Shaw Akula, Ph.D.: HHV-8 envelope glycoprotein gB as a potential target for anti-viral agents
The goal of the COBRE project is to conduct a thorough structural and functional analyses of HHV-8 glycoprotein gB molecule and determine the feasibility of developing novel strategies to block HHV-8 gB interactions with host cell receptor and thus block HHV-8 infectivity. We hypothesis that small molecules binding to HHV-8 envelope gB have the potential for use as anti-HHV-8 agents. Towards this, we successfully produced the soluble form of gB in insect cell culture system, and purified it. We used the phage display library to identify small peptides that binds soluble gB protein. Panning experiments yielded 3 phage-expressed small peptides binding specifically to gB. Binding of phage-expressed small peptides to gB was competitively blocked by addition of increasing quantities of soluble gB, further confirming the specificity of the peptide-gB interactions. Recently, we synthesized these 3 phage-expressed small peptides and tested their ability to inhibit HHV-8 infection of target cells. Preliminary evidence suggests that one of the peptides significantly lowered GFP-HHV-8 infection of human foreskin fibroblast (HFF) cells. These results need to be confirmed by other assays. As we had standardized the protocol for the use of phage-display library in our lab, we also used this powerful tool to identify small peptides that bound to another unique, virion envelope associated and an immunogenic glycoprotein called gpK8.1A. Panning experiments yielded several phage-expressed peptides binding specifically to gpK8.1A and 83% of them sharing a common motif; none of them had sequence similarity with what was identified for gB. These results further confirmed the specificity of the phage-display library as a tool to identify the small peptides in this study. In addition, for the studies in the future, we cloned gB in a prokaryotic expression vector with an aim to express gB with a HIS-tag at the amino terminal, purify, and use it for structural analyses. Future studies will be directed towards testing and comparing the ability of the synthetic peptides corresponding to the phage-expressed small peptides identified using both gB and gpK8.1A to inhibit, the function of the respective proteins and virus infection as a whole.
Geraldine Arrode, Ph.D.: Characterization of immune T cells induced by a unique HIV DNA vaccine
The need for an effective prophylactic vaccine against HIV-1 is imperative. In addition to broadly neutralizing humoral immunity, a successful HIV-1 vaccine must likely induce strong, durable T cell immunity. The DNA based vaccine strategy has proven to be effective in mice and non human primates models. However, we lack a detailed understanding of the nature of cellular immunity induced by such vaccination. In addition, the current gamma interferon (IFN-G) ELISPOT assay mostly used to detect cellular immunogenicity in T cell based vaccine may not measure true components of protective immunity. We have developed a DNA vaccine that expresses six proteins of HIV. Immunization of macaques with this DNA resulted in induction of small numbers of virus-specific IFN-G producing CD8+ T cells. These responses were nevertheless protective against HIV-like disease. We have used this DNA to immunize mice in order to dissect the identity of cells participating in the CMI response against the virus. By developing multicolor flow cytometry assays to evaluate the cellular immune response, we identified HIV specific CD8+ T cells with unique proportions (strong proliferative capacity, IFN-G secretion only in 20% of the responsive T cells, granzyme B production in most of the cells, and cells with long-lived memory phenotypes). This broad response lasted more than half of the lifespan of the immunized mice. The experiments proposed herein will use tetramer technology to further characterize the population of non IFN-G producing CD8+ T cell induced by the vaccine. We hypothesize that these cells are memory like T cells without immediate effector functions. In addition, we will examine whether the promoter used in the DNA based HIV vaccine (SIV 5'LTR promoter versus CMV promoter) may govern this type of T cell responses after immunization. The information gained will help optimize the design of future HIV-1 DNA based vaccines.
Cory Berkland, Ph.D.: Biodegradable Nanoparticles for AIDS Gene Therapy
Human immunodeficiency virus (HIV) infection is centered around infection and replication of the virus in activated CD4+ T cells, but the rate of the replication is modified by host responses to the infection, some of which cause inhibition of virus replication and others, enhancement of replication. Traditional intervention strategies focus on reducing viral replication with antiretroviral drugs, but the infection may also be impeded by curtailing the exaggerated production of the host factors that promote virus replication. Published reports by the project mentor (Buch) have indicated that antisense DNA of IL-4 (AS IL-4) inhibits virus replication in vitro and in vivo. However, delivery of this therapeutic gene remains problematic since most gene delivery vehicles (i.e. viral and liposomal) are immunogenic or even toxic. Alternatively, poly(DL-lactic-co-glycolic acid) (PLGA) particles are biodegradable and exhibit low toxicity and immunogenicity, depending on the physicochemical properties of the particle. Our lab possesses the unique ability to control the size and surface chemistry of PLGA particles with high precision. Therefore, we propose to synthesize a library of PLGA particles containing AS IL-4 that exhibit discrete size and surface chemistry and screen for effective gene delivery by applying in vitro and in vivo performance criteria. We will use the mouse model of HIV infection in which the human immune response becomes reconstituted in NOD/SCID mice that are repopulated with human CD4+ T cells and monocytes and then inoculated with HIV. Specific Aim 1: Formulate a library of PLGA AS IL-4 delivery vectors designed for low immunogenicity and low toxicity. Specific Aim 2: Identify vectors that efficiently mitigate IL-4 production in human CD4+T cells and macrophages. Specific Aim 3: Identify vectors selected from aim 2 that reduce HIV replication in the human cells transplanted into the mice. We will create a comprehensive library of PLGA gene delivery vectors leveraging known strategies to minimize particle toxicity and immunogenicity. We also expect the proposed screening process will facilitate identification of gene delivery vectors that can reduce IL-4 production, thus, mitigating viral replication in the CD4+ T cell and macrophage cultures. The proposed research is significant because a non-immunogenic gene delivery system capable of abrogating HIV replication could provide useful adjunct therapy and may even be preferred to currently available antiretroviral drugs, which are expensive, poorly tolerated and often toxic. Finally, the proposed research will augment current efforts to effectively implement gene therapy as a treatment for AIDS, thus, directly addressing the goals of the NIH and COBRE for improving human health through a multidisciplinary effort to genetically treat infectious disease.
Indranil Biswas, Ph.D.: Identification and Regulation of Stress Response Genes in Group A Streptococcus
Streptococcus pyogenes, the group A streptococcus (GAS), is an important and common human pathogen which causes a wide variety of diseases including relatively mild, self-limiting infections of the throat and skin as well as severe invasive necrotizing fasciitis and streptococcal toxic shock syndrome. Bacterial pathogens encounter a number of stresses that are either due to attack by the immune system or result from bacterial entry into tissue sites that inhibit growth. Exposure of bacteria to these adverse environments can induce two kinds of stress responses: general stress response and specific stress response. The general stress response provides cross-protection against diverse environments by inducing a wide variety of genes regardless of initial stimulus and is controlled by a single or few master regulators. Specific stress responses facilitate a cell's survival under a particular stress condition by inducing a subset of genes appropriate for that physiological condition and are regulated by unique groups of repressors. Very little is known about stress response genes and their regulation in GAS. Unlike other pathogens, GAS does not encode an alternate sigma factor that controls general stress response genes. In addition, general stress response genes have not been identified in GAS. However, Clp family of proteases that are involved in thermal and oxidative stress responses in other bacteria, are present in GAS. In this work, we propose to study stress response regulation in GAS. Specifically, we want to identify general stress response genes and study the role of two Clp proteases in general stress responses, with the following Specific Aims. In Aim 1, we will identify the genes that are required for survival and are induced by thermal stress, oxidative stress, and osmotic stress conditions. We will use transposon mutagenesis, DNA microarrays and proteomic approaches to identify differentially expressed genes/proteins under various stress inducing conditions. In Aim 2, we will study the role of ClpE and ClpP proteases in the regulation of stress responses and virulence expression. GAS encodes five Clp proteins that have close homologs in other bacteria. Among them, ClpE is uniquely present in gram-positive bacteria. The role and regulation of ClpE in general stress response and in pathogenesis has not been investigated in streptococci including GAS. In contrast, ClpP protease is ubiquitously present in all bacteria and is involved in general stress responses; however, its role in pathogenesis and in regulation varies significantly among bacteria. We will study the role of ClpE and ClpP in stress survival and identify various proteins and virulence factors that are regulated by ClpE and ClpP by proteomic analysis. We will use Pclp-reporter fusions and transposon mutagenesis to identify genes required for PclpE and PclpP expression (Aim 2C). In summary, this study will identify groups of stress response genes and virulence genes in this important human pathogen. In addition, the results can be extended to other pathogens like S. pneumoniae and may possibly identify new and potentially broad-spectrum drug targets.
