Shelley M. Payne - US grants

The effect of endotoxin and glucocorticoid antagonizing factor (GAF) on the hormonal regulation of two hepatic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and tyrosine aminotransferase (TAT), is to be examined. The objective is to explain how GAF blocks the glucocorticoid induction of PEPCK but not that of TAT and why it has no effect on the induction of either enzyme by cAMP. Mice and hepatoma cells in culture will serve as models for this research. Three remaining sites where blockage of induction could arise are to be explored. They are transcription, post-transcriptional modification and translational events. The rate of change in enzyme mRNA synthesis during induction under control conditions and when endotoxin or GAF is present is to be evaluated. With GAF, both PEPCK and TAT will be studied since induction of the former but not the latter is inhibited. Enzyme mRNA formation will be measured by hybridization using the dot-blot and Northern transfer procedures and the results compared to those obtained by Northern transfer when nuclear mRNA is hybridized. This visualizes the processing of the primary transcript into mRNA. This will estimate total hybridizable mRNA present in the hepatoma cells or mouse liver. The methods can be used only with PEPCK since no cDNA probe is available for TAT. Translatable mRNA measured during in vitro protein synthesis will determine whether the change in translatable mRNA occurs in the same proportion as the change in hybridizable mRNA. One is by activity measurement and the other by radioimmunoassay. If the two change by equal amounts and agree with the mRNA changes, a reasonable estimate of the site of action of endotoxin or its mediator can be achieved (namely, transcription). If more mRNA appears by hybridization than by in vitro translation, it might be due to interference with normal post-transcriptional modification to active mRNA. Two dimensional gels will show how many cellular proteins fail to be induced by glucocorticoids when endotoxin is present. Purified GAF and antiserum to it will be used to assess the biological significance of GAF in endotoxemia. This research should advance insight into Gram-negative infections.

Iron is an essential, but elusive element for most cells, and high affinity transport systems are usually required for its acquisition. The long range goals of this project are to characterize the regulation of the iron acquisition systems and other iron regulated genes of the enteric bacterial genus, Shigella. These studies will include (1) characterizing the mechanisms of regulation of enterobactin expression in Shigella flexneri, (2) measuring the expression of iron-regulated genes in an in vivo system, and (3) characterizing the positive and negative regulatory elements which affect expression of the gene for Congo red/heme binding (crb). Experimental approaches will utilize genetic and biochemical techniques. In vivo expression of the genes will be investigated using fusions to reporter genes, such as lux, whose expression can be readily monitored, and an in vivo labeling system has been developed to compare proteins synthesized by bacteria inside host cells to those observed in vitro. Characterization of mutants with altered gene regulation is also proposed. These studies will help define mechanisms of gene regulation both in vivo and in vitro.

Dr. Payne is a leading investigator in the area of siderophore- mediated iron uptake in bacteria. The problem addressed is how enteric bacteria utilize heme as a source of iron. This award is a National Science Foundation Faculty Award for Women Scientists and Engineers. The proposal fulfills the FAW objectives, which are: 1, to recognize outstanding and promising women scientists and engineers in academic careers of research and teaching and 2, to facilitate the further development of their careers.

