INBRE Faculty Associates in Infectious Disease

CREIGHTON UNIVERSITY COLLEGE OF ARTS AND SCIENCES

"Roles of Type III Chaperones in the Type III Protein Secretion System of Pseudomonas syringae "
Karin V. van Dijk, Ph.D.
Collaborator: James R. Alfano, Ph.D. (UNL)

Abstract: Many Gram-negative plant and animal pathogens depend on a Type III protein secretion system (TTSS) that injects proteins called effectors into eukaryotic cells, to cause disease. Although all effectors contain N-terminal secretion signals, a subset require the binding of specialized proteins, type III chaperones (TTCs), to become secretion competent. Much research has been carried out on TTCs, but their essential function remains a matter of controversy and studying this topic will promote our understanding of TTSSs and may allow for novel strategies to control plant and animal diseases caused by TTSS-containing pathogens. Experiments outlined in this proposal are designed to explore the role chaperones play in the TTSS system of Pseudomonas syringae by investigating the contribution of TTC-binding domains to effector secretion and translocation, by determining protein interactions between TTCs and effectors and/or the secretion apparatus proteins, and by exploring whether TTCs contribute to regulation of this secretion system.

NEBRASKA WESLEYAN UNIVERSITY

"Novel Gene Discovery from Nucleocytoplasmic Large DNA Viruses (NCLDV)"
Garry A. Duncan, Ph.D.
Collaborator: James Van Etten, Ph.D. (UNL)

Abstract: The major goal of this research endeavor is to search for novel genes among the Nucleocytoplasmic Large DNA Viruses (NCLDV). The model virus of this group, Paramecium bursaria chlorella virus-1 (PBVC-1), which infect a green alga, has been sequenced, as have 7 other viruses of this group. These viruses contain many novel genes/proteins, some of which are the smallest known, several of which have commercial applications (next paragraph and section B.3.). PBCV-1 belongs to an ancient family of eukaryotic viruses that infect a chlorella-like green alga, a symbiont of Paramecium bursaria. PBVC-1 is a plaque-forming virus that has proven to be very amenable to laboratory study. James Van Etten, (JVE) the mentor/collaborator for this project (see section E below), has 25+ years working on this virus and has current NIH funding to investigate the DNA replication and gene expression of this virus. Furthermore, JVE has a network of collaborators who are studying a wide variety of PBVC-1 properties. This virus will be used as a reference against all newly sequenced viruses.

The reasons for focusing on the NCLDV group of viruses are that they have, by definition, large genomes (the mimivirus encodes over 1000 proteins) and they are ancient. Their large genomes have come about by high jacking genes from a variety of sources and by gene duplication. Their ancient history and short life cycles have enabled them to evolve some of the smallest functional proteins, while other proteins have been shown to have medical importance (see section B.3.). For example, PBCV-1 has genes that encode proteins involved in DNA replication, recombination and repair, cell signaling, DNA restriction/modification, to name a few. The PBCV-1 type II DNA topoisomerase is the smallest one to be described to date and yet has very high activity levels (Lavrukhin et al., 2000). Topoisomerase II from PBCV-1 has been of interest in the study of cancer cells. Its restriction and methylation enzymes are commercially sold by New England Biolabs. This virus also encodes the smallest K+ channel protein. Plugge et al. (2000) have extensively characterized this protein from PBCV-1. Ion channel proteins affect ion concentrations and are the target of channel blockers for muscles, including the heart. As well, potassium ion (K+) channels play a key role in the proliferations of breast cancer (Abdul, Santo, and Hoosein, 2003).

The primary objectives towards this proposal are: (i.) sequence additional viruses from this group; (ii.) conduct genomic, bioinformatic and phylogenetic analyses of these newly sequenced genomes. The analyses will be ongoing as new databanks and methods emerge. (iii.) Upon identification of novel or unusual genes, hand them off to others in the Van Etten lab and their colleagues who will clone, express and characterize them.

