COBRE Phase III Pilot Projects

 Year 1 Pilot Projects

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.

Revathi Govind, Ph.D., Kansas State University
Role of TcdR, the alternate sigma factor in Clostridium difficile virulence

Clostridium difficile is the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease (CDAD) because it disrupts normal protective gut flora and enables C. difficile to colonize the colon. Toxigenic C. difficile strains produce two toxins, toxin A and toxin B that are considered to be the major virulence factors. The toxins encoding genes, tcdA and tcdB are part of a pathogenicity locus, which also carry the gene encodes for the toxin genes positive regulator tcdR. TcdR is an alternate sigma factor that binds with RNA polymerase core enzyme to make the holoenzyme that initiate transcription at tcdA and tcdB promoters. Alternate sigma factors are known to regulate virulence and virulence associated genes in many pathogenic bacteria. Including toxin genes, TcdR may regulate other virulence-associated genes in C. difficile. We have created and characterized, tcdR mutant in two different C. difficile strains. Mutation in tcdR affected both toxin production and sporulation in C. difficile. Microarray analysis revealed many differentially expressed sporulation-associated genes in tcdR mutant. In this project in our first aim, we propose to test the role of TcdR in C. difficile sporulation. In our second aim, we are proposing to monitor TcdR dependent promoter expression at cellular level, using a novel reporter system. During the current decade there has been a dramatic increase in the incidence and severity of C. difficile infections due to the emergence of hypertoxinogenic C. difficile strains. Our long- term goal is to unravel pathogenic mechanisms of C. difficile, thus new strategies to prevent, treat and manage C. difficile infection can be developed.

Jianming Qiu, Ph.D., University of Kansas Medical Center
Mechanisms of the cell cycle arrest induced during parvovirus B19 infection

Human parvovirus B19 (B19V) infection causes severe hematological disorders that in some cases can be fatal, including hydrops fetalis in pregnant women, transient aplastic crisis in patients with a high rate of red blood cell turnover, and chronic anemia in immunodeficient and immunocompromised patients. Currently, there are no specific antiviral drugs available to treat patients with B19V infection, and an effective vaccine to prevent B19V infection in high-risk individuals has yet to be developed. B19V replication is highly restricted to human erythroid progenitor cells (EPCs) in bone marrow and fetal liver. B19V-casued hematological disorders are largely due to direct killing of the EPCs, in which B19V replicates. B19V infection induces a DNA damage response (DDR) that is mainly mediated by activation of ATR. The B19V large non-structural protein NS1 is essential for B19V DNA replication and induces infected cells arrested at a phase with a 4N DNA content (4N phase). Replication of the B19V linear single-stranded (ss)DNA genome occurs in host cells arrested at the 4N phase, and is facilitated by the activation of ATR. Thus, B19V does not use the host double-stranded DNA replication machinery for replication of its ssDNA; rather, it appears to induce a DDR and subsequently to co-opt the host mechanism of DNA repair for its own replication. We hypothesize that during early infection, the ATR-mediated DDR induces intra-S phase arrest that facilitates viral DNA replication (repair) through inhibiting cellular DNA replication, and that in contrast, during late infection, the G2/M arrest induced by B19V NS1 promotes cell death.            

We have established two experimental cell systems that will allow us to dissect the mechanism(s) of the cell cycle arrest during B19V infection: an efficient system of productive B19V infection involving the ex vivo-expansion of EPCs under conditions of hypoxia, which mimics the microenvironment of EPCs; a reverse genetics approach that involves transfection of a replicative form of B19V DNA into megakaryoblastoid UT7/Epo-S1 cells cultured under hypoxia. We will first explore a role of the ATR-Chk1 activation in inducing intra-S phase arrest during B19V infection and understand how the intra-S phase arrest facilitates B19V DNA replication. Second, we will characterize the B19V NS1-indcued cell cycle arrest at G2/M phase and understand the mechanism of how NS1 induces the G2/M arrest. Our studies will delineate the key molecular mechanisms of B19V replication and pathogenesis, which can be applied to develop anti-virus strategies for treating patients with B19V-casued hematological disorders.