DESCRIPTION (adapted from investigator's abstract): Iron acquisition is essential for pathogenic bacteria. In the vertebrate host, however, iron is not readily available, and this scarcity restricts the growth of pathogenic microorganisms and limits their ability to infect and invade. Bacteria have evolved multiple high affinity iron acquisition systems to cope with iron-limiting environments. In spite of considerable characterization of these systems in the laboratory, for most pathogens, the sources of iron used in the host and the relative importance of the different acquisition systems within different niches of the host are poorly understood. The objectives of this study are to determine mechanisms by which gram-negative enteric pathogens obtain iron in the host and to study the possible transmission of iron-acquisition genes and their role in evolution of emerging pathogens. This study will focus on heme transport systems in Shigella and selected Escherichia coli, pathogens which are responsible for considerable morbidity and mortality throughout the world. The first Specific Aim is to continue the characterization of the heme transport locus from Shigella dysenteriae type 1. The PI has found that a nearly identical locus is present in many pathogenic E. coli, including E. coli 0157:H7, and the phylogenetic distribution of this locus is suggestive of horizontal gene transfer. Therefore, the second Specific Aim is to determine whether these genes are mobile and how they may be spreading within the enteric pathogens. The data and strains developed in characterizing the heme transport systems of Shigella and E. coli, together with previous data on high affinity iron transport systems, have placed the PI in the position to now accomplish the third Specific Aim, to determine how Shigella and E. coli acquire iron in vivo. The fourth Specific Aim is to use genetic approaches to understand which genes Shigella specifically expresses in vivo, and to elucidate the role of these genes in Shigella infection. Results obtained from this study will provide basic information on genetics and regulation of potential virulence factors in an important group of pathogens and will provide data useful in design of potential vaccine strains.

DESCRIPTION (provided by applicant): Most bacterial pathogens have an absolute requirement for iron. The low availability of iron in most environments has led to the evolution of high affinity iron transport systems. Although iron acquisition systems have been identified in several gram negative organisms, the sources of iron used and the relative contribution of the different systems in their growth and survival in the host and in different niches in the external environment are not understood. Vibrio cholerae, the causative agent of cholera, is responsible for considerable morbidity and mortality worldwide. This organism is amenable to genetic manipulation, and several iron acquisition systems have already been identified. However, genetic analysis indicates that there are additional high-affinity iron transport systems in V. cholerae. The recent completion of its genome sequence will allow us to identify the remaining iron acquisition systems and to rigorously examine the roles of the systems in different environments and during exposure to different environmental stresses. Our first Specific Aim is to complete our characterization of V. cholerae heme transport and utilization. Our genetic data indicate that this pathogen expresses multiple heme transport systems, and we will define which genes are required for heme transport. We will also continue characterization of genes that function in the utilization of heme after it has been transported into the cell. Our second Specific Aim is to identify the transport systems used for the uptake of two exogenous siderophores used by V. cholerae, enterobactin and schizokinen. The third Specific Aim is to use our mutant collection, together with other reagents, to determine which transport systems are used during specific environmental conditions, and during growth in the vertebrate host.

Most bacterial pathogens have an absolute requirement for iron. The low availability of iron in most environments has led to the evolution of high affinity iron transport systems. Although a variety of iron transport systems have been identified in Shigella and pathogenic E. coli, the sources of iron used by the pathogens when growing within the host and the specific iron transport systems involved in growth and survival in vivo are not known. Shigella species, the causative agents of dysentery, are closely related to E. coli and share many of the same iron transport systems. However, differences have been noted among this group of pathogens and those differences may relate to differences in sources of iron at various sites within the host or in the environment. Because Shigella spp. have the ability to invade host cells and grow with the cytosol, there may be specific iron transport systems associated with iron acquisition in the intracellular environment. Our first specific aim is to complete the characterization of the Shigella heme transport systems. Many shigellae and pathogenic E. coli, including O157:H7, have specific receptors for heme. Although receptors and other proteins involved in transporting heme across the bacterial cell wall have been identified, other steps in heme acquisition and its use as an iron source are not understood. The second aim is to use genetic and genomic approaches to identify the additional iron uptake systems in these pathogens. We will then apply these data and use the mutants created in these studies to help understand the role of the iron transport systems in growth and survival in vivo and in the environment. Thus our third specific aim is to determine which systems are used under specific environmental conditions and during intracellular growth. Our fourth specific aim is to assess expression of iron transport and other genes during intracellular growth and during infection by using microarray technology.