Shilpa Buch, Ph.D.: Gene Therapy of TB Granulomas in Mice
This is an application to use A SHIV/cynomologous macaque model of HIV-TB disease to explore the feasibility of using therapy with antisense DNA of host response genes. The therapy is aimed at reducing virus replication in macrophages in lung tissue, and inhibiting chemotaxis of monocytes to the lungs. We will target monocytes because when these cells differentiate into macrophages in the lungs, they not only contribute to the inflammatory process but also provide host cells for replication of the virus. We will use cynomolgus macaques and a macrophage-tropic strain of SHIV, recently adapted to this macaque species in our lab, for these studies. The research plan calls for a sequential series of studies establishing first, creation of conditions of infection in macaques that will favor predictable reproduction of the synergistic interaction between the SHIV & Mycobacterium antigen, purified protein derivative (PPD) before extending the study to SHIV/M.TB model. Second, we will use liposome technology to deliver the antisense DNA, and use the mouse system to refine methods of formulation, delivery, targeting of the liposomes to lungs, and later, to evaluate the duration of expression of the genes in vivo. We will next implement this technology in macaques infected with the SHIV and co-injected with M.bovis PPD to further refine the therapeutic algorithm. Having done this, we will transfer the study to the University of Pittsburgh, where, in collaboration with us, Dr JoAnne Flynn at the Univ. of Pittsburgh will supervise the implementation of the protocol for establishing the dual infections with the SHIV and M.TB in the animal BL3 suite at her institution. Collaboratively, she will then implement the gene therapy protocol and evaluate efficacy of the treatment strategy aimed at interrupting the synergistic interaction between the SHIV and the Mycobacterium.
Shilpa Buch, Ph.D.: Immunogenicity of HIV DNA vaccines and Cytokines in Mice
Control of replication of HIV replication in infected persons is associated with development of memory cytotoxic (CTL) and helper T (HTL) lymphocytes and neutralizing antibodies against the virus. Currently, there is no safe vaccine that is known to induce these responses. DNA vaccines using the CMV promoter to drive expression of selected viral genes induce CTL responses but require boosting regimens with vectors expressing HIV proteins to induce neutralizing antibodies. We explored an alternative strategy to develop a DNA vaccine and used the genome of SHIV, a chimera of SIV and HIV, as starting material. We retained the 5'LTR housing the SIV promoter to regulate viral gene expression, the regulatory genes tat and rev to regulate transcription, and the vpu and nef to regulate viral protein production, but deleted the 3'LTR to minimize the ability of the DNA to integrate into host DNA, and the reverse transcriptase coding sequence to abolish infectivity of assembled virions. Three macaques injected three times with this DNA developed both arms of the immune response and resisted replication of a highly pathogenic challenge virus. In this grant application, we will use BALB/c inbred mice to determine whether the immune responses induced by the vaccine could be enhanced further by increasing the vaccine dosage and the frequency of administration, and determine whether supplemental use of cytokines as adjuvants would further enhance the immunity. The experimental strategy is divided into 4 Specific Aims and involves intramuscular injections of the DNA followed by measurement of CTL, HTL, and neutralizing antibody responses early and several months later; correlation of these responses with the numbers and duration of accumulation of phenotypes of mononuclear cells (dendritic cells, T lymphocytes, B-lymphocytes, macrophages) recruited to the injection sites; the identity of cells that become transfected with the DNA at the intramuscular sites of injection, and the duration of expression of viral proteins in these cells. These data will provide guidelines for design of optimal immunization protocols aimed at induction of long-term immunity against HIV.
Kyeong-Ok Chang, D.V.M., Ph.D.: Replication of Noroviruses in Cell Culture
Although noroviruses (NIAID category B priority pathogen) are a major public health concern with their ability to cause large outbreaks of acute gastroenteritis, the lack of a cell culture system greatly hinders research on preventive measures, such as the development of vaccines and anti-viral drugs. The long-term goal of this application is to isolate the fasitidious viruses using the recently developed norovirus replicon harboring cells, and identification of bile acids as an active growth factor for the growth of porcine enteric calicivirus (PEC), a norovirus, in cell culture. We have developed a novel cell-based norovirus replication system (Norwalk virus [NV] replicon harboring cells), and found that type I interferon (IFN) significantly inhibited the expression of NV proteins and genome in the replicon harboring cells. Furthermore, we demonstrated that bile acids mediated the replication of PEC by inhibiting IFN pathway in cell culture. Using the NV replicon harboring cells and the bile acid-mediated PEC replication, we plan to study the replication of noroviruses in cell culture. Our specific aims are: (1) to search for norovirus strains that could replicate efficently in replicon harboring cells, (2) to elucidate the detailed role of IFNa in the replication of NV, and (3) to explore the effects of bile acid signaling transduction on the replication of noroviruses. We plan to generate additional norovirus replicon-harboring cells using different norovirus strains by adapting the same strategy used in the NV replicon-harboring cells. The detailed role of a in the regulation of NV replication will be examined in the replicon-harboring cells by inducing and/or inhibiting a pathways. We hypothesize that noroviruses are susceptible to the inhibitory effects of a, and consequently are difficult to grow in cell culture, because they induce a. We plan to study if the inhibitors of a or bile acid signaling transduction can provide a mechanism to allow norovirus replication in cell culture. The effects of different bile acids, in the presence of the G-protein coupled receptor for bile acids on the replication of noroviruses, will be examined for the purpose. These studies should contribute to the isolation of noroviruses in cell culture, which is a key for development of preventive strategies to control diarrheal disease caused by noroviruses.
Yahia Chebloune, Ph.D.: CAEV/SHIV Chimera for Studies on Bystander Death of CD4+ T cells in Goats
The natural course of HIV-1 infection in human is characterized by an initial acute transient loss of CD4+ T lymphocytes, followed years later by irreversible progressive loss of this cell population, ending in death of infected patient. Early studies established that the loss of CD4+ T lymphocytes was mediated by the virus. However, the number of HIV-infected CD4+ T lymphocytes was found to be at least 100 times lower than the number of cells that were lost. This led to the conclusion that HIV induces depletion not only of infected CD4+ T cells but also non-infected CD4+ T cells involving bystander effects. Newer data have shown that CD4+ T lymphocytes could be killed by fusion of normal CD4 T cells with infected cells, and by induction of apoptosis by the diffusible HIV Tat. More recent data have suggested implication of other viral proteins, Nef, Vpr and Vpu in the depletion of bystander CD4+ T cells. However, whether these data, obtained in cell cultures, are relevant in infected animals is not known. In this project, we aim to develop lentivirus chimeras between primate and goat lentiviruses, and a goat animal model to study this type of pathogenesis. Caprine arthritis encephalitis virus (CAEV) is the natural lentivirus of goats that causes chronic inflammatory lesions in target organs of infected goats, but does not cause AIDS. The virus replicates exclusively in cells of macrophage lineage and its genome lacks the primate lintiviral genes that encode accessory proteins of HIV. These accessory proteins were shown to have toxic effects on bystander CD4+ T cells of both human and goat origin in cell culture studies. The envelope coding sequence of SHIVKU2 will be deleted and replaced with the homologous sequences of CAEV. The new recombinant virus will therefore be expected to switch its species tropism from primate to goats, and cell tropism to monocyte/macrophage cell lineage but not CD4+ T cells of the goat. The ability of the recombinant virus to induce death of CD4+ T cells without causing infection will be confirmed in cultures of goat CD4 T cells. The virus will then be used to inoculate goats. Here, we expect that the virus will replicate efficiently in goat macrophages, and by virtue of its toxic potential for CD4 T cells, will cause elimination of the latter, possibly leading to onset of AIDS in the goats. Infected animals will be monitored for progression of the infection in the macrophage population, using infectious cell assays, and potential loss of CD4 T cells measured by flow cytometry.
