Terje Dokland - US grants

A grant has been awarded to the University of Alabama at Birmingham (UAB) under the direction of Dr. David Chaplin for partial support of the purchase of a 200 kV field-emission gun cryo-electron microscope (cryo-EM). Contemporary research in a broad range of scientific disciplines ranging from nanotechnology, to biochemistry, virology, and cell biology relies on defining molecular structures to understand biophysical and biochemical mechanisms. Electron microscopy is one of the most important techniques in modern life science research and the only one that is able to directly image structures ranging in size from individual atoms to whole cells and tissues. Cryo-electron microscopy is a technique that allows biological material to be imaged in its native, hydrated form, in the absence of the damaging stains and fixatives required by conventional electron microscopy. Due to its high quality imaging system, the cryo-EM can achieve a resolution that approaches that of X-ray crystallography, allowing structure determination of proteins and protein/nucleic acid complexes. Using tomographic approaches, the three-dimensional organization of whole cells and organelles can be visualized in their native, functional states. The acquisition of this cryo-EM is a central component of campus-wide structural biology initiatives that link the UAB School of Medicine and School of Natural Sciences & Mathematics. The instrument will be available on a shared basis to researchers throughout the southeast United States, expanding its impact to that of a regional resource.

Establishment of the 200 kV cryo-EM at UAB will dramatically accelerate research and promote teaching, training, and learning at UAB. Several research programs, ranging from the structure and assembly of bacteriophages and animal viruses to cell wall pore proteins and the analysis of nanoparticles used in preparation of prosthetic joints will benefit from the availability of this instrument. In addition to these ongoing research projects, the cryo-EM will stimulate the development of new research initiatives and will support the expansion of training programs leading to careers in structural biology research. These training initiatives will include laboratory courses available to both undergraduate and graduate students at UAB. The cryo-EM will also be used as part of the BioTeach program offered through UAB's Center for Community Outreach and Development. BioTeach provides basic knowledge and laboratory skills to high school science teachers.

The acquisition of this 200 kV cryo-EM will dramatically advance the scientific discovery process and will promote teaching, training, and learning at UAB. Local availability of this cryo-EM will accelerate progress in research focused on structural aspects of virus assembly and maturation, and in nanostructures for bioengineering and biomedicine. Lastly, the cryo-EM will be used in an educational program to exposes Alabama K-12 teachers to high-end instruments and modern approaches used in scientific research. This experience is expected to stimulate their students, particularly underserved minorities and females, to consider post-secondary educational training and careers in science.