DESCRIPTION (provided by applicant): The American Society for Microbiology (ASM) requests $24,800 in 2003, $23,700 in 2004, and 27,800 in 2005 for three Summer Institutes in Preparation for Careers in Microbiology (SI), a five-day program for 24 graduate students and post-doctoral fellows in the microbiological sciences. The overall objective of the five-day Institute is to provide participants with intensive training in five major areas: career development; grant preparation, communications, teaching, and ethics. At the conclusion of the Institute, participants will be able to: 1. Prepare a well-organized research proposal that clearly articulates the specific aims of the project and the plan to address the problem, 2. Deliver a clear and concise oral presentation of their research project to the scientific community, 3. Examine a number of career opportunities and pathways available to microbiologists-in-training, 4. Recognize and effectively meet the educational needs of undergraduate microbiology students in a variety of different settings, and 5. Describe the ethical implications regarding scientific research as encountered by the scientific community. The ASM Education Board's Committee on Graduate Education sponsors the Institute. In August 2001 and 2002, the ASM offered two Institutes at the University of Wisconsin, Madison. The Institute is the first and only program of its kind providing students with hands-on, practical training in grant preparation and review, scientific communications and teaching, career development, and ethics. The dates of the Institutes are: August 2-6, 2003, August 7-11, 2004, August 6-10, 2005.

The American Society for Microbiology annual Summer Institute for Graduate Students and Postdoctoral Scholars in Preparation of Careers in Microbiology was envisioned in 2000. The first Institute was offered in 2001 at the University of Wisconsin with 19 participants. In 2001 and 2002 respectively, the ASM sought funds from NIH to support the Institute (1R13 AI52120-01 and 1R13 AI055488-01-03). In this proposal (cycle 3), the ASM requests $141,600 for one summer institute per year for five years beginning in 2006 and ending in 2010. Students have limited and inconsistent training in grant preparations, communications, teaching and mentoring, career planning, and ethics even though they have numerous opportunities to conduct scientific research during their graduate training. To address these shortcomings, the ASM Committee on Graduate and Postdoctoral Education proposes an annual Institute. The goal of the Institute is to provide up to 30 graduate students and postdoctoral scientists in the microbiological sciences with intensive and closely guided instruction and mentoring in five key areas important for selecting and preparing for a career as a microbiologist. The specific aims of the Institute are to help participants: 1. Prepare a well-organized research proposal that articulates clearly the specific aims of their project and a plan to address the problem. 2. Deliver a clear and concise presentation of their research project to the scientific community. 3. Examine a number of career opportunities and pathways available to microbiologists-in-training and plan their own pathway. 4. Incorporate effective teaching and mentoring strategies to meet the educational needs of microbiology students in a variety of different settings. 5. Recognize ethical implications regarding scientific research as encountered by the scientific community. The public will benefit from the Institute by empowering graduate students and postdoctoral scientists with skills necessary to become excellent research scientists, teachers and mentors, and thereby, shortening the time from education and training to productive employment in the microbiological sciences.

Vibrio cholerae, a major human pathogen responsible for both endemic and epidemic cholera, has an absolute requirement for iron. Because V. cholerae must be able to obtain iron in a variety of different environments in and outside of host organisms, it has evolved multiple iron acquisition systems and can use iron from a variety of sources. These iron transport systems include the endogenous siderophore vibriobactin, receptors for exogenous siderophores and for heme, and ferric and ferrous iron uptake systems. Iron transport systems are tightly regulated to avoid either iron starvation or iron toxicity, and there is both transcriptional and post-transcriptional regulation of iron transport and iron metabolism. Regulation is mediated by iron and the global regulator Fur and by the small RNA RyhB. There is evidence for additional levels of regulation. We will characterize the V. cholerae iron transport systems, the regulation of their expression, and their role in survival of V. cholerae in the environment and in the host. These systems will be characterized using molecular biology, genetics, and biochemical techniques. As we complete the characterization of all the iron transport systems that this pathogen uses, we can determine their roles in pathogenesis and further dissect the complex regulatory network by which environmental signals regulate the expression V. cholerae iron transport genes. The specific aims of this grant are: 1) Identify the complete complement of V. cholerae iron transport systems. 2) Characterize the ferrous iron transporter Feo and the novel VciB iron acquisition system. These systems are of particular interest since they are both expressed by bacteria during infection of the host and are likely to be major iron uptake systems in the microaerobic environment of the intestine. 3) Determine the mechanism of regulation of V. cholerae iron transport genes in response to environmental signals. The systems respond to multiple, different signals to optimize iron acquisition and growth of V. cholerae in the variety of environments in which it is found, and we propose to define these regulatory networks. It is important to understand bacterial iron transport systems and their regulation, because the ability of pathogens to compete with their host for this essential element is a critical component of the host-pathogen interaction. Defining and characterizing the V. cholerae iron transport systems at the molecular level and understanding their expression in response to different environmental conditions will provide the basis for developing strategies to control this major human pathogen. The results of these studies are likely to be broadly applicable to human pathogenic bacteria, since the majority of them must acquire iron during infection.