David Cue, Ph.D.: Antimicrobials targeted to M protein of streptococci
The overall goal of our original proposal was to test the hypothesis that small molecules capable of binding to M proteins of group A streptococci (GAS) could be useful as antimicrobial agents. During the last funding period, we used phage display to identify peptides capable of binding type 1 M protein and tested the peptides as inhibitors of M1 function (i.e. binding of human plasma proteins, adherence to and invasion of human epithelial cells). Although we identified several M1-binding peptides, none of the peptides significantly inhibited M1 function. Because identification of an inhibitory peptide/compound is essential in order to produce publishable results, or to acquire NIH funding, we have temporarily ceased working on this project and are now focusing our efforts on more promising research, as described below.
The overall goal of our current research is to develop a fuller understanding of the roles of human regulators of complement activation (RCAs) in the pathogenesis of Group A streptococci (GAS). Activation of the complement system in response to infectious agents is a key component of the innate immune response, leading to opsonization and/or lysis of invading pathogens. Uncontrolled complement activation, however, results in damage to host tissues and complement depletion. Nearly a dozen plasma and cell-membrane RCAs function to tightly regulate complement activity. Many pathogens exploit human RCAs for host colonization. GAS are important human pathogens known to utilize RCAs for cellular adhesion and for evasion of complement-mediated opsonization. GAS recruitment of RCAs (e.g. factor H (FH), factor H-like protein 1 (FHL-1) and C4b binding protein) has been regarded as a function of the streptococcal M proteins, a family of antiphagocytic, cell-surface proteins. Recently, we discovered a high-affinity receptor for FH/FHL-1, expressed by the clinically important, M1 serotype GAS. The receptor, designated Fba, contributes to the resistance to phagocytosis of GAS. Additionally, we discovered that binding of FHL-1, via Fba, promotes entry of GAS into the cytoplasm of human epithelial and endothelial cells. The latter findings are novel and argue that bacterial acquisition of RCAs may contribute to pathogenesis via a previously unrecognized mechanism. Furthermore, although purified M1 protein can bind FH, we observed that M1 GAS do not efficiently bind FH or FHL-1 when streptococci are incubated with human serum, suggesting that M1 protein-mediated resistance to opsonization may not involve RCA acquisition. Our study will focus on two important areas of GAS pathogenesis. The first is the functional interaction between Fba and FHL-1 and the role of Fba in cellular invasion and resistance to phagocytosis. The second area is determining the mechanism, whereby, M1 protein confers resistance to opsonization and phagocytosis.
David Davido, Ph.D., University of Kansas
HSV-1-mediated proteolysis of cellular targets
Herpes simplex virus (HSV) infections, typically characterized by physiologically-distinct lytic and latent phases, result in intermittent mouth (cold) and genital sores and are the primary cause of infectious blindness in western industrialized countries. Virus-host interactions dictate whether HSV initiates a lytic infection, establishes latent infection, or reactivates from latency by a variety of stress stimuli (e.g., heat shock). A key player in determining whether an infection will be lytic or latent is the immediate-early (IE), infected cell protein 0 (ICP0), which is an E3 ubiquitin (Ub) ligase activity that transactivates the expression of viral genes. E3 Ub ligases are components of pathways that typically attach and polymerize Ub (a 76 amino acid protein) to target proteins, marking them for proteolysis. Current data suggests that ICP0 directs the degradation of several cellular proteins that are, in turn, required for its transactivating activity. Efforts to identify specific targets of ICP0-mediated degradation by proteomic approaches have been challenging given the limitations in the purity, solubility, and detection of proteins. While several functions of ICP0 during viral infection have been characterized, the identities of cellular proteins marked for degradation by ICP0 are largely unknown. Until these new targets have been identified, it is unclear the exact role ICP0's E3 Ub ligase activity plays in its transactivating activity.
The long-term objective of our research is to determine how virus-host interactions, at the molecular level, control the HSV-1 life cycle. The objective of this proposal is to identify cellular proteins whose degradation is directed by ICP0. The central hypothesis of this pilot grant is that ICP0 mediates the degradation of specific cellular proteins. While unable to perform additional experiments in the time frame of this application (an 8-month time frame), we propose in future studies that degradation of these targets by ICP0 is essential for efficient HSV replication. Our approach is to identify functional targets of ICP0- mediated degradation in a novel screen. Our rationale for carrying out these studies is that by understanding how ICP0 facilitates productive infection via proteolysis, targets of ICP0 degradation could be used to develop new therapies to impair HSV replication and its associated diseases. Given the time frame of this application, we propose the following specific aim:
Specific Aim #1: Isolate novel targets of the HSV-1 E3 Ub ligase, ICP0. Our working hypothesis is that ICP0 directs the proteolysis of specific cellular proteins.
The contribution of this proposal is that we expect to identify cellular proteins involved in the biology of ICP0 using an innovative approach, which is a significant contribution that can applied to isolate and identify genes and pathways that play important roles in viral infections and proteolysis.
Apurba Dutta, Ph.D.: Synthesis of Novel C-5' Modified Nucleoside Analogs
Abstract: Nucleoside analogs as inhibitors of the reverse transcriptase (RT) enzyme of HIV-1 were the first class of compounds to be used in anti HIV-1 therapy. However, despite the development of several nucleoside HIV-1 RT inhibitor drugs, and the effectiveness of these compounds in combination regimes, prolonged treatment with these drugs often results in development of viral resistance or long-term toxicity. For this reason, design and development of new agents that can address the above concerns continues to be an urgent need. Although, a variety of structurally modified nucleosides have been designed, synthesized and evaluated for their biological activity, these studies were directed mainly towards modifications of the furanose core and the nucleobase component. Surprisingly, SAR studies involving the C-4' hydroxymethylene side chain of these nucleosides have remained largely unexplored. In the present research, design and synthesis of a new class of nucleoside analogs have therefore been targeted, wherein a unique one-carbon homologation of the hydroxymethyl side chain at C-4' position (nucleoside numbering) and incorporation of an additional nucleophilic group at C-5' in the form of a stereodefined amine functionality has been undertaken. The C-4' primary hydroxy group of nucleosides play a pivotal role in many biological transformations involving initial phosphorylation at this site. Thus, it will be interesting and informative to study the effect of the proposed side chain elongation on the phosphorylation of the terminal hydroxy group and the consequent biological activity of these modified nucleosides. Also, the hitherto unexplored addition of the secondary amino group at the C-5' position incorporates a more nucleophilic alternative site and opens up the possibility of additional or competitive phosphorylation at this group. Accordingly, initial stereoselective synthesis of a strategically functionalized chiral gamma-butyrolactone, starting from D-serine, followed by subsequent elaboration of this versatile synthon afforded various side-chain modified nucleosides. HIV RT inhibition assays of these analogs however indicated no biological activity. In future studies, screening of the above compounds for kinase activity and further modification of the side-chain amine moiety to reduce the steric bulk around C-5' will be undertaken.
Apurba Dutta, Ph.D.: Synthesis of Novel C-5' Modified Nucleoside Analogs
Opportunistic fungal infections are responsible for a significant portion of systemic infections in the immunosuppressed, such as AIDS patients, organ transplant recipients, and cancer patients undergoing chemotherapy. With a rapidly growing population of such individuals, systemic mycotic infections are becoming a major cause of suffering and death. The limited drug choice, development of resistance, and somewhat narrow spectra of the currently available antifungal agents, as well as their toxicity, often complicate treatment of deep-seated mycoses. Thus, there is an urgent need for the development of new and more efficacious antifungal agents that can address the clinical limitations of the present day drugs.