[unreadable] DESCRIPTION (provided by applicant): Staphylococcus aureus produces a number of toxins of great medical importance, including toxic shock syndrome toxin (TSST-1) and enterotoxins B and C. Several of these toxins are carried on so-called pathogenicity islands, genetic elements that resemble prophages, but do not carry most of the genes normally associated with phage activities. These elements are normally highly stable, but specific phages have the ability to excise them and package them into phage particles that can transfer the toxin genes to new cells. SaPI1 is an S. aureus pathogenicity island that carries the genes for TSST-1 as well as enterotoxins Q and L. SaPI1 is mobilized by phage 80?, and efficiently packaged into phage-like particles, using 80? structural proteins. However, the SaPI1 capsid is only 1/3 the size of the normal 80? capsid, commensurate with its smaller genome. It is not known what the mechanism of size determination is, but the system resembles the E. coli phage P2/P4 system, in which the satellite phage P4 hijacks the structural proteins from P2 and uses them to package a smaller capsid. The overall objective of this study is to understand the mechanism of size determination in the S. aureus phage 80?/Sal1 system and its role in the development of bacterial pathogenesis. In this study we will use cryo-electron microscopy and three-dimensional reconstruction methods to determine structures of phage 80? and SaPI1 particles. The specific aims of this study are: (1) Determine the 3D structure of S. aureus phage 80? capsids; (2) Determine the 3D structure of SaPI1 capsids; (3) Determine the structure of procapsids produced by co-expression of 80?/SaPI1 structural proteins. RELEVANCE TO PUBLIC HEALTH: Staphylococcus aureus has become a major health problem in hospitals, especially with the emergence of multiple antibiotic resistant strains. Several of the toxins produced by pathogenic S. aureus cause severe systemic infections, including the toxic shock syndrome toxin TSST-1 and enterotoxins B and C. Recently, S. aureus toxins have become a particular concern as potential bioterrorism agents. The bacteriophage/pathogenicity island system is of special interest for its role in transferring toxin genes and conferring pathogenicity on non-pathogenic strains. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Staphylococcus aureus produces a number of toxins of great medical importance, including toxic shock syndrome toxin (TSST-1) and enterotoxins B and C. Several of these toxins are carried on so-called pathogenicity islands (SaPIs), genetic elements that resemble prophages, but lack most of the genes normally associated with phage production. These elements are normally stable, but can be mobilized by infection with certain phages or by the induction of indigenous prophages, upon which the SaPIs get excised and packaged into phage-like particles that can transfer the toxin genes to new cells. The type member of the SaPI family, SaPI1 can be mobilized by phage 80? and is efficiently packaged into phage-like particles using 80? structural proteins. However, the SaPI1 capsid is only 1/3 the size of the normal 80? capsid, commensurate with its smaller genome. With the emergence of antibiotic-resistant Staphylococcus strains and the renewed interest in therapeutic use of bacteriophages to treat such infections, the phage-induced mobilization of SaPI1 will be an important consideration. The overall objective of this study is to understand the structural basis for the phage-induced mobilization and spread of S. aureus pathogenicity islands and its role in the development of bacterial pathogenesis. In this project, we address two specific questions in relation to the SaPI1 mobilization: How does SaPI1 induce the formation of small capsids from 80?-encoded proteins? How is the SaPI1 DNA selected for packaging into these capsids? Understanding these questions will have a bearing on understanding the evolution of pathogenicity in S. aureus. To this end, we will use a combination of genetic, biochemical and structural approaches, including cryo-EM, X-ray crystallography and Mass Spectrometry. The specific aims of this study are: (1) Define the role of the 80? structural gene products in capsid formation (2) Determine the mechanism of SaPI1-induced capsid size determination (3) Identify the determinants for DNA discrimination by SaPI1. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus has become a major health problem in hospitals, especially with the emergence of multiple antibiotic resistant strains. Pathogenic S. aureus may cause severe systemic infections by producing several toxins from genes carried on so-called pathogenicity islands in the bacterial genome. The bacteriophage/pathogenicity island system under study in this project is of special interest for its role in the horizontal spread and long-term establishment of pathogenicity in the population.

PROJECT SUMMARY Staphylococcus aureus is an opportunistic bacterial pathogen involved in severe infections in humans. Most virulence determinants in S. aureus are carried on mobile genetic elements (MGEs), such as plasmids, bacteriophages and genomic islands. Transduction by bacteriophages (phages) represents the main mechanism by which MGEs are transmitted horizontally in S. aureus. Among these MGEs are the S. aureus pathogenicity islands (SaPIs), which carry genes encoding superantigen toxins and other virulence factors. SaPIs are normally stably integrated into the host genome, but become mobilized at high frequency by specific helper phages, resulting in packaging of the SaPI genomes into transducing particles made from helper-encoded structural proteins. SaPIs have evolved the ability to sense the presence of a lytic phage, exploit phage functions and interfere with phage multiplication, in order to promote their own dissemination. SaPIs this play important roles in S. aureus evolution and pathogenicity. The overall aim of the current project is to understand the structural basis for SaPI mobilization, helper-SaPI specificity, and the factors involved in their spread and establishment. Our specific aims are: (1) Determine the mechanism of SaPI-induced capsid size redirection; (2) Understand the function of the phage baseplate in infection and host specificity; (3) Elucidate the role of minor capsid protein gp44 in the lytic/lysogenic switch. These three aims focus on different aspects of the mobilization process and will be studied by a combination of genetic, biochemical and structural methods. All three aims are based on a solid premise set by our previous studies and extensive preliminary data. Upon completion of these aims, we will have gained new insights into the process of capsid assembly and size redirection, the infection and transfer process, the mechanisms by which SaPIs and their virulence factors are transmitted and established in the bacterial population.