DESCRIPTION (provided by applicant): Vibrio species are predominantly aquatic bacteria. While many of them are non-pathogenic, some vibrios are associated with severe disease in humans. Vibrio cholerae, in particular, has a long history of causing epidemic and pandemic cholera. Cholera results from colonization of the small intestine by V. cholerae, where it produces a potent toxin that results in massive water loss and electrolyte imbalance. In nature, V. cholerae may be found free-living, but its survival appears to be favored by specific associations with other organisms. Some of the genes required for V. cholerae to form these associations and persist in its aquatic ecosystem are also involved in promoting disease in humans. In vitro studies have suggested that protozoans may both use V. cholerae as a food source and promote V. cholerae survival, but the nature of these interactions is not well understood. Our studies will focus on the relationship between V. cholerae and the model protozoan amoeba Dictyostelium discoideum, and our laboratories have combined expertise in these two organisms. Our hypothesis is that V. cholerae is subject to predation by amoebae in the environment and that production of virulence factors such as cholera toxin helps protect V. cholerae from predation and enhances its survival. Further, we propose that D. discoideum can be used as a model for interaction of V. cholerae with amoebae. We will use genetic, biochemical, and cellular biology approaches to dissect the relationship between D. discoideum and V. cholerae. Analysis of mutants and genetic screens will be used to identify the V. cholerae genes required for survival and persistence in the presence of D. discoideum, and we will use cellular biology techniques to begin to elucidate the interactions between D. discoideum and V. cholerae. The specific aims are to (i) identify V. cholerae genes involved in resistance to predation by amoebae, (ii) determine the mechanism of survival of V. cholerae within D. discoideum, and (iii) determine whether V. cholerae grown in the presence of the amoeba are infectious or hypervirulent. Understanding this predator/prey relationship will not only provide basic information about V. cholerae in the environment, but also may provide clues to methods for better controlling the environmental sources of V. cholerae and thereby help prevent epidemics of cholera. PUBLIC HEALTH RELEVANCE: Vibrio cholerae is a major cause of morbidity and mortality worldwide. This bacterium has a reservoir in nature and periodically re-emerges to cause epidemics and pandemics of cholera. In order to persist in the wild, V. cholerae must be able to resist predation by amoebae. The proposed studies to determine the mechanisms of resistance to predation will lead to a better understanding of the ecology of V. cholerae. This may allow the development of strategies to control the source of epidemic V. cholerae.

