Esther Bullitt - US grants

The aim of this research is to understand the structure and assembly of adhesion pili found on pathogenic bacteria, thereby providing insight into how the architecture of these pili supports their role as a virulence factor. Bacterial survival and colonization require attachment of the bacteria to hosts. In many stains, this process is initiated and maintained by pili; in Escherichia coli that cause pyelonephritis, adhesion and virulence depend on P-pili. Hib-pili expressed on the surface of Haemophilus influenzae mediate H. flu's colonization of the upper respiratory tract, and thus its ability to cause diseases such as childhood meningitis, otitis media, and pneumonia of the elderly. As bacteria become more resistant to traditional antibiotics, it is important to develop new therapies against bacterial infections. Structural information about adhesion pili will provide a basis for future rational design of new therapies to prevent bacterial binding or to remove pathogenic bacteria bound to the human host. The proposed research addresses this long-term goal through structural studies of bacterial adhesion pili. These studies focus on: 1) electron microscopy and three-dimensional (3-D) helical reconstruction of P-pili preserved in vitreous ice and of Hib-pili negative stain, 2) controlled damage/recovery of pili to investigate the possibility of re-formation of intact helical filaments, 3) investigation of the 3-D structure of P-pili with mutant structural proteins (pilins), to examine regions of the PapA pilin essential for their assembly into tightly coiled helical filaments, 4) bacterial attachment assays, to assess the effect of mutations and the effect of damage on bacterial binding, and 5) in vitro reconstitution of hetero-pilin polymers from chaperone-pilin complexes, to improve our understanding of the bioassembly process of a prototypical macromolecule.

DESCRIPTION (provided by applicant): Survival of pathogenic bacteria in their host is correlated with their capacity to maintain attachment to host tissue. Often this adherence is facilitated by the presence of filamentous adhesion pili on the bacterial surface, as in ETEC (enterotoxigenic Escherichia coli). ETEC cause severe diarrhea, presenting a significant worldwide health risk, particularly for infants and small children. In addition, the ease with which ETEC-caused diarrhea spreads via tainted food or water, places international travelers at high risk when entering regions where infection is endemic. In order to prevent or limit outbreaks, it is vital that we understand the mechanism of sustained bacterial attachment that can lead to colonization and illness. Structural information about adhesion pili will provide a basis for rational design of new therapies for prevention of bacterial binding or for removal of pathogenic bacteria already bound to the human host. The long-term goal of this project is to elucidate how the structure of pili supports their role as a virulence factor for pathogenic bacteria. In the proposed project period, studies on the structure and function of ETEC pili, in-depth computer modeling of P-pili expressed on the surface of pyelonephritic Escherichia coli (which cause urinary tract infections involving the kidneys), pilus damage/recovery experiments, and localization of type 1 adhesins will be used to examine the relationship between the structure and the function of adhesion pili. A combined approach will be employed to 1) elucidate the structural features of virulent ETEC pili that enable them to withstand peristaltic motility and other intestinal cleansing systems. Studies will include electron microscopy and image processing of negatively stained and frozen-hydrated ETEC pili. 2) examine the mechanism by which the P-pilus helical filament can unwind to a thin fibrillar structure five times its original length. Energy minimization and spatial constraints will be used with genetic algorithms to model, from individual monomeric subunits, this prototypic pilus filament into both intact and damaged pili. 3) investigate a means for reducing bacterial binding through damage to pili, with the aim of reducing the bacterial load and thus permitting the body's natural defenses to eradicate the remainder. Studies will use optical tweezers to measure the forces necessary to damage P-pili expressed on uropathogenic bacteria, and to investigate whether recovery occurs. 4) localize the adhesins on type 1 pili using immunoelectron microscopy.