Abstract The Southeastern Center for Microscopy of MacroMolecular Machines (SECM4) is a consortium of 15 Universities/Medical Centers with a total of 19 investigators throughout the Eastern United States studying a wide range of important biomedical projects as variable as high resolution virus structure, membrane protein structure, macromolecular complexes of various types, some isolated in active form from cells, bacterial ultrastructure, muscle filaments, spliceosomes, ribosomes complexes all of which will benefit from ready access to a high resolution electron microscope such as a Titan Krios equipped with a direct electron detector (DED). Human health implications extend from virus and bacterial pathogens to the understanding of diseases resulting form genetic mutations. The basic biology of cancer and heart disease is being studied in several member laboratories. The Titan Krios at Florida State University has been in operation since 2009 and recently has had its image recording device upgraded from CCD camera to a Direct Electron LLC, DE-20 direct electron detector positioned ahead of an existing imaging filter which removes inelastically scattered electrons thereby improving the image quality. Although we propose a robust plan to enable members to come to Florida State University, we propose creating a facility based on the synchrotron template currently in use at multiple sites X-ray crystallography beam lines around the country whereby users ship specimens to us and watch the data being collected as it comes off the microscope from the familiar confines of their own laboratories. We will provide sufficient preprocessing that consortium members can evaluate the prospects for obtaining a final high resolution structure from damage and motion corrected ?movie? images of their samples. The result will be a model for high throughput structure determination utilizing high-end instrumentation that can reveal the inner workings of complex macromolecules and subcellular structures.

PROJECT SUMMARY Recently, single particle cryo-electron microscopy (cryo-EM) combined with 3-D reconstruction has emerged as a revolutionary tool for solving high-resolution 3-D structures of viruses and macromolecular complexes. The rapidly increasing number of near-atomic resolution (3-4?) structures solved using cryo-EM has allowed unprecedented atomic level understanding of fundamental cellular processes and viral infections. To obtain near-atomic resolution cryo-EM structures, requires the collection of high-resolution image data using state-of- the-art imaging resources, including both a high-end transmission electron microscope and a direct electron detector. The direct electron detectors not only improve image resolution and contrast, but also record movies for subsequent computational correction of electron beam-induced, sample movements during exposure. The increased image contrast and resolution are essential for solving near-atomic resolution structures of small protein complexes. However, the high cost to purchase and maintain a state-of-the-art cryo-EM resource, with both a high-end electron microscope and a direct electron detector, precludes many cryo-EM investigators from having access to these new techniques. Here, the creation of a Midwest Consortium for High- Resolution Cryo-electron Microscopy is proposed to provide access to such high-resolution data collection capability for cryo-EM laboratories without access to such resources. This consortium will consist of 4 investigators from the host institution (Purdue Univ.) which will maintain the high-resolution data collection resource (Titan Krios 300kV FEG microscope with phase plate, energy filter, and direct electron detector) and will provide services to 11 investigators from 10 partnering institutions (Boston Univ. School of Medicine, Michigan State Univ., Penn State College of Medicine, Rutgers Univ., Univ. of Alabama at Birmingham, Univ. of Colorado Boulder, Univ. of Kansas, Univ. of Missouri, Univ. of Vermont, and Virginia Commonwealth Univ.). The collective experience of the Purdue facility staff, faculty and onsite service engineer, in high-resolution cryo-EM imaging, will ensure that the facility operates at peak performance with minimal service interruptions. The high-resolution data collection capabilities established at Purdue and accessible to the partnering labs, will allow these investigators, who are all, except for the 3 new investigators, NIH-funded, to overcome the resolution barrier and facilitate discovery within their own cryo-EM projects on a range of structures, such as, bacterial pathogen adhesion proteins, human viruses, Huntington's Disease proteins, synaptic vesicle proteins, chemoreceptor signaling complex, etc. The Consortium will provide comprehensive support to the cryo-EM projects, including shipping samples, preparing sample grids, collecting high-resolution single particle and tomography images, processing raw movies, and transferring data back to the partners' labs. The data collection services will range from full data collection where partners simply mail in the samples and then receive the image data, to hands-on operations of the cryo-EM by partners trained by the host facility's staff.