In this context, microbial derived complex peptidyl nucleoside antibiotics represent a new and promising class of antifungal agents of potent biomedical significance. Many of these natural products are among the most effective inhibitors of Chitin synthase, the fungal glycosyl processing enzyme responsible for the formation of chitin, an essential fungal cell wall component. The gross structural similarity between these antibiotics and uridinediphosphate-N-acetylglucosamine (UDP-GIcNAc), the natural substrate for chitin synthases, is thought to be responsible for the antifungal activity of these compounds. As the chitin biosynthesis pathway is absent in humans and other vertebrates, inhibition of chitin biosynthesis is considered to be a novel and attractive target for the potential development of safe and effective therapeutic agents against opportunistic fungal infections. Accordingly, the present research is directed towards design, synthesis, pharmacophore modification and biological evaluation of selected complex peptidyl nucleoside antibiotics as potential antifungals. The primary health significance of the proposed research is the development of approaches and methods for the easy accessibility of a biologically important class of compounds with demonstrated antimicrobial potential.
Patrick Fields, Ph.D.: Early Gene Expression During T cell Activation
Appropriate T cell differentiation is a critical component of a normal immune response and insures sufficient immunity to a variety of insults, most notably microbial pathogens. During T cell differentiation, the cytokine loci undergo significant changes in chromatin structure that facilitate coordinated expression of the cytokine genes. These changes include altered DNAseI hypersensititvity, histone acetylation, and DNA methylation. We recently found that altered histone acetylation, coupled with cytokine gene transcription, occur within hours of T cell stimulation. Interestingly, the patterns of transcription and chromatin remodeling were independent of Th1/Th2 polarization. The early changes in histone modifications at the promoters of the cytokine loci were plasic and could be maintained or reversed depending on the cytokine environment that the T cells encountered subsequently. The goal of this proposal is to examine early cytokine transcription as well as changes in the local chromatin environment around the cytokine loci in order to determine whether these early changes represent global alterations in gene expression patterns or whether they are part of a specific genetic program that is initiated upon T cell activation. In either instance, the changes may set the stage for subsequent effector differentiation and thus may be an important determinant of cell fate.
The scientific aims of this proposal are to study the regulation of early transcription of T helper cytokines in uncommitted T cells. We hypothesize that this early transcription is a critical step in determining the ultimate differentiated state of the effector cells. We will assess the role of chromatin remodeling within the cytokine loci on this early transcription, in an effort to distinguish these events from those in the differentiated effector cells. Inappropriate T cell differentiation has been implicated in a number of pathological states and is a critical determinant of immune responsiveness. The information gleaned from these studies will contribute to our understanding of Th1/Th2 differentiation and will hopefully enable the rational design of therapeutics that will enhance our ability to control microbial pathogens.
Severin Gudima, Ph.D.: Hepatitis B virus/hepatitis delta virus infection, its relation to pathogenesis
Human hepatitis B virus (HBV) infection is a major health risk. There are approximately 400 million carriers of persistent infection worldwide, of whom about 1 million die annually from HBV-induced liver cancer and chronic liver disease. Co- or super-infection with the sub-viral agent hepatitis delta virus (HDV) can lead to more severe pathology, increasing liver damage and risk of cirrhosis and hepatocellular carcinoma (HCC). This proposal is aimed to ascertain molecular mechanism of HBV/HDV infection with emphasis on pathogenesis.
Aim 1. The goal of this aim is to understand how the virus-induced pathogenesis is linked to a particular genotype of HBV. The project will compare aspects of replication, assembly, infectivity in primary human hepatocytes and host response for all known 8 HBV genotypes. The studies proposed will: (i) facilitate the understanding of the molecular mechanism of HBV genotype-specific pathogenesis; (ii) ascertain how HDV chronic infection is maintained; (iii) have a serious impact on the understanding of virus-host interactions. The finding of the most infectious HBV genotype will lead to three major applications: development of more potent peptide antivirals; performance of microarray studies using the system of in vitro infection of primary human hepatocytes; and establishment of HBV-susceptible cell lines.
Aim 2. To delineate the significance of the balance between HBV and HDV for outcome of infection, as will it become transient or chronic with possible risk of liver cancer, it is proposed to quantitatively examine what regulates competition or homeostasis between the two viruses during co-assembly, since both viruses share the same envelope. Also, using cultures of primary human hepatocytes, the patterns of co- and super-infection will be analyzed, addressing if the two viruses compete for susceptible cells, and if their replication rates are mutually affected. These studies will: (i) facilitate building the model of HBV-HDV homeostasis; (ii) enhance our understanding of virus-virus interactions in relation to pathogenesis; (iii) have significance in terms of the chronic infection mechanism by addressing the role of re-infection.
Curt Hagedorn, M.D.: Hepatitis C Virus NS5B Polymerase
Estimates are that a minimum of four million people in the United States have chronic hepatitis C. The high rate of progression to chronic infection, liver disease, and hepatocellular cancer make it a major public health problem. The production of HCV vaccines and new molecular based HCV therapeutics represent unsolved challenges need to meet this problem. The central hypothesis is that mutant forms of the HCV NS5B polymerase, that develop and are maintained during chronic HCV infection, will provide isoforms of the protein with more favorable properties for determining the structure of the ternary complex of NS5B, a template, and an inhibitor.The Specific Objectives are: To analyze NS5B mutations that occur during chronic HCV infection and select several mutant forms of the protein to study; To develop methods of expressing and purifying sufficient quantities of recombinant HCV NS5B polymerase proteins, representing selected mutations, for crystal growth studies; and To initiate crystal growth studies of the NS5B polymerase, an oligonucleotide template, and an inhibitor. This will permit structure based approaches to be used in developing therapeutic NS5B inhibitors and links strengths regarding HCV proteins (Hagedorn Lab) with expertise in Medicinal Chemistry in future NIH grant applications.
Curt Hagedorn, M.D.: HCV NS5B Polymerase Mutations: Biology/Pharmacology
At least four million people in the United States have chronic hepatitis C. A recent NIH Consensus Conference concluded that the number may actually be two-three fold higher than this estimate. The high rate of progression to chronic infection (70-80%), liver disease (>50%), and the worldwide distribution of chronic hepatitis C make it a major cause of morbidity and mortality. The production of new molecular based HCV therapeutics represents an unsolved challenge that needs to be intensely pursued to solve this major public health problem. The goal of this project is to study mutations of the hepatitis C NS5B polymerase that occur in vivo during chronic infection of the host. The aims of this study will include improving the methods to express and purify large quantities of these proteins, identifying forms of the hepatitis C virus NS5B polymerase that have more desirable crystal growth properties so that studies to define how NS5B interacts with the HCV RNA template and a nucleotide can be performed, and to conduct high through put screening tests of wt and mutant forms of NS5B to identify non-nucleoside inhibitors of hepatitis C virus RNA replication. The long term goal is to develop structure based drug design approaches to produce small molecule inhibitors that are effective against wild-type and mutant forms of the HCV NS5B polymerase. These studies will link strengths regarding HCV proteins (Hagedorn Lab, KUMC) with expertise in high throughput screening and Medicinal Chemistry (KU Lawrence) to accomplish major goals and produce new NIH grant applications.
Lynn Hancock, Ph.D.: Genetics of Capsule Synthesis in Enterococcus faecalis
Entercocci are leading causes of hospital-acquired infections, accounting for nearly 10 % of all nosocomial infections. Infections caused by enterococci constitute a significant treatment challenge due to the presence of multi-drug resistance. The goal of this study is to follow up the recent observation that capsular material is expressed by Enterococcus faecalis in vivo, and that production of this material affects the course of enterococcal infection. The proposed studies will determine how capsule diversity is achieved at the genetic level, as well as identify commonalities shared between the respective capsular serotype biosynthetic pathways. The proposed studies will also determine the contribution of the capsule biosynthetic genes to the synthesis of an emergent mucoid phenotype prevalent among chronic urinary tract infections, as well as determine the genetic basis for the mucoid trait. As biofilm formation represents an important developmental switch in the lifestyle of E. faecalis as a nosocomial pathogen. these studies will determine the contribution of the capsular polysaccharide in biofilm formation and identify the genetic pathways responsible for the production of the exopolymer matrix encasing enterococcal biofilms. The results from these studies should identify microbial components that can be targetted for antimicrobial therapy to treat and prevent infections caused by multi-drug enterococci.