DESCRIPTION (provided by applicant): This proposal requests funding for the purchase of a TVIPS 4K x 4K CCD camera for our FEI Tecnai F20 (TF20) electron microscope, with a 1K x 1K Fastscan CCD accessory. Research by the major users of the TF20, including six NIH-funded projects, provide cutting edge, state-of-the-art advances in the fields of bacterial pathogenesis, apoptosis, cardiovascular disease, viral replication, protein biogenesis, and muscle regulation. Minor users broaden our research further with four NIH-funded studies on the glycolytic pathway, nitrogen metabolism in bacteria, and the mitochondrial respiratory pathway. The purchase of a new CCD camera for the TF20 will allow us to collect data efficiently and accurately for the following major projects: 1) comparative studies on the specialization of pathogenic bacterial adhesion pili for colonization of specific organs in their human hosts, 2) studies on the apoptosome, a macromolecular assembly that is critical to the mitochondrial apoptosis pathway, 3) studies on the role of `bad cholesterol'in cardiovascular disease: the structure and function of LDL low density lipoproteins alone and in complex with its binding receptor, LDLR, 4) studies on the role of protein oligomerization for the replication of poliovirus RNA, including studies on the 3Dpol polymerase in isolation and tethered to membranes via the protein 3AB, 5) comparative studies of ribosome-Sec61 and ribosome-SecY complexes that mediate co-translational translocation of nascent proteins in mammals and bacteria, and 6) studies on the regulation of skeletal muscle contraction by thin filaments. The addition of a large format CCD to our existing TEM will allow us to remain at the forefront of our respective fields. The 1K x 1K CCD that is currently on theTF20 is an excellent tool for astigmatism correction, focus adjustment, and for checking a sample after a low dose image has been recorded on film. The requested large format CCD captures images of an area that is ten times larger than our current CCD. This increase in the amount of sample imaged is essential, as the limits imposed by our current CCD exclude the possibility of collecting the large datasets required for high resolution image reconstructions. However, the existing CCD will be a significant asset for routine work that is done on our Philips CM12 electron microscope Thus, an additional key benefit of this proposal will be the transfer of our existing TVIPS 1K x 1K CCD to our Philips CM12 electron microscope, for which no funding is requested. The CM12 has a large user base that currently includes the five major TF20 users and seven additional projects, six of which are NIH-funded. There is also a steady influx of new users. Having a CCD on this microscope will be extremely beneficial both as a training tool and for data collection on the CM12. Public Health Relevance: This proposal requests funding to upgrade our FEI Tecnai F20 electron microscope with a digital imaging system. Research by users of the electron microscope, including 10 NIH-funded projects, provide cutting edge, state-of-the-art advances in the fields of cardiovascular disease, bacterial pathogenesis, cell death (apoptosis), viral replication, muscle regulation, protein biosynthesis, and energy generation. Addition of this hardware will significantly improve the productivity of all these research projects, advancing research toward the prevention or elimination of human disease.

DESCRIPTION (provided by applicant): Diseases caused by positive-sense single-stranded RNA viruses, including poliovirus, rhinovirus, hepatitis C virus, and SARS, are critical health issues worldwide. Progression of disease requires replication of the virus genome by oligomeric complexes assembled on virally-induced membrane structures in the host cell. Design of drugs that interfere with protein-protein interactions has made considerable progress in recent years, making disruption of the viral replication complex an attractive target for antiviral intervention. This proposal focuses on poliovirus (i) in order to use the extensive knowledge of the structural and functional properties of poliovirus as a basis for detailed characterization of these protein-protein interactions, and (ii) to address the need for new antiviral strategies against poliovirus that a 2007 National Academy of Sciences panel asserted would significantly strengthen the eradication effort, and prevent its threat as a bioweapon against an unvaccinated population. Assemblies of poliovirus RNA polymerase, the functional centerpiece of the replication complex, will be analyzed in terms of the intermolecular interactions responsible for their stability and interpreted on the basis of high resolution crystallographic data and extensive mutational and biochemical data. As the field of structural electron microscopy advances toward routine sub-nanometer resolution, it is biological questions such as those asked here that continue to motivate technical innovations. In Specific Aim 1, methods for the routine use of cryo-electron microscopy to solve structures of nanocrystals as small as 10 unit cells on a side will be developed and used to analyze the structure of nanocrystals of RNA polymerase and characterize the intermolecular interactions therein. This will be of value far beyond the proposed studies of polymerase, enabling structural analyses of the 'shower' of protein nanocrystals that often appear in crystallization trays, that until now have been discarded as failed experiments, and that recently have been attracting greatly increased interest. In Specific Aim 2, a mechanism for stabilizing polymerase-RNA complexes and supporting RNA replication via a positively charged channel across the polymerase interface will be tested by structural and functional analyses of complexes comprising wild-type and mutant polymerases. In Specific Aim 3 transfection of viral replication proteins into mammalian cells will be used to (i) define th minimum viral component required for the membrane reorganization of cellular membrane into double-bilayers, and (ii) characterize the protein-protein interactions that stabilize polymerase-containing oligomers, as these interfaces represent sites of vulnerability for disruption of viral replication and for development as potential drug targets.

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.