Staphylococcus aureus is an opportunistic bacterial pathogen involved in severe infections in humans. Most virulence determinants in S. aureus are carried on mobile genetic elements, such as plasmids, bacteriophages and genomic islands. Among these are the S. aureus pathogenicity islands (SaPIs), which carry genes encoding superantigen toxins and other virulence factors. SaPIs are normally stably integrated into the host genome, but become mobilized by specific helper phages, resulting in packaging of the SaPI genomes into transducing particles made from helper-encoded structural proteins. Many SaPIs redirect the assembly pathway of their helpers to form capsids that are smaller than those normally made by the phage. We recently described a new class of SaPIs (including SaPIbov5) that are mobilized by prolate cos phages, which package units of DNA delimited by cos sites, rather than headfuls of DNA. The mechanism of capsid size redirection is completely different from the SaPIs that are mobilized by headful packaging phages, and depends on a SaPI-encoded capsid protein (CP) homolog called Ccm, but the process is not understood. In this developmental project we will investigate this process by a combination of staphylococcal genetics, biochemistry and high resolution cryo-EM. There are three aims: (1) Determine whether Ccm is incorporated into small phage capsids; (2) Determine structures of ?12 (large) and SaPIbov5 (small) capsids; (3) Define the roles of CP and Ccm during capsid assembly. Upon completion of these aims, we will have gained new insights into the process of assembly in prolate phages, mobilization of cos SaPIs, SaPI evolution and the role of SaPIs in the emergence of staphylococcal virulence.

ABSTRACT The Southeastern Consortium for Microscopy of MacroMolecular Machines (SECM4) comprises 10 Universities/Medical Centers throughout the Eastern United States with a total of 13 cryoEM investigators studying a wide range of important biomedical problems as variable as high resolution virus structure, membrane protein structure, macromolecu- lar complexes of various types, some isolated in active form from cells, bacterial ultrastructure, spliceosomes, ribosome complexes all of which benefit from access to Florida State Universities (FSU) Titan Krios and its DE-64 direct electron detector. Recently the FSU Titan Krios was upgraded through the addition of a FEI Volta phase plate and a Gatan BioQuantum/K3 imaging filter which facilitate imaging of small molecules using single particle methods as well as thicker specimens that are imaged using cryoelectron tomography. The upgrades expanded the range of medically related structural biology problems to which SECM4 members can contribute. These upgrades also have made the FSU Titan Krios, which was one of the earliest ones installed in the US, comparable to recently installed Titan Krios microscopes, except for one feature. Newer Titan Krios microscopes have a more robust Autoloader than the early version currently operating on The FSU microscope. The Autoloader is the device that facilitates exchange of frozen hydrated specimens from the outside world into the high, contamination free environment of the Titan Krios. The current Autoloader, installed in August 2011, is currently responsible for more than 50% of the operational down time due to instrument failure. This Administrative Supplement seeks funds to replace the current Autoloader with the most recent version with the goal of reducing the greatest cause of instrument down time. SECM4 operates on the synchrotron template currently in use at sites having X-ray crystallography beam lines around the country. SECM4 members ship specimens to FSU and watch the data being collected as it comes off the microscope from the familiar confines of their own laboratories. SECM4 provides sufficient preprocessing that consortium members can evaluate the prospects for obtaining a final high-resolution structure from damage and motion corrected ?movie? images of their samples. SECM4 will become a model for high throughput structure determination utilizing high-end instrumentation to reveal the inner workings of complex macromolecules and subcellular structures.