Wolfram Zueckert, Ph.D., University of Kansas Medical Center
Mechanism of Borrelia surface lipoprotein secretion

Borrelia spirochetes, the causative agents of arthropod-borne Lyme borreliosis and relapsing fever, are often described as gram-negative bacteria due to their Gram stain properties and diderm, i.e. double-membrane envelopes.  Yet, a closer examination reveals significant differences in cell envelope composition and architecture.  Of particular importance for transmission and human disease is the unique Borrelia-vector/host interface, which is dominated by surface lipoproteins.  Despite the emergence of these peripherally membrane-associated proteins as major virulence factors, targets of the immune response and premier vaccine candidates, the processing and targeting pathways that guide them to their sites of biological activity have been only partially defined. The overall objective of our research is to gain an understanding of spirochetal envelope biogenesis, with a focus on determining the secretion and sorting mechanisms of spirochetal lipoproteins.  Our seminal studies using Borrelia burgdorferi as a model spirochete have shown that (i) surface lipoprotein localization determinants commonly localize to N-terminal tether peptides, (ii) translocation through the outer membrane (OM) requires an at least partially unfolded lipopeptide, (iii) accordingly, dimeric lipoproteins assemble into their final quarternary fold after reaching the bacterial surface, and (iv) translocation through the OM can be initiated by an unfolded C terminus.  Preliminary studies also suggested that at least one of the predicted inner membrane Lol pathway orthologs is not directly involved in surface lipoprotein localization.  We therefore hypothesize that surface localization requires maintenance of a translocation-competent intermediate, likely by interaction with a periplasmic holding chaperone, which may work in concert with a so far unidentified OM lipoprotein translocon.  To test these hypotheses, we have formulated the following independent but synergistic specific aims:

1.   To identify and define the periplasmic and OM pathway components governing Borrelia surface lipoprotein secretion by reverse and forward genetics approaches, using conditional knockouts of candidate periplasmic chaperones and a powerful combination of established FACS-based lipoprotein localization and novel suppressor screens.

2.   To define the Borrelia OM lipoprotein translocation mechanism using in vivo site-specific photocrosslinking and pulse-chase experiments.

These studies will (i) achieve further milestones in our investigation of Borrelia lipoprotein secretion, (ii) shed more light on the evolution of bacterial protein export mechanisms, (iii) significantly increase our understanding of spirochetal virulence, and may (iv) translate into the design of future intervention strategies.


Year 2 Pilot Projects

Bhaskar Das, Ph.D., University of Kansas Medical Center
Edward Stephens, Ph.D., University of Kansas Medical Center
Nanoparticle conjugated retinoids as effective therapeutic agents against HIV-infection

AIDS is still a pandemic that afflicts nearly 34 million people worldwide. Despite the development of highly active antiretroviral therapy (HAART) it has been not possible to eradicate AIDS because of lack of a vaccine and a lack of effective therapy against latently infected viruses. Furthermore, with the advent of HAART more individuals are living with AIDS, and the prevalence of cognitive impairment resulting from chronic CNS HIV exposure is increasing (Sacktor et al., 2001; Langford et al., 2003). Novel approaches are needed to develop drugs that may reach the latent reservoir of HIV-1 and that may penetrate the blood brain barrier to reduce the burden of chronic CNS HIV-1 replication. The long term goal of this application is to develop novel nanoparticle conjugated libraries to enhance nanoscience and nanotechnology research approaches that have the potential to make valuable contribution to biology and medicine and particularly giving emphasis to develop novel therapeutic agents against HIV-infection. We propose to develop nanoparticle conjugated libraries of drugs to utilize as therapeutic agents for HIV-infection, especially focusing on N-(4-hydroxyphenyl) retinamides (4-HPR) and its derivatives. 4-HPR or fenretinide is a synthetic derivative of retinoic acid or vitamin A (also called retinoid) and is an FDA approved drug under phase II clinical trials for many cancers. 4-HPR has been shown to be highly tolerable with minimal toxicities in humans. Our previous efforts to further modify 4-HPR led to the identification of an active moiety and allowed the synthesis of derivatives and peptidomimetic that retain the activity (Das et al and Das, Kalpana). 4-HPR was previously shown to be effective against HIV-1 replication (Blumenthal PNAS,2004,101(43), 15452-15457). Our preliminary studies have indicated that some of the derivatives of 4-HPR are more active in inhibiting the growth of HIV-1 than the parent 4-HPR. Based on these findings, we propose to first synthesize diversity oriented chemical libraries of 4-HPR. We will attach these functionalized 4-HPR molecules with ironoxide and trimethoxy silane based surface modified nanoparticles. We will evaluate the toxicity and efficacy of 4-HPR derivatives and nanoparticle libraries compounds using an in vitro cell culture system by using survival (MTS) assay. Starting with the lead compounds, we will iterate the process till we get a compound active at nanomolar level. Identification of new 4-HPR derivatives will lead to development of novel drugs against HIV-1. Our goal is to conduct a pilot study that can lead to a combined R01 application for future development of these compounds as therapeutic agents against HIV-1.