Philip R. Hardwidge, Ph.D.: Modulation of NF-κB signaling by E. coli protein kinases
Enterohemorrhagic E. coli (EHEC) O157:H7 contributes greatly to the enormous economic and health burden of food borne disease. EHEC causes severe diarrhea through contamination of beef and vegetable products and also causes a fatal kidney disease for which there is no effective treatment or prophylaxis. The central hypothesis for the proposed research is that the EHEC effector proteins NleH1-1 and NleH1-2 bind to the non-Rel NF-κB subunit Rps3 to disrupt specific host transcriptional responses to bacterial infection. We have formulated this hypothesis based on our strong preliminary findings that demonstrate NleH binding to Rps3 in vitro, alteration of NF-κB dependent transcription following NleH1-1/2 transfection, and hypervirulence of EHEC strains lacking either nleH1-1 or nleH1-2 in a gnotobiotic piglet model of EHEC infection. The specific aims are to:
1. Map the NleH-Rps3 binding domains. Our working hypothesis is that NleH1-1 and NleH1-2 bind the mammalian non-Rel NF-kB subunit Rps3 to subvert its normal function.
2. Quantify the influence of NleH1-1/2 translocation on host transcription. Our working hypothesis is that NleH1-1 represses NF-κB-dependent host transcription, whereas NleH1-2 stimulates NF-κB.
3. Measure the contribution of NleH1-1/2 to bacterial virulence in animal models of attaching/effacing pathogens. Our working hypothesis is that NleH1-1/-2 promote bacterial transmission by maintaining an optimal balance between bacterial colonization vs. host inflammatory responses.
The proposed research is innovative because it will test the hypothesis that translocated bacterial protein kinases subvert the host innate response to infection by disrupting a novel molecular 'specifier' of selected host transcriptional responses to external stimuli. These studies are expected to have a significant positive impact on the design of new strategies to combat diarrheal pathogens, as they may identify host proteins indispensible for bacterial infection as novel therapeutic targets for broad control of enteric pathogens.
Helmut Hirt, Ph.D.: Characterization of the Novel Enterococcus faecalis Protein EbsG in Lipoteichoic Acid (LTA) Structure and Function
Enterococci have become important nosocomial pathogens in the recent decade. In particular the rise in antibiotic resistant strains is troublesome and new approaches and novel targets to combat these infections are needed. Previously, a mutant in Enterococcus faecalis was isolated with transposon mutagenesis that resulted in altered lipoteichoic acid (LTA). LTA is important in the initial adhesion of bacteria and can trigger septic shock. The genetics of LTA are largely unknown. The mutant was avirulent in a rabbit endocarditis model. The transposon inserted into four distinct loci on the chromosome. One of these loci encodes for a novel surface protein (EbsG) and an enzyme (EbsJ) that is thought to be involved in LTA turnover. An insertion mutant in ebsG results in altered cell surface properties and a change in LTA. The hypothesis postulates an interaction of the two proteins EbsG and EbsJ on the cell surface that allows LTA turnover. The interaction will be investigated by generating antibodies against the purified proteins and labeling of the proteins on the cell surface. For that purpose, fragments of the genes have been cloned in expression vectors. A fragment of ebsG has been cloned in the vector pET28b and fused to a His-tag. The purification is currently under way. A fragment of ebsJ was cloned in the vector pTBY4 and fused to an intein with a chitin binding domain. Purification of the protein has also started. The antibodies against EbsG can also be used for further characterization of the protein, which contains a von Willebrand factor domain, shows homology to a collagen binding protein and could thus be a virulence factor for Enterococcus faecalis.
Kee Jun Kim, Ph.D.: Role of CNF1 in Escherichia coli meningitis
Despite advances in antimicrobial chemotherapy and supportive care, the mortality and morbidity associated with Escherichia coli meningitis remain significant due to incomplete understanding of the pathogenesis of this disease. E. coli K1 is the major cause of neonatal Gram-negative bacterial meningitis and its invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for its penetration of blood-brain barrier (BBB) in vivo and in vitro. My colleagues and I have shown that cytotoxic necrotizing factor 1 (CNF1) is a major bacterial determinant contributing to E. coli K1 invasion of HBMEC and that laminin receptor (LR) is the cellular receptor for CNF1, which induces host cell actin cytoskeleton rearrangements through activation of RhoGTPases. Further characterization of CNF1-LR interaction suggests that LR plays essential role in CNF1-mediated E. coli K1 internalization into HBMEC, but it is incompletely understood how CNF1-LR interaction modulates actin cytoskeleton rearrangements in HBMEC, resulting in E. coli K1 invasion of HBMEC. Therefore, I hypothesize that E. coli K1 CNF1 interaction with its receptor (LR) triggers downstream signal transduction pathways responsible for actin cytoskeleton rearrangements and, in turn, E. coli K1 entry into HBMEC. To test my hypothesis, I propose to (1) determine whether activation of RhoGTPases is a proximal downstream of CNF1-laminin receptor (LR) interaction in HBMEC; (2) determine signaling molecules downstream of RhoGTPases activated through CNF1-LR interaction; (3) determine the role of ezrin in CNF1-mediated E. coli K1 invasion of HBMEC. The information derived from this study should enhance our understanding of the pathogenesis of E. coli meningitis, i.e., molecular mechanisms underlying CNF1-mediated E. coli K1 internalization into HBMEC and lead to the development of novel strategies to prevent E. coli meningitis.
Alexey Ladokhin, Ph.D.: Effect of Hemifluorinated Surfactants on Membrane Insertion/Folding of Diphtheria Toxin T-Domain
The function of diphtheria toxin T-domain (DTT) is to translocate the catalytic domain of the toxin across the lipid bilayer in response to acidification of the endosome, a mode of entry shared by a number of bacterial toxins, including the potential bioweapon botulinum. Despite the progress in characterization of membrane interactions of DTT, the molecular mechanism of its action and the structure of DTT in its functional membrane-inserted form remain unknown. Hemifluorinated compounds, such as HF-TAC, are a novel class of non-detergent surfactants designed to amend solubilization of membrane proteins for functional and structural studies. Because DTT, depending on pH, can be found in both an insertion-competent and water-soluble form, it presents a convenient model for studying the general effects of these surfactants on the membrane protein insertion/folding pathway. The objective of this grant is to determine the mode of interaction of surfactants with DTT using site-directed fluorescence labeling and other spectroscopic approaches. The specific aims are: (1) determine the effect of HF-TAC on the aggregation state of DTT; (2) determine the effect of HF-TAC on the free energy of membrane association of DTT; (3) determine the effect of HF-TAC on DTT-induced membrane disruption; (4) determine the direct effect of HF-TAC on the integrity of the lipid vesicles. This study will lay the groundwork for subsequent high resolution structural studies of DTT in membrane environment.
Audrey Lamb, Ph.D.: Siderophore Production and Import in Pseudomonas aeruginosa
The life expectancy of patients with Cystic Fibrosis (CF) has increased dramatically over the last few decades. This increase is due not to the treatment of the genetic disorder itself, but to improved antibiotic therapy for bacterial airway infection in these patients. Chronic lung infection by Pseudomonas aeruginosa, the main pathogen in patients with CF, causes acute pneumonia that leads to debilitating lung malfunction. Even with aggressive antibiotic therapy, chronic infection is generally inevitable, and results in premature death of these patients. In response to iron-limiting conditions in the human host, P. aeruginosa synthesize and secrete pyochelin and pyoverdin, two low molecular weight chelating agents known as siderophores. These siderophores form a complex with iron and deliver this essential cofactor to the bacterial cell through specific membrane receptors. Pyoverdin and pyochelin have been shown to contribute to the virulence of P. aeruginosa. Therefore, the enzymes and channel proteins required for production and uptake of these two siderophores are potential drug targets in the fight against early mortality in CF patients. The primary research goals of this lab begin with the determination of the x-ray crystallographic structures of the biosynthetic enzymes of pyochelin. Using this information and a variety of biochemical and biophysical techniques, we will investigate the protein-protein complexes that are formed for efficient siderophore production.