Staphylococcus aureus is an opportunistic bacterial pathogen involved in severe infections in humans. S. aureus pathogenicity islands (SaPIs) are mobile genetic elements that carry genes encoding superantigen toxins and other virulence factors, and are mobilized at high frequency by specific ?helper? bacteriophages. Many SaPIs redirect the assembly pathway of their helpers to form capsids that are smaller than those normally made by the phage. This request is for a diversity supplement to R01 AI083255 (parent grant) to support Ms. N'Toia Hawkins, a graduate student in my lab who is currently funded by a UAB diversity fellowship that expires in July 2019. The parental R01 project is focused on understanding the mobilization and size redirection process for one class of SaPIs?including SaPI1 and SaPIbov1?that are mobilized by headful packaging helper phages with isometric capsids, such as 80?. We recently described a new class of SaPIs (including SaPIbov5) that are mobilized by prolate cos phages, such as ?12. The mechanism of capsid size redirection is completely different from the SaPIs that are mobilized by headful packaging phages, and depends on a SaPI-encoded capsid protein (CP) homolog called Ccm. The overall objective of the N'Toia's project is to understand the mechanism of Ccm-mediated capsid size redirection and, more broadly, the mobilization of cos type SaPIs. We propose to incorporate this project as a supplemental aim to the parent R01 with N'Toia taking the lead, with funding from the proposed diversity supplement. The supplement project has three specific aims: Aim 1: Determine structures of ?12 (large) and SaPIbov5 (small) capsids Aim 2: Identify the location of Ccm in SaPIbov5 capsids Aim 3: Define the role of the CP and Ccm N-terminal domains in the size redirection process These aims will be addressed by a combination of genetics, biochemistry, cryo-EM and three-dimensional reconstruction approaches, and will lead to new insights into the process of prolate phage assembly and genetic mobilization in S. aureus. Furthermore, the project will serve as an excellent vehicle for Ms. Hawkins' training and development into an independent scientist and to help her reach her career goals.

? DESCRIPTION (provided by applicant): This application seeks funding to establish the West/Midwest Consortium for High-Resolution Cryo Electron Microscopy (cryoEM). The goal of the consortium is to offer 19 cryoEM users from 10 regional institutes free access to a high-end cryoEM facility with proven high-resolution capabilities located in the California NanoSystems Institute (CNSI) at University of California, Los Angeles (UCLA). UCLA will act as host institute and provide investigators in these cryoEM laboratories access to its highly productive Titan Krios cryo electron microscope recently upgraded with a Volta phase plate, a Gatan imaging filter (GIF), and pre- and post-GIF direct electron detectors. A highly experienced staff with proven records of cryoEM-derived atomic structures will provide on-site assistance to collect data and provide streamlined movie pre-processing for our consortium user laboratories. The critical need for this consortium for recording atomic-resolution cryoEM images of a broad range of bio-medically significant macromolecular complexes is justified by the immediate benefits to the 19 consortium users. Of particular note, we have demonstrated that, once trained by our staff, users no longer have to be present at the microscope and can remotely control the microscope and perform high-resolution cryoEM imaging after our staff have aligned the instruments for top performance, thus significantly improving accessibility to the high-end instrument. A three-member supervisory committee representing non-consortium users, regional consortium users and the UCLA facility staff will provide evaluation and approval of user projects, adding and dropping users. Under our typical operation, a user group with approved projects requests research instrument time with an existing professionally-designed online reservation system, sends cryoEM grids by overnight courier (such as FedEx) in a liquid nitrogen-cooled dry shipper, and, after their grids are loaded by our staff, image their sample remotely for 3-7 days on average, and up to 2-3 weeks if necessary. The 10 institutes are concentrated on the West coast (University of Washington in Seattle, University of California at San Diego, Scripps) and Midwest (Case Western Reserve University, Purdue University, The University of Kansas, University of Alabama at Birmingham) except for three (University of Texas at El Paso, University of Texas Medical Branch in Galveston, and University of Florida at Gainesville). With the exception of three new investigators, all these users are funded by the NIH or other federal agencies to pursue biomedical research, ranging from biology, biochemistry, virology and microbiology. The establishment of this consortium will immediately empower our users with high-resolution cryoEM capabilities for a broad range of biological samples, enabling them to understand mechanisms of action and identify new targets for the development of new therapeutics.