DESCRIPTION (provided by applicant): Iron participates in a wide variety of cellular reactions and is an essential element for nearly all organisms. Iron acquisition is problematic for pathogenic bacteria, which must compete with the host for limited amounts of available iron, and bacteria typically express high affinity transport systems that are specific for their ferric or ferous iron ligands. Feo is the major ferrous iron transport system in prokaryotic organisms. It is widely distributed in eubacteria and in the Archaea, suggesting that it is a very ancient class of iron transporter. Further, the FeoB protein has GTPase and GDI domains similar to eukaryotic G proteins, and Feo may represent a primitive ancestor of modern G proteins. Despite its ubiquitous nature and potential role in bacterial pathogenesis, little is known about its structure, mechanism of transport, and biological role. Thus, it is critical to characterize this protein and its role in ferrous iron transport. These studies will be done in the pathogen Vibrio cholerae, a major cause of human morbidity and mortality. V. cholerae is an ideal model for these studies as we have the reagents and genetic tools to answer basic questions about Feo. Further, we can extend these studies to include questions about the role of Feo in host colonization and disease, which will lay the groundwork for developing new methods of blocking infection by V. cholerae. Our first specific aim is to use genetic and biochemical approaches to characterize Feo structure and its mechanism of transport. These studies will help define the essential protein components of the Feo transporter and the critical regions and amino acids within each protein. Based on these results, we will be able to test models for Feo structure and function. Our second specific aim is to characterize the accessory ferrous iron transport protein, VciB. This may provide insight into how Feo obtains its ferrous iron ligand in the periplasm. Third, we will define the mechanism of regulation of expression of the feo operon and determine its pattern of expression when the bacteria are within the host. These data will provide new information about how pathogenic bacteria coordinate the expression of their iron transport genes to allow optimal utilization of available iron sources and will also help define the environmental signals the bacteria encounter in the host.

DESCRIPTION (provided by applicant): The ASM Kadner Institute for Graduate Students and Postdoctoral Scientists in Preparation for Careers in Microbiology is a five-year initiative to (1) empower graduate students and postdoctoral scientists with skills necessary to become excellent research scientists and teachers and (2) provide information about diverse career opportunities and the resources and skills needed to pursue these opportunities. The program's overall goal is to provide up to 24 graduate students and postdoctoral scientists in the microbiological sciences annually with intensive and closely guided instruction and mentoring in five areas important for selecting and preparing for a career as a microbiologist. Recognizing that students have limited and inconsistent training in grant preparation, communications, teaching and mentoring, career planning, and ethics, the ASM Committee on Graduate and Postdoctoral Education founded the ASM Kadner Institute for Graduate Students and Postdoctoral Scientists in Preparation for Careers in Microbiology to focus on these career aspects. The specific aims of the Institute are to help participants: 1. Prepare a well-organized research proposal that articulates clearly the specific aims of their project and a plan to address the problem. 2. Deliver a clear and concise oral presentation of their research project to the scientific community. 3. Examine a number of career opportunities and pathways available to microbiologists-in-training and plan their own pathway. 4. Incorporate effective teaching and mentoring strategies to meet the educational needs of microbiology students in a variety of different settings. 5. Recognize ethical implications regarding scientific research as encountered by the scientific community. Kadner participants who aspire to become career microbiologists and scientists represent a broad spectrum of subdisciplines, including microbial structure and physiology, microbial genetics, clinical microbiology, medical microbiology, applied and environmental microbiology, virology, immunology, and public health. Microbiology research advances the sciences as a vehicle for understanding basic life processes and promoting knowledge gained for improved public health as well as economic and environmental well-being.