Susan Egan, Ph.D., University of Kansas
Inhibitors of AraC family virulence activators in Enterotoxigenic E. coli and Shigella

Many AraC-family transcriptional activators are required for the expression of virulence factors in bacteria that cause human disease.  Loss of AraC-family activator function, by either genetic deletion or chemical inhibition, dramatically reduces disease in a large number of different pathogens.  Among the pathogens that require AraC-family activators for disease are numerous examples that show rapidly increasing resistance to currently available antibiotics.  The long-term goal of our work is to identify inhibitors of AraC-family proteins that have potential to be developed into novel antibacterial agents.  The focus of the current proposal is the AraC-family activators Rns from Enterotoxigenic

E. coli [ETEC], and VirF from Shigella; both of which cause enormous worldwide morbidity and mortality and exhibit increasing resistance to antibiotics.  Rns and VirF are required for the expression of virulence factors that are necessary for these two important human pathogens to cause disease.  Therefore, our central hypothesis is that inhibition of transcription activation by Rns and VirF will prevent the expression of genes that encode critical virulence factors in ETEC and Shigella, and thereby reduce the ability of these pathogens to cause human disease.  The objectives of this proposal will be met through two specific aims:  Aim 1 will investigate a small molecule inhibitor, SE-1, that blocks the function of both Rns and VirF [as assayed in heterologous and in vitro systems].  We will test a set of chemical analogs of the inhibitor as a first step toward determining the structure activity relationship for the compound and optimizing the inhibitor.  Our assays will include assay of the interactions of ETEC and Shigella with host epithelial cells in the presence of the inhibitors and use of the flow cytometry core facility.  This chemical optimization could potentially be greatly enhanced through the use of structure-based design principles.  Toward this end, Aim 2 will identify the binding site of SE-1 on one or more AraC family proteins, preferably through a high-resolution structure of the protein-inhibitor complex.  Given that SE-1 is active against multiple AraC family activators, we propose the following alternative approaches: obtaining co-crystals of SE-1 with either ToxT [the master virulence regulator from Vibrio cholerae; has been successfully crystallized] or the RhaS DNA binding domain, or mutational analysis and inhibitor-binding assays.  We expect to identify the binding site of SE-1 and to use this information in the design of more potent inhibitors of AraC family virulence regulators.  The ultimate goal of this work is to identify inhibitors of AraC-family virulence regulators with the potential to be developed into antibacterial agents targeting pathogens that are responsible for massive worldwide illness and death.


Year 3 Pilot Projects

David Davido, Ph.D., University of Kansas
Viral and host factors regulate HSV-1 infection

The specific events that dictate herpes simplex virus type 1 (HSV-1)-cell interactions critically affect the outcome leading to either lytic or latent infection. An HSV-1 immediate-early (IE) regulatory protein that plays a key role in this process is infected cell protein 0 (ICP0). The ICP0 gene encodes a 775 amino acid (aa) protein characterized as a phosphorylated, nuclear E3 ubiquitin (Ub) ligase that activates transcription of all classes (IE, early (E), and late (L)) of HSV-1 genes. It transactivates these viral genes via its E3 Ub ligase activity, in part, by counteracting host cellular defenses. ICP0 functions to disrupt nuclear domain 10 (ND10), a component of intrinsic defenses, which is comprised of cellular proteins including promyelocytic leukemia (PML) and Sp100, their SUMO-modified isoforms, and high molecular weight SUMO-conjugated proteins. Recent data strongly suggest that two very specific domains of ICP0 phosphorylation (aa 224-231 and aa 365-371) contribute to two of its known activities (i.e., E3 Ub ligase and ND10-disrupting). One phosphorylation domain (aa 224-231) may facilitate its interactions with E2 Ub conjugating enzymes (Ubc), which modulate ICP0's E3 Ub ligase activity. The other domain (aa 365-371) is adjacent to a known SUMOinteracting motif (SIM), which may be regulated by phosphorylation. Exactly how this region, through specific phosphorylation sites, and their interactions with components of the cellular factors function to impair host defenses and influence the outcome of HSV-1 infection, however, is largely unknown. Our long-term research goal is to elucidate the molecular interactions between HSV-1 and its host that function to modulate the HSV-1 life cycle. Our objective in this proposal is to identify and define mechanisms of ICP0's interactions with cellular components to promote HSV-1 productive infection. Our central hypothesis is that phosphorylated regions on ICP0 facilitate its interactions with specific cellular proteins, which will examine in the first year of this application. In future studies, we propose that ICP0's interactions with these cellular targets inactivate the host's antiviral responses to enhance viral replication. Our rationale for these studies to identify pathways in HSV-1 infectious cycles that may lead to the development of new anti-HSV therapies. Upon the completion of this research, we expect to have identified specific components of the host's cell factors and specific phosphorylation motifs on ICP0 that interact with one another. We propose that these interactions influence ICP0 counter-defense functions and viral replication and will have a significant positive impact on mechanisms of viral replication and pathogenesis.