Mohammad A. Mir, Ph.D.: Mechanics of hantavirus nucleocapsid protein mediated translation initiation of viral mRNA
Eukaryotic mRNA translation initiation is a very complex process involving multiple translation initiation factors. Translation begins with recruitment of eIF4F complex at mRNA cap which engages 43S pre-initiation complex at mRNA 5' terminus. Another well characterized mechanism utilized by several viruses includes IRES translation initiation strategy that internally loads ribosomes on mRNA, independent of 5' cap. Hantaviruses, members of the Bunyaviridae family are emerging viruses that initiate mRNA translation by a different novel mechanism, using viral capsid protein (N) to engage the ribosome at mRNA cap, independent of eukaryotic eIF4F complex. We will further characterize N mediated translation initiation mechanism and illustrate possible benefits of this novel strategy that favor virus replication in infected cells. We will identify the components of 43S pre-initiation complex that interact with N. N specifically binds the viral mRNA 5' UTR with high affinity and preferentially facilitates the translation of viral mRNAs in vitro. We will identify and characterize the binding site for N on viral mRNA 5' UTR and will determine whether N preferentially facilitates the translation of viral mRNAs in host cells. N is also an RNA chaperone that unwinds RNA duplexes. However, this RNA chaperone activity is not involved in N mediated mRNA translation. Secondary structures in mRNA 5' UTR are removed by eIF4A (a component of eIF4F complex) during ribosome scanning and identification of AUG codon. Since N functionally supplants eIF4F complex, we hypothesize that N translocates the loaded ribosomes from 5' cap to the AUG codon, avoiding the regular scanning of 5' leader. Multifaceted experimental approaches have been designed to test this hypothesis. Host cells design various strategies to prevent virus replication, such as, interferon induced PKR over-expression leads to the phosphorylation of eIF2a, and transient shutdown of host mRNA translation. N significantly inhibits INF induced phosphorylation of eIF2a, suggesting that N likely blocks the virus induced host translation shutoff and prevents the cells from entering into an antiviral state. We will use multiple experimental approaches to check whether N mediated translation strategy is a viral counter measure against host cell antiviral response.
Manuel Moro, D.V.M., M.P.H., Ph.D.: Lyme Borreliosis and Babesial Coinfection
The blood parasite, Babesia microti and the spirochetal agent of Lyme disease, Borrelia burgdorferi, are both transmitted by the deer tick, Ixodes scapularis and are transmitted in arease that are currently endemic for Lyme disease. In humans, infection with these two organisms may occur alone or in combination. Prospective studies indicate that persistence of infection with B. burgdorferi and B. microti are enhanced by concurrent infection and result in an increased severity of symptoms. Our laboratory has demonstrated increase arthritis severity associated with downregulation of IL-10 and IL-13 cytokines in a murine model of coinfection. From these data we hypothesize that, during coinfection, B. microti downregulates the expression of IL-10 and IL-13 which in turn increases inflammation of joints and create and adequate milieu for B. burgdorferi persistence. To test this hypothesis, we propose to (1) determine if downregulation of IL-10 and/or IL-13 cytokines directly results in an increase of arthritis during coinfection; (2) identify the temporal expression of key inflammatory chemokines directly from the arthritic joints in a murine model coinfection; (3) determine in vivo gene expression of selected prototype genes from B. burgdorferi from joints during coinfection. Theses tudies will lead ultimately to a better understanding of the mechanisms by which coinfected individuals may be at higher risk of Lyme arthritis and may identify new therapeutic targets for Lyme borreliosis and bebesiosis coinfection.
Amit Mukherjee, Ph.D.: Bacterial Cell Division Proteins as Targets for Anti-Microbials
Bacterial cell division is a complex process involving several proteins among which FtsZ, a prokaryotic homologue of tubulin, plays a central role. Prior to the onset of division or septation, FtsZ, a cytoplasmic protein, assembles at the middle of the cell into a dynamic ring-like structure (Z ring) and remains at the leading edge of the constricting septum until its completion. The Z ring acts as a scaffold and recruits other cell division proteins in a linear order to form the division machinery. In E. coli, the endogenous cell division inhibitors, SulA and MinC, prevent Z ring formation which is lethal to the cell. In vitro, both block FtsZ polymerization. Thus FtsZ and other cell division proteins are attractive targets for novel anti-microbials. The long term objective of this proposal is to develop new anti-microbials that target FtsZ and other cell division proteins. More detailed study of the interaction of FtsZ and SulA will shed light on how FtsZ function can be blocked. High throughput screening of a library of natural compounds for FtsZ GTPase inhibitors and identifying potential inhibitors as lead compounds will be helpful in designing new anti-microbials.
Christophe Nicot , Ph.D.: Breaking HTLV-I latency: p30-RNA interactions a novel therapeutic target
The human T-cell leukemia virus type 1 (HTLV-I) is associated with an aggressive and fatal T-cell type leukemia known as Adult T-cell Leukemia/ Lymphoma (ATLL). We have found that the HTLV-I p30II protein is a negative regulator of virus expression from infectious molecular clones and chronic virus-producer cell lines. Based on our preliminary findings, we believe that p30II is required to support replication by reducing the levels of Tax and Rex expression and direct the virus to a latent replication cycle. Therefore, by reducing viral gene expression and immune recognition of infected cells, p30II may serve to promote viral persistence and clonal expansion of infected cells through cellular replication and may represent a target for the eradication of latent viral reservoir. Because we have found that p30 specifically interact with the viral tax/rex mRNA identification of the p30 response element (RE) and p30-p30RE interactions are very important to understand the regulation of HTLV-I replication. The goals of this project are to characterize p30II RNA-binding activities, to this end we will purify recombinant p30II protein expressed from bacteria and perform in vitro RNA binding assays and will investigate the different domains of p30 involved.
Christophe Nicot , Ph.D.: Roles of P30 in HTLV-1 Latency
The human T-cell lymphotropic virus type 1 (HTLV-I) is epidemiologically associated with an aggressive and fatal T-cell type leukemia/ lymphoma designated Adult T-cell Leukemia/ Lymphoma (ATLL). HTLV-I infection is also associated with a progressive myelopathy designated Tropical Spastic Paraparesis/ HTLV-I-Associated Myelopathy (TSP/HAM) of probable immune-mediated pathogenesis. It is estimated that 20 to 30 million people worldwide are infected with HTLV-I. HTLV-I possesses unusual features that would not predict its survival in an immune-competent host: The virus is poorly infectious but elicits a vigorous humoral and cellular host immune response. In addition, HTLV-I is mainly replicated in vivo through division of infected cells and therefore presents a very low antigenic variability. However, in spite of these apparent disadvantages the virus has persisted in humans for more than 100,000 years, indicating that HTLV-I has efficiently adapted to its host. We have recently found that HTLV-I has evolved a protein p30 that interacts specifically with the tax/rex viral RNA encoding positive regulators of virus expression. Because p30 is unable to shuttle out of the nucleus, tax/rex RNA is trapped in the nucleus and expression of these proteins is inhibited. The goals of this study are to investigate the molecular mechanisms involved in p30-viral RNA interactions and p30 effects on transcription leading to inhibition of virus replication. Three specific aims are proposed. Aim 1) Investigate post transcriptional regulation of p30 to design small inhibitors. Aim 2) Investigate expression of p30 in ATL or TSP/HAM patient samples infected with HTLV-I at different stages of the disease. Aim 3) Identify cellular proteins interacting with HTLV p30 and involved in retention pathways.