DESCRIPTION (provided by applicant): Shigella spp. cause disease by invading and multiplying within human colonic epithelial cells. Successful infection requires appropriate timing of virulence gene expression and the efficient acquisition of nutrients within the host; however, the sensing and acquisition of carbon sources and other nutrients by Shigella during infection is poorly understood. This application focuses on the role of carbon metabolism pathways in the virulence of Shigella flexneri. Our data suggest that S. flexneri uses information about the available carbon sources to determine the attachment and initial steps in invasion of host epithelial cells. Once inside the host cells, the bacteria must adjust their metabolism to take advantage of the different carbon sources available in the intracellular environment. The carbon and other nutrient sources present in the host cell cytoplasm and the pathways used by intracellular Shigella to obtain these sources are largely unknown. The first specific aim is to define the carbon and nutrient sources available to S. flexneri in the intracellular environment of the host. Using metabolomics, we can assess the nutrients present in the cytoplasm of uninfected cells and follow changes in the metabolome during the course of infection. Once we have identified the carbon sources that are present, our second aim is to determine the pathways the bacteria use to assimilate these nutrients. We will use proteomic and transcriptomic analysis of the intracellular bacteria to define the carbon metabolism genes expressed by intracellular S. flexneri. These will be complemented by genetic analysis to determine which of the expressed pathways are required for, or contribute to, intracellular growth. We will construct mutants that are defective in one or more of the carbon metabolism genes expressed intracellularly and test their ability to invade and cause plaques. The third aim is to determine the mechanism by which a regulator of central carbon metabolism, Cra, is linked to S. flexneri invasion and cell-to-cell spread. A mutation in cra markedly increased S. flexneri adherence to epithelial cells but limited cell-to-cell spread, pointing to a link between S. flexnei carbon metabolism and virulence. We will use genetic and biochemical characterization of the cra mutant to determine the mechanism of Cra's effect on virulence. Taken together, the data generated in this study will provide essential information on carbon metabolism and its role in Shigella virulence, and will contribute significantly to our broader understanding of the physiology and metabolism of S. flexneri in the host cell environment. Such data are applicable to the design of therapeutics targeting intracellular S. flexneri, as well as to the design of vaccines based on antigens expressed when the bacteria are growing within host cells.

Zika virus (ZIKV), a flavivirus, has recently spread to the Americas and is currently associated with a major pandemic in human populations in South, Central, and North America. ZIKV infections in pregnant women has been associated with severe birth defects, notably microcephaly; thus, it is critical to understand the epidemiology and spread of this viral infection. ZIKV, which can be spread by mosquitos, was first isolated in Africa from non-human primates, suggesting that these animals may serve as a reservoir for the virus. However, much less is known about the reservoirs or spread of the virus in the Americas. Countries where outbreaks are occurring, including Brazil, Columbia, Ecuador and Mexico, are home to a number of species of Neotropical primates, and the human and non-human primate populations occupy overlapping areas. This suggests that there is the potential for these animals to be a reservoir for maintenance and spread of the virus in human populations. Our first aim is to determine whether Neotropical primates are a potential reservoir for ZIKV. We have assembled an expert team of primatologists, virologists, enteric infectious disease researchers, and epidemiologist who will work collaboratively to answer this question. We will sample 12 species of Neotropical primates at 8 field sites in 4 countries. We will determine the presence of active infection or carriage of ZIKV by quantitative RT-PCR, and we will test for antibodies against the virus as an indicator of prior exposure. Additionally we will obtain blood samples from human volunteers in proximal geographic areas to determine if human infection positively correlates with ZIKV presence in Neotropical primates. Molecular phylogenetic analysis of ZIKV gene sequences from human and non-human primates will help determine the epidemiology and spread of the virus in these populations. Because obtaining blood samples from the animals is a difficult and invasive procedure, our second aim is to develop a rapid, noninvasive screen for the virus. Some flaviviruses are shed in the feces of primates, therefore we will analyze fecal samples from primates that have ZIKV-positive blood cultures or serology to determine whether the virus is present. Urine, which has been used to detect ZIKV, will be an alternative method. We will modify the protocol to optimize ZIKV detection from this non-invasive sampling method. The results of this study will provide critical information on the role of Neotropical primates in the spread of Zika in the Americas.