Steven M. LeVine, Ph.D., University of Kansas Medical Center
Genetic modifiers of sepsis

Sepsis is a major cause of mortality in the US accounting for ~66 deaths/100,000 individuals. This statistic demonstrates that current therapies are inadequate. A successful intervention strategy targets a critical component of the disease mechanism, but for sepsis the understanding of pathogenesis is incomplete. The host response to infection is a critical factor affecting outcome, and genetic variability can be a key determinant for susceptibility or resistance. Previously, we utilized congenic mice to screen for varying host responses to anthrax lethal toxin. Congenic mice had chromosomal segments from the wild inbred CAST/Ei strain on an otherwise C57BL/6 genetic background (e.g., B6.CAST.11M mice). Using these congenic mice, we found that a region on chromosome 11 (43-107 Mb) controlled the host immune response to anthrax lethal toxin. Since these findings, varying degrees of resistance to other pathogens have been observed between B6.CAST.11M and C57BL/6 mice, and multiple quantitative trait loci (QTL) appear to control these responses. Also, QTL controlling cytokine and hypothermic responses to lipopolysaccharide (LPS) (or muramyl dipeptide plus LPS) have been mapped within this chromosomal segment. Based on these observations, modifier genes controlling the host response to sepsis should be located within chromosome 11, and we hypothesize that B6.CAST.11M mice will display disease resistance relative to C57BL/6 mice during sepsis. The primary aim will be to characterize the differing host responses by B6.CAST.11M and C57BL/6 mice using a cecal ligation and puncture (CLP) model of sepsis. The Flow Cytometry Core Facility will be used to evaluate the cytokine profile and inflammatory cell responses over the disease course. Pathology, blood chemistry, coagulation profile, and clinical signs will also be assessed. The short-term objective will be to identify relevant host responses that account for disease resistance in a CLP model. Midterm objectives, which will be part of a future R01 proposal, will be to identify the gene(s) and detailed molecular mechanisms accounting for disease resistance in a CLP model. A long-term goal would be to use the information gained from the short- and mid-term objectives to develop a new intervention strategy.

Year 4 Pilot Projects

Jeffrey Bose, Ph.D., University of Kansas Medical Center
Alternate mechanisms behind AtlA-dependent biofilm formation in S. aureus

Staphylococcus aureus is a leading public health threat with the capacity to cause a variety of diseases. Simplistically, S. aureus infections can be viewed in two categories; 1) invasive diseases that rely on the production of various surface-associated and secreted virulence factors, and 2) chronic infections as the result of forming biofilm communities adherent to host tissues and implanted devices. Biofilm infections are difficult to treat due to their resistance to antibiotics and the immune system, and are generated in a complex process that begins with adherence, requiring a combination of proteins and extracellular DNA. Previously, we and others have shown that the enzyme activity of the peptidoglycan hydrolase AtlA is important for initial cell adherence and biofilm formation due to DNA release. However, adding DNA to atl mutants cannot rescue loss of biofilm formation, suggesting that AtlA plays other roles during biofilm initiation. Furthermore, because of its role in peptidoglycan cleavage, the activity of AtlA must be tightly controlled to prevent accidental     lysis of the cell. Yet, little is known about how the cell regulates the activity of this important enzyme. The central hypothesis for this study is that AtlA enzymatic activity is controlled by multiple cellular processes and contributes to biofilm formation beyond the known release of DNA. To test this hypothesis, two specific aims are proposed. Aim1 examines the contribution of AtlA enzymatic activity in biofilm formation by determining if: 1) the large clusters of atl mutants affect surface attachment, 2) AtlA releases additional cellular components that contribute to biofilm formation and 3) surface proteins are not exposed in atl mutants. Aim 2 characterizes mutants identified from a transposon mutant library screen for mutations affecting AtlA activity. As a result of this screen, 16 mutants have been selected for further investigation and will be put through a battery of AtlA-related tests to determine at what point AtlA is altered. 