This proposal requests support for the purchase of an Thermo Fisher Glacios electron microscope equipped with a Falcon 3EC direct electron detector and EPU data collection software to be shared by several users at the University of Alabama at Birmingham (UAB). Recent technical developments in cryo-EM have revolutionized structural biology, allowing structures of proteins as small as 100 kDa to be determined to near-atomic resolution, without the need to crystallize. These developments include mechanically and optically stable microscopes with automatic sample changers able to run continuously for several days with minimal supervision, and direct electron detectors (DEDs) with greatly improved resolution performance and low noise (high detective quantum efficiency, DQE) compared to traditional CCD detectors. UAB is currently equipped with an FEI F20 electron microscope with a Gatan K3 direct electron detector. While this instrument is theoretically capable of reaching ?4Å resolution, it is incapable of high-throughput operation needed for sample screening and high-resolution data collection. The proposed Glacios instrument will enable high-resolution/high-throughput cryo-EM at UAB. Many investigators at UAB who use structural approaches for their research will be able to take advantage of the new capabilities, leading to accelerated progress on many medically important projects in key areas such as antibacterial drug discovery, emerging and biodefense- related viruses, vaccine development, cystic fibrosis, Parkinson's disease, cancer, and others. The Glacios microscope, the Falcon DED and embedded EPU software constitute a fully integrated system that will be easy to operate and maintain by a variety of users. With the new instrumentation, UAB will be able to attract new investigators who need high-resolution cryo-EM for their research. In The proposed instrumentation will have a transformative impact on research, training and recruitment at UAB.

The emergence of antibiotic resistance combined with a paucity of new antibiotics under development have sparked a renewed interest in phage therapy as a treatment strategy for infectious diseases caused by common pathogens like Staphylococcus aureus and Staphylococcus epidermidis. However, several challenges remain before phage therapy can become a viable treatment option, including the narrow host range of the phages and concerns about genetic mobilization. Most current therapeutic strategies rely on cocktails of poorly characterized phages with uncertain interaction with the human microbiome. There is therefore a need for a more rational approach to phage therapy based on well-characterized components with defined and programmable host specificities. The bacteriophages of the Picovirinae subfamily of the Podoviridae (picoviruses) are attractive candidates for therapeutic applications due to their small (<20 kbp) genomes and strictly lytic lifestyle. The overall objectives of this exploratory/developmental (R21) project are (1) to understand the determinants for host range and specificity in staphylococcal picoviruses, and (2) to uncover the rules for manipulating this specificity to enable the rational design of therapeutic phages with tunable host range. In this proposal we will test our central hypothesis that picovirus receptor binding protein structures correlate with host cell wall teichoic acid composition. This will be accomplished through a combination of phage discovery and sequence analysis, cryo-electron microscopy, and CRISPR- based genome editing, via three specific aims: (1) Determine the genetic basis for host attachment by staphylococcal picoviruses; (2) Define the structural determinants for picovirus host range and specificity; and (3) Engineer phages with altered host ranges. This work will establish a predictive framework for determining the sensitivity of a pathogen to a specific set of picoviruses. In doing so, this research will provide a versatile toolkit for the rational design of therapeutic phages with pre-determined host specificities against pathogenic staphylococci. This novel approach can also be broadly applied to target other Gram-positive pathogens.