Cholera is a disease of pandemic proportions, and its health, economic, and political impacts cannot be overstated. V. cholerae is primarily associated with aquatic habitats; however, under appropriate conditions, V. cholerae can infect the human host and grow to very high densities within the human intestine. The transition to the human host represents a major environmental shift for V. cholerae. V. cholerae must sense and respond to this new environment in order to survive, colonize, and produce disease. Regulation of colonization and virulence factors in response to environmental cues is mediated through several regulatory factors, including the highly conserved RNA- binding global regulatory protein CsrA. CsrA plays a role in quorum sensing, biofilm formation, virulence gene expression, carbon metabolism, and environmental fitness, both in V. cholerae and in other bacterial species. We recently demonstrated that V. cholerae CsrA regulates the master virulence gene regulator ToxR in response to particular amino acids in the medium, consistent with a role for CsrA in incorporating environmental cues into the complex regulation of virulence gene expression. CsrA is essential for the growth of V. cholerae, making it difficult to study in the laboratory; however, we have isolated a csrA point mutant strain that is viable, but defective for ToxR regulation. Using this mutant, we showed that csrA is critical for virulence in the infant mouse model of V. cholerae infection. Despite its importance as regulator of virulence and other processes, there is little or no information about what genes are directly targeted by CsrA in V. cholerae. The goal of this proposal is to define the direct targets of CsrA regulation. Because csrA is essential, and because CsrA is a posttranscriptional regulator, traditional genetic approaches are limited. We have developed a novel technique involving in vivo CsrA-RNA co-immunoprecipitation followed by deep sequencing of the immunoprecipitated RNA species to identify the direct targets of CsrA regulation. This information, combined with RNA-Seq transcriptome analysis, will be critical for understanding the larger network of genes controlled by CsrA in this pathogen. We also propose to solve the crystal structure of V. cholerae CsrA bound to target RNA. There are currently no published structural analyses of CsrA among the vibrionaceae. Understanding the structural features of V. cholerae CsrA will greatly increase our knowledge of how CsrA post-transcriptionally regulates its RNA targets.

The diarrheal disease cholera poses enormous health, economic, and political burdens worldwide. The causative agent of cholera, Vibrio cholerae, is endemic in aquatic habitats, but it is capable of infecting the human small intestine, causing a massive, life-threatening diarrheal response. To colonize the host and cause disease, V. cholerae must acquire essential nutrients, including iron, in the host environment. The predominant form of iron present in the small intestine is ferrous iron. Thus, it is critical to understand the mechanisms of ferrous iron uptake in V. cholerae. The major ferrous iron transporter in V. cholerae is Feo. The Feo system is widely distributed among all bacterial species, and has important functions in the virulence of several pathogenic species; nevertheless, very little is known about its structure and mechanism of transport. In V. cholerae, the Feo transporter is composed of three proteins, FeoA, FeoB, and FeoC. The membrane-embedded C-terminal domain of FeoB is likely to form the pore for iron transport, and, interestingly, its N-terminal domain has homology to small eukaryotic G proteins, suggesting a novel mechanism of transport. The roles of FeoA and FeoC are unknown. We recently demonstrated that the three Feo proteins associate to form a higher order complex in vivo. This represents the first structural analysis of the mature, membrane- embedded, active Feo complex in vivo. The goal of this proposal is to determine the overall structure of this large complex in order to build and test models for the mechanism of iron transport. In our first specific aim, we will determine the mass and stoichiometry of the native Feo complex. This will lay the groundwork for a structural model of the active Feo transporter. In aim 2, we will refine this model by delineating the sites of interaction within and between the members of the complex. We will then test the functional significance of these interactions for complex formation and iron transport activity. In aim 3, we will determine the source of energy for transport through Feo. These studies will significantly advance our knowledge of the structure and function of this important and unique iron transporter. Significantly, all our studies will be carried out using active, membrane-associated Feo complexes in vivo, giving our results an undeniable relevance over the in vitro studies that currently dominate the Feo field. As our previous work shows, V. cholerae is an ideal model organism for the study of Feo. We have already assembled most of the strains and reagents needed, and we, and our collaborators, have the expertise required to carry out the proposed experiments.