The completion of these studies will shed new light into the mechanism by which AtlA contributes to biofilm formation

Mary Markiewicz, Ph.D., University of Kansas Medical Center
The role of NKG2D in immunosurveillance of spontaneous lymphoma

Follicular lymphoma is the second most common form of non-Hodgkin's lymphoma worldwide. Despite new therapies, patients are rarely cured, with a median survival of 10 years. Therefore, studies aimed at developing new therapies for this disease are needed. There is evidence that the immune system can detect and eliminate nascent B cell lymphomas under the correct conditions, suggesting immunotherapeutic strategies may be useful in treatment of the disease. However, to develop such strategies we must first understand the signals involved in effective elimination of tumor cells by the immune system. One signal that is implicated in tumor immunosurveillance is NKG2D engagement on immune cells by NKG2D ligands expressed on tumor cells. Here the role of NKG2D receptor-ligand interaction in the control of spontaneous B cell lymphoma will by assessed using a novel mouse model.

Preliminary data demonstrate that B cell lymphoma development is enhanced in transgenic mice with ubiquitous expression of the NKG2D ligand RAE1epsilon compared with wild-type mice and suggest that these mice have a defect in controlling the outgrowth of indolent lymphoma. Correspondingly, NKG2D surface expression is low on immune cells in the transgenic mice. This NKG2D deficiency is hypothesized to result in decreased tumor immunosurveillance, particularly at an indolent stage, and increased lymphoma development. The hypothesis that NKG2D-mediated immunity is important in controlling B cell lymphoma

outgrowth will be tested in a novel mouse model. It will be determined whether NKG2D ligands are expressed by spontaneously developing lymphomas in mice and whether ubiquitous RAE1epsilon expression enhances the growth of nascent B cell lymphomas in mice.

Jianming Qiu, Ph.D., University of Kansas Medical Center
Airway Epithelium Response to Parvovirus Infection

Human bocavirus type 1 (HBoV1) is an emerging respiratory pathogen that causes lower respiratory tract infections in young children. Neither an effective vaccine to prevent HBoV1 infection nor a specific antiviral drug is available. HBoV1, a human parvovirus, belongs to the genus Bocavirus of the family Parvoviridae. HBoV1 replication is highly restricted to human airway epithelium (HAE) and appears persistent in in vitro culture as well as in infected individuals.

 The airway epithelium maintains intimate contact with the environment and constitutes the primary participant in innate immunity. Although host innate response against virus infections are actively studied, how the host cells defend ssDNA virus invasion, e.g., parvovirus, remains an enigma. In recent years, we have established an HBoV1 reverse genetics system and a system of HBoV1 infection of in vitro well differentiated human airway epithelium (HAE), well positioning us in understanding the responses of the airway epithelium to DNA virus infection. Our preliminary results have demonstrated that HBoV1 infection of HAE induces a DNA damage response (DDR), which is mediated by all the three related phosphatidylinositol 3-kinase-like kinases (PI3KKs). Though the host HAE cells are non-dividing cells, HBoV1 DNA replicates well in them. We also observed that upon HBoV1 infection, the infected HAE responds to the infection immediately by secreting proinflammatory cytokines. Of note, application of INFα on the HAE significantly diminishes HBoV1 replication.

 We hypothesize that during early HBoV1 infection, the DNA damage machinery first senses the incoming viral ssDNA genome and initiates a DNA repair-facilitated DNA replication in non-dividing airway epithelial cells, whereas the replicating viral genomes are detected by a DNA sensor, a pathogen recognition Receptor

(PRR), which initiates an innate immune response to combat virus replication. Eventually, the two cellular responses reach a balance that maintains a persistent HBoV1 infection in HAE. In this proposal, we will first explore which PI3KK is involved in the initiation of the DNA repair-facilitated viral DNA replication; second, we will identify the ssDNA PRR; third, we will test the role of the IFNα-induced ISG (Interferon Stimulated Gene) APOBEC3A in the inhibition of HBoV1 replication in HAE.

 Our discovery that HBoV1, a small ssDNA virus, autonomously replicates in non-dividing cells is novel. Thus, our proposed study will not only delineate the key molecular pathways underlying HBoV1 replication but will also unveil novel mechanism of how ssDNA is recognized by the host innate immunity machinery.

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.

Last modified: Jul 31, 2015