A. Lorena Passarelli, Ph.D.: Characterization of a viral fibroblast growth factor
Fibroblast growth factors (FGFs) are a large family of polypeptide growth factors widespread in organisms ranging from nematodes to humans. FGFs are key regulators in cell differentiation, cell proliferation, and cell motility. In addition to having effects on normal cell development, they are also implicated in the development and progression of tumorigenesis. In Drosophila, a single fgf, branchless, regulates tracheal cell migration determining the pattern of its main branches and fine terminal branching in response to signals from oxygen-deficient cells. Eukaryotic viruses encode many genes that allow them to manipulate host programs including immune responses, gene transcription and translation machinery, and cell cycle. Baculoviruses are large DNA-containing viruses that, similar to other eukaryotic viruses encode genes that affect the development of their host. This proposal will functionally characterize a baculovirus gene with homology to FGFs (vfgf) and will assess how vfgf allows the virus to spread infection throughout its host. We will determine if this factor is functionally similar to other FGFs in cell culture and in vivo, and more importantly, what its function is in the context of baculovirus infections of their invertebrate hosts. In the short run, this proposal will specifically (1) analyze the regulation and protein expression of vfgf; (2) measure fundamental activities exhibited by FGFs such as cell motility, cell proliferation, extracellular activity, and heparin binding, and identify possible host vFGF receptors; and (3) engineer recombinant viruses deficient in vfgf and evaluate their phenotype in cell culture and the insect host. In the long run, we expect that our system will contribute valuable information to virus-host interactions, branching morphogenesis and how it is affected during virus infection, and tumor progression mediated by disregulation of fgf.
Jianming Qiu, Ph.D.: Post-transcriptional Regulation of Parvovirus B19 Capsid Gene Expression
Parvovirus B19, the only parvovirus so far known to be pathogenic in humans, causes a variety of diseases, including erythema infectiosum in children, aplastic crisis in patients with chronic hemolytic anemia, persistent bone marrow failure in immunocompromised patients, and fetal hydrops in pregnant women. B19 is unique among animal DNA viruses in that it has a single promoter, and so its genetic diversity is controlled exclusively by post-transcriptional mechanisms. A single class of pre-mRNA molecules generated by B19 virus undergoes extensive alternative splicing and polyadenylation that generates sub-genomic mRNA molecules which program this diversity. Control of the synthesis of the capsid proteins is one of the key regulators of B19 tissue tropism. B19 permissive infection is characterized by a switch to increased capsid production. Other parvoviruses have an internal promoter that regulates production of capsid-encoding mRNA during the synthesis phase of viral replication, however, this mechanism is not available to B19. Control of expression of B19 capsid-coding genes by post-transcriptional mechanisms is the focus of this application. B19 has an efficient polyA site in the center of the genome. Use of this site precludes inclusion of the capsid coding ORFs into mRNA, and an understanding of how the choice is made to either polyadenylate, or read-through this site, is the top of Specific Aims. We propose to identify both the cis sequence and trans-factors that control these events, the mechanisms that govern their selective use, and exam how this choice is differently made in cells permissive or no-permissive for replication. How these post-transcriptional processes generate appropriate levels of the capsid proteins from the single pre-mRNA molecules encoded B19, is critical to our understanding of parvovirus gene expression, and the biology of parvovirus infection. However, in addition, these viral systems provide very tractable models with which to learn much about these basic cellular mechanisms in general.
Ernst Schönbrunn, Ph.D.: Molecular mode of action of novel MurA inhibitors
Bacterial survival strictly depends on the functionality of the cytosolic enzyme MurA. MurA belongs to the small enzyme family of enolpyruvyl transferases; it catalyzes the first committed step in the biosynthesis of the bacterial cell wall. Since this enzyme is absent from mammals but essential for bacterial growth, MurA is a prime target for the development of novel antibacterial agents effective against a broad range of pathogenic bacteria. By high-throughput screening (HTS) using a 50,000 compound library, we have recently identified six new MurA inhibitors with unique scaffolds. Their IC50 values ranged from 2 μM to 12 μM. The central goal of this proposal is to thoroughly evaluate the mode of action of these new lead structures on MurA. The specific aims, which integrate enzyme kinetics and protein crystallography techniques are to perform inhibition kinetics and determine the crystal structure of MurA bound with these inhibitors in order to resolve the structure-activity relationships in atomic detail. The long-term goal of this proposal is to provide a basis for the rational design of novel potent broad-spectrum antibacterial drugs that specifically target Mur A.
Liang Tang, Ph.D.: High Resolution of Herpesvirus
Herpesvirus is one of the largest families of human and animal viruses. Infection by these viruses can cause a variety of diseases including disseminated disease in the newborn, cold sores, cutaneous lesions, mononucleosis, non-epidemic encephalitis in adults, retinitis, and cancer. DNA packaging is a key step during the replication of herpesvirus and many double-stranded DNA (dsDNA) bacteriophages. The DNA packaging machinery consists of two basic components: a terminase and a portal protein. The terminase catalyzes the DNA packaging by cleaving genome-length DNA from concatemers and providing the energy for the packaging reaction. The terminase contains two subunits: a DNA-recognition subunit that specifically binds to the viral concatemeric DNA, and a catalytic subunit that cleaves the genome-length DNA from the cancatemer. The DNA is then injected into a pre-formed capsid through a conduit formed by the ring-like portal protein complex, powered by the catalytic subunit of the terminase. The role of the terminase in herpesvirus replication has made it an attractive drug target. High resolution structural information about the terminase is lacking. The proposed research is aimed at high resolution structural studies on the terminase of HSV-1 by X-ray crystallography and cryoEM. The interactions between the terminase/portal and the capsid will be explored. These structural and functional studies will establish a solid basis for our understanding of the molecular mechanisms of this complex molecular motor which is required for the replication of herpesvirus and dsDNA bacteriophages. The structural information about the terminase and its interactions with the portal and the capsid to be derived from the proposed research will open avenues to novel measures to control and prevent infection and diseases caused by herpesvirus. The proposed research will: (1) determine the X-ray structure of pUL15 of the HSV-1 terminase; (2) perform the cryoEM image reconstruction of the HSV-1 capsids; (3) investigate the assembly of the terminase, portal complex, and the capsid; (4) propose a detailed model for the DNA packaging mechanism in HSV-1.
Xiaoyan Tang, Ph.D.: The Function of the Pseudomonas Type III Effector Protein AvrPto in Pathogenesis and Host Defense
Many Gram negative bacterial pathogens rely on type III secretion system for pathogenicity. Through the type III secretion system, bacteria inject an array of virulence proteins, termed type III effectors, into host cells to interfere host defense and metabolism. In host plant carrying a disease resistance or R gene, the R gene recognizes the effector and triggers disease resistance. My COBRE project is to study the structure and function of AvrPto, a type III effector of Pseudomonas syringae pv. tomato. AvrPto activates disease resistance in tomato plants carrying Pto, a ser/thr protein kinase, and tobacco plants carrying an uncloned R gene. The disease resistance reaction on tomato plants requires direct interaction of Pto and AvrPto proteins. AvrPto also enhances the virulence of P. s. tomato on tomato plants lacking Pto. The progress we made towards understanding the structure and function of AvrPto include: 1. A small central region in AvrPto was identified that determines the specific interaction with Pto. 2. A small region was identified at the C-terminal region that determines the specific recognition with tobacco R gene. 3. The virulence and avirulence functions of AvrPto can be separated by mutations in the AvrPto protein. 4. Over 80 tobacco genes were isolated that are induced by AvrPto-mediated resistance. 5. Additional signal transduction components in the Pto/AvrPto mediated resistance have been identified genetically, and the corresponding genes are being mapped in tomato. 6. I have extended my research on type III secretion system of Xanthomonas campestris pv. campestris. Over ten putative type III effector proteins have been identified in X. c. campestris. I have made significant progress on characterization of the type III effector locus avrBs1 encoding a tyrosine phosphatase and an Avr protein. I was awarded with a USDA grant (12/2003-11/2005) to further characterize the avrBs1 locus.