ABSTRACT The enteric, intracellular human pathogen Shigella causes hundreds of millions of cases of the diarrheal disease shigellosis per year worldwide, which results in as many as one million deaths. Currently, there is no approved vaccine for the prevention of shigellosis, and increasing drug resistance complicates treatment of the disease. The bacteria are acquired by ingestion of contaminated food or water, and upon reaching the colon, Shigella invade the colonic epithelial cells, replicate intracellularly, spread to adjacent cells, and provoke an intense inflammatory response. While much is known regarding the mechanism of pathogenesis, there are still gaps in our knowledge. Progress in closing these gaps has been hampered by the lack of a small animal model that accurately mimics the human disease. Cell culture has been used to study aspects of invasion and replication in the cell, but the use of transformed cells in culture does not provide the same environment as the normal human epithelium. A potential solution to this problem is the development of enteroids derived from human intestinal stem cells as a model for Shigella flexneri infection. In this study, we will use human intestinal enteroid monolayers derived from colon tissue to replicate aspects of the bacterial-host interaction that currently cannot be emulated in cell culture. We will use enteroids to test the current paradigms of S. flexneri invasion, intracellular replication, and spread, in order to determine how S. flexneri responds to its normal host environment. In the first Aim, we will establish the basic parameters for colonoid infection by S. flexneri and test the hypothesis that M-cells are required in the invasion process in human intestine. In Aim 2 we will focus on the dynamics and kinetics of intercellular spread of the bacteria in the enteroid monolayers. One aspect of Shigella infection that is poorly understood is the basis for its tropism for colonic epithelium; in Aim 3 we will use human intestinal enteroids derived from duodenal, jejunal, ileal and colonic tissue to determine whether there is tissue specificity for S. flexneri invasion. This will be critical in designing future experiments to determine the host receptors for S. flexneri and the environmental factors that influence S. flexneri invasion efficiency. Completion of this project will not only allow us to examine basic assumptions about Shigella pathogenesis but will also result in the development of a model system for future studies of the interaction between Shigella and the human epithelium.

Shigella species are invasive human pathogens that cause bacillary dysentery, or shigellosis, a potentially fatal diarrheal disease. The global burden of shigellosis is estimated at more than 200 million cases per year. There is currently no effective vaccine against Shigella, and drug resistance is widespread and on the rise; thus, there is a critical need to identify novel Shigella targets for immunization and new antibiotics. One promising approach is to target metabolic processes that are important for pathogenesis, but not for survival of Shigella in the host environment. Inhibiting such pathways would be less likely to select for resistant Shigella or to disrupt the normal gut microbiota. Our work has shown that Shigella flexneri uses mixed acid fermentation to break down glycolysis intermediates during growth within host cells. This process is critical for S. flexneri pathogenesis, but is not required for growth of the bacteria, either inside or outside host cells. Mixed acid fermentation leads to the production of formate, which is excreted by S. flexneri into the host cell cytosol. Formate induces expression of S. flexneri virulence genes that are required for cell-to-cell spread. Our hypothesis is that, as the bacteria multiply, formate levels reach a threshold that can be sensed by S. flexneri as a signal to begin the process of spreading to neighboring cells, thus linking cell density with the need to move deeper into the intestinal epithelium to find new resources. The goal of this study is to investigate how formate is sensed by the bacteria, and how this signal leads to changes in gene expression that promote cell-to-cell spread and evasion of host immunity. We propose to identify key players in the formate sensing pathway in order to derive a model for how the formate signal is relayed from the cell surface to its target genes. We will also determine the downstream effects of formate signaling on both S. flexneri and host cell gene expression. Historically, Shigella studies have been hampered by the lack of a physiologically relevant host-pathogen model system. We have recently demonstrated that critical aspects of Shigella pathogenesis are faithfully reproduced in human intestinal enteroids (HIEs), ?mini-intestines? derived from human intestinal biopsies. We propose to use HIEs to determine both the Shigella and host cell transcriptomes in response to formate. This will allow investigation of gene expression and metabolism during the course of a Shigella infection in fully differentiated, non-transformed, native human tissue. We have assembled the necessary strains and reagents, and we have the expertise to carry out these experiments, which we predict will lead to vital new information in the search for novel shigellosis treatment and prevention strategies.