? DESCRIPTION (provided by applicant): The National Center for Macromolecular Imaging (NCMI) at Baylor College of Medicine is the host laboratory offering to provide a portion of the time on their JEM3200FSC and JEM2200FS electron microscopes for a cryo-electron microscopy (cryoEM) and tomography (cryoET) data acquisition facility Consortium. Both instruments are equipped with a field emission gun and an in-column energy filter, while the JEM2200FS also has a Zernike phase plate attachment. Both instruments are currently equipped with DE20 and DE12 cameras operated in integrating mode. These microscopes have been used productively for a variety of biological specimens, including macromolecules, molecular machines and cells, at state-of-the-art resolution. Our facilities will be allocated for he proposed Consortium while taking into account the ongoing research in the host institution. Our lead PI, Wah Chiu is a recognized expert in pushing cryoEM and cryoET beyond current boundaries and has decades of experience in leading several NIH funded research centers and academic training programs. There are 11 participating institutions across the USA, led by investigators who have a research track record in cryoEM and/or cryoET. Day-to-day operation will be carried out by a part-time cryoEM/ET scientist and IT staff. Both in person and remote data collection protocols will be used for distant users. The Consortium will provide travel funds for the distant users. We request funding for a Direct Detection Device (DDD) operated in counting mode to meet the needs of all types of specimens. The host Institution will provide an administrative assistant to support user visits and various Consortium activities, and matching funds to purchase the DDD and adequate hard drives for short-term data storage. The participating PIs will be involved in formulating the specific policies for Consortium governance and developing training activities. Scheduling will be allocated evenly in the first year among participants with some flexibility based on specimen readiness. Allocation of microscope times and priorities will be assigned in the second year and beyond based on recommendations of an external microscope allocation committee appointed by all the PIs to avoid conflict of interest. Al PIs will meet quarterly on WebEx to discuss technical and administrative issues, and we will hold an annual user meeting. An annual review of all aspects of the Consortium operation and the data outcomes of the users will be conducted by an advisory committee to assure the highest productivity and usage of the proposed facility. The membership of the participating institutions will be dynamically reviewed by all the PIs with the inputs from the external advisory committee after the second year of our operation.

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.

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.

The ability of salivary peptides to inhibit diarrhea-causing bacteria from binding to the gut is newly discovered, and not well characterized. The innovative long-term goal is to realize the full potential of salivary components as both prophylactic and therapeutic agents against gastrointestinal and respiratory diseases. The objectives of this proposal are to define the efficacy of specific salivary components as pathogen inhibitors, and to determine the structure and mechanism by which one these peptides, Histatin-5, binds to pili that are external filaments on enterotoxigenic Escherichia coli (ETEC). This pilus/peptide interaction provides the dysfunction that inhibits bacterial binding, and knowing the mechanism of this dysfunction will lead to novel therapeutic approaches against ETEC and other pathogens. The central hypothesis is that enhanced utilization of the innate immune system to combat disease can be achieved through therapeutics developed from components of saliva. The rationale for this proposal is that completion of this research provides a path forward for utilizing salivary components to fight diarrheal diseases. In addition, determination of the mechanism of Histatin-5's action will permit creation of therapeutics with even greater efficacy. To achieve our goals, we will pursue the following two specific aims: 1. Define the capacity of selected salivary peptides to inhibit bacterial binding of enterotoxigenic Escherichia coli (ETEC) to target cells; 2. Determine the mechanism by which the salivary peptide, Histatin-5, inhibits bacterial binding via pili, an essential virulence factor of enterotoxigenic Escherichia coli (ETEC). These aims will be achieved using 1) bacterial adhesion studies on cell cultures and primary human intestinal cultures (?organoids?) and 2) structure determination at high resolution using electron cryomicroscopy and cryotomography (cryo-EM and cryo-ET). The proposed research is significant, because it will open a new avenue for development of therapeutics against diarrhea-causing bacteria. Unlike traditional antibiotics that broadly target enzymes involved in nucleic acid, protein, and cell wall synthesis, here, we explore a novel aspect of the host's innate immune system: the ability of salivary components to inhibit bacterial adherence to the host. The expected outcome of this research is a list of salivary components that can be exploited as therapeutics against diarrhea-causing pathogens, and the definition of the mechanism by which one component creates dysfunction of a critical virulence factor. The results will have an important positive impact immediately as a first step in defining and understanding the role of saliva in fighting GI disease, and long-term because they will lay the groundwork for bringing new researchers into this emerging field, and for development of saliva-based GI and respiratory therapeutics.