases into host cells. Among the most widespread is the newly recognized Type VI secretion system (T6SS) which is composed of 15-20 proteins whose biochemical functions are not well understood. Using crystallographic, biochemical, and bioinformatic analyses, we identified 3 T6SS components, which are homologous to bacteriophage tail proteins. These include the tail tube protein; the membrane-penetrating needle, situated at the distal end of the tube; and another protein associated with the needle and tube. We propose that T6SS is a multicomponent structure whose extracellular part resembles both structurally and functionally a bacteriophage tail, an efficient machine that translocates proteins and DNA across lipid membranes into cells.

National Academy of Sciences2009

Assembly Mechanisms and Cellular Effects of Bacterial Pore-Forming Toxins

Mirko Bischofberger

EPFL2011