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.
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.
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.