Charlotte Vines, PhD: Regulation of CCR7 Mediated Adhesion of T cells through LFA-1
In response to invasion by foreign pathogens, naïve T cells home to the lymph node, to interact with antigen presenting cells and mediate the resultant immune response. Homing of naïve T cells from blood to lymphoid organs is regulated, in part, by activation of chemokine receptors by distinct chemokines found in the local environment. These activated chemokine receptors trigger signaling pathways that can lead to firm adhesion of T cells through the integrins, a family of heterodimeric, transmembrane, adhesion proteins. The distinct molecular events that are initiated at each of these steps are still being defined. We propose to examine the signaling pathways, which are initiated by chemokine binding to the CCR7 G protein-coupled receptor, that lead to cell adhesion via activation of the leukocyte functional antigen (LFA-1) integrins. CCR7 is activated by binding to two different endogenous ligands, CCL19 (MIP3β/ELC/Exodus-3) and CCL21 (6Ckine/SLC/TCA4/ Exodus-2), which are differentially expressed in secondary lymphoid tissues. Naïve T cells and certain populations of central memory cells (Tcm) employ the CCR7 chemokine receptor and LFA-1 to target T cells to secondary lymphoid compartments enabling their association with and priming by mature dendritic cells. Interestingly, T cells respond differently to CCL19 than they do to CCL21. While both ligands can mediate activation of G protein signaling and adhesion of T cells to LFA-1, only CCL19 promotes receptor phosphorylation/sensitization, and robust protein kinase C (PKC) activation. In contrast, CCL21 promotes PKC independent activation of the Rap1/RAPL ras family members, which increases the avidity (local increase in concentration) of LFA-1, and thereby up regulates adhesion to via LFA-1. Furthermore, while CCL19 enhances proliferation of NK cells, CCL21 can inhibit proliferation of certain myeloid progenitors. These observations suggest the molecular events triggered in response to the different ligands are distinct. This proposal will examine the signaling events involved in CCR7 mediated adhesion, proliferation and cell migration by its two ligands CCL19 and CCL21 and will lead to a better understanding of the molecular events that are required to recruit T-cells during an immune response.
Tom Yankee, Pharm.D., Ph.D.: Gads regulates the signaling threshold through the T cell receptor
The T cell repertoire must be sufficiently diverse so that every pathogen we might encounter can be recognized and targeted for destruction. This great diversity must be regulated so that T cells do not recognize and destroy our own tissues and cause autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and type 1 diabetes. The balance between broad immunologic diversity and restriction of the T cell repertoire to avoid autoimmunity is struck during the processes of positive and negative selection. When developing T cells first express a mature T cell receptor (TCR) complex, the cells are tested for their ability to recognize self-MHC complexes. Thymocytes expressing a TCR that binds MHC with high affinity undergo apoptosis via negative selection. Conversely, T cells that are unable to bind MHC undergo apoptosis via "death-by-neglect". Only T cells expressing a TCR that can bind MHC with moderate affinity are positively selected and continue to mature. It is unclear how intracellular signaling pathways relay the relative affinity of the TCR-MHC interaction. The goal of this research project is to understand how TCR-proximal signaling events regulate downstream signaling pathways. We hypothesize that the Gads adaptor protein, via post-translational modifications, regulates the signaling threshold through the TCR. This hypothesis is based on previous observations showing that Gads-deficient mice have defects in positive and negative selection. Despite these defects, Gads-/- mice can generate mature T cells, indicating that Gads is not required for TCR-mediated signaling. Further, our studies using peripheral T cells demonstrate that Gads-/- T cells can proliferate in response to antigenic stimulation, but they require antigens of higher affinity for the TCR to induce proliferation than wild-type cells. To test our hypothesis, we will identify the Gads-dependent signaling pathways that are required for positive and negative selection. Then, we will determine how Gads regulates the signaling threshold through the TCR and, in turn, regulates the downstream signaling pathways. We propose that Gads is regulated via post-translational modifications that control the duration and intensity of TCR-mediated signaling pathways.
Tom Yankee, Pharm.D., Ph.D.: The effects of morphine on the immune responses against HIV in vivo
In addition to being a risk factor for HIV infection, drug abuse can have a profound impact on the immunologic responses necessary for control of HIV replication. For example, CD8+ T cell-mediated immunity, which is critical for suppression of productive infection by HIV, is severely hampered by morphine. The goal of this research is to lay the foundation for mechanistic studies into how morphine regulates CD4+ T cell activation and differentiation. CD4+ T cell differentiation into the Th1 lineage and the subsequent activation and differentiation of HIV-specific CD8+ T cells is required for successful vaccination against HIV. We hypothesize that morphine induces permanent changes within naïve CD4+ T cells such that, upon antigenic stimulation, the cells are "pre-programmed" to differentiate into the Th2 lineage. This hypothesis is based on our work shwoing that morphine-treated rhesus macaques are unable to mount a CD8+ T cell-mediated immune response. In addition, morphine can augment IL-4 production by CD4+ T cells in culture. To examine the effects of morphine on CD4+ T cell differentiation, we are combining the use of transgenic T cell receptor models with direct examination of the effects of morphine on immune responses to an HIV vaccine. In this way, we will be able to relate detailed mechanistic studies of the effects of morphine on T cell-mediated immune responses to clinically relevant effects of morphine on HIV vaccine efficacy. In specific aim 1, we will use a mouse line expressing an MHC class II-restricted T cell receptor (TCR) to examine the effects of morphine on Th1/Th2 lineage commitment. We will examine how concurrent and prior administration of morphine with antigenic challenge affects Th1/Th2 differentiation in vivo. We will also examine whether the changes in Th1/Th2 lineage commitment can be reversed by altering the cytokine environment. In specific aim 2, we will examine CD8+ T cell-mediated immune responses to a DNA vaccine against HIV. Currently, DNA vaccines against HIV are the most likely candidates for human use. We will examine the effects of concurrent or previous use of morphine on the immune responses elicited by a DNA vaccine against HIV. We will also determine the minimal dosing interval of morphine required to impair CD8+ T cell-mediated immunity. Accomplishing these aims will lay the foundation for detailed mechanistic studies into the effects of morphine in vivo.
Wolfram Zückert, Ph.D.: Structure-Function Analysis of Borrelia Surface Lipoproteins
Surface-exposed lipoproteins of relapsing fever (RF) and Lyme disease (LD) Borrelia spirochetes mediate certain interactions of the bacteria with their arthropod and vertebrate hosts. RF spirochaetes such as Borrelia hermsii serially evade the host's antibody response by multiphasic antigenic variation of Vsp and Vlp proteins. Furthermore, the expression of Vsp1 and Vsp2 by RF Borrelia turicatae is associated with neurotropism or higher blood densities, respectively. Collaborative efforts with crystallography groups at Texas A & M, Houston TX and Rutgers University, Piscataway NJ to determine the structures of several Vsp and Vlp proteins are ongoing and have so far supported our hypothesis of overall structural conservation among Vsps and Vlps, with variation occurring mainly in surface exposed loops. We are now employing a novel lipoprotein surface display system using "transgenic" LD Borrelia burgdorferi to study the potential function of these variable loops in the evasion of the humoral immune response as well as host tissue tropism. Experiments leading to the development of this surface display system have suggested that lipoprotein export pathways in Borrelia are conserved, yet different from the ones described in other diderm bacteria such as Escherichia coli. We have thus begun using B. burgdorferi as a model organism to characterize spirochetal lipoprotein sorting signals and to identify components of the spirochetal lipoprotein transport and sorting machinery. We are initially focusing on a putative chaperone facilitating the transport of lipoproteins through the periplasm.
Previous Cores Supported
Protein Screening Core - Jeffrey Aube, Ph.D. and Helena Mailnakova, Ph.D., Core Directors
X-Ray Crystallography Core - Todd Holyoad, Ph.D., Core Director
Luminex Core - Shilpa Buch, Ph.D., Core Director
Signal Transduction Core - Christophe Nicot, Ph.D., Core Director
This grant was made possible by NIH Grant Number P20 RR016443 from the COBRE program of the National Center for Research Resources and NIH Grant Number P30 GM103326 from the COBRE program of the National Institute of General Medical Sciences.