"Bioprospecting for Medicinally Important Compounds using Endophytic Organisms"
Jerald S. Bricker, Ph.D.
Collaborator: Gary Strobel, Ph.D. (Montana State University)

Abstract: The focus of this project is the search (better known as "bioprospecting") for biologically active endophytes (definition: an organism, such as a fungus, that grows within the tissues or cells of another plant). My primary goal is to identify organisms that produce novel compounds that exhibit biological activity, hopefully against human diseases. Endophytes are organisms that hold significant potential as sources of new drug therapies. Even though nearly every plant species on the planet harbors one to thirty novel endophytes (Strobel, personal communication), very few higher plants have been screened for presence of these organisms. This situation is particularly interesting in light of the fact that 80% of the antibiotics currently used in medicine are derived from the prokaryotic genus Streptomyces (Castillo et al. 2006) and that Streptomyces are fairly common endophytes. Thus, a screening program searching for endophytes in local environments should turn up new sources and genera of Streptomycetes. Not all endophytes produce antibiotics. Thus, it is logical to assume that many other active compounds will be identified with activity to organisms or diseases other than bacteria. Bacteria, viruses, protozoa, helminths, malaria, and cancer (to name a few) are all threats to human health. Each of these conditions requires a novel pharmaceutical compound to fight it. As a result, anyone interested in endophytes quickly understands the potential benefit to human and global health. New sources of antimicrobials must be discovered. Endophytes hold the promise of providing a compound (as anticancer/chemotherapy agents, antivirals, or antibiotics) that is beneficial to human health.

UNIVERSITY OF NEBRASKA AT OMAHA - BIOLOGY

"Virulence Determinants in the Coxsackievirus B3 Genome"
William E. Tapprich, Ph.D.
Collaborators: Steven Tracy, Ph.D. (UNMC) and Nora M. Chapman, Ph.D. (UNMC)

Abstract: Coxsackievirus B3 (CVB3) is the leading cause of viral myocarditis, causes pancreatitis and plays a role in type I diabetes. Like all picornaviruses, CVB3 translates its RNA genome using an internal ribosome entry site (IRES), whereby ribosomes are recruited directly to an initiation codon by recognizing a highly structured RNA element in the 5' nontranslated region (5’NTR). The overall goal of this research is to understand the structure and function of the 5’NTR and its associated IRES. The specific objective of this proposal is to explore the RNA elements and the RNA-protein interactions that determine virulence in CVB3. The two specific aims of the previous grant were accomplished, confirming the hypothesis that the IRES folds into a stable structure that is required for proper function. As proposed in specific aim 1, the structure of a wild type CVB3 IRES was determined. As proposed in specific aim 2, differences that mediate the virulence phenotype have been mapped. The goal of the current research is to define the minimal RNA elements that determine virulence and explore the RNA-protein interactions that underlie IRES function. Two specific aims will test the hypothesis that small, independently folding RNA structures in the 5’NTR provide recognition features for cellular and viral proteins that together determine viral virulence. In specific aim 1, the solution structure of RNA domains previously identified to be virulence determinants will be determined in virulent and non-virulent variants of the genome. These variants will include naturally occurring sequences, site-directed mutants and chimeric constructs. Our established methods of base-specific chemical modifying agents will probe the accessibility of nucleotides in the folded RNA domains. In specific aim 2 the molecular mechanism of IRES function will be explored by studies of RNA-protein interactions. Probing studies of RNA-protein complexes using our established chemical modification techniques will provide insight into the conformational dynamics and functional states of the 5’NTR. These results will aid in the search for effective antivirals and vaccines, not only for CVB3 but also for a host of other disease-causing picornaviruses.

"Investigation of Early Lead Anti-Toxoplasma Compounds"
Paul H. Davis, Ph.D.
Collaborator: TBD

Abstract: Toxoplasma gondii is a zoonotic human parasite with worldwide distribution. In addition to its classical association with fetal malformation (a leading cause of congenital neuropathy, affecting >1/1000 live births in the US) and abortion, toxoplasmosis also afflicts the growing ranks of immunocompromised individuals (cancer and transplant patients, as well as victims of HIV). Primary infection (acquired via ingestion of cysts in contaminated water, soil, or undercooked meat) leads to an initial systemic spread of tachyzoite parasites with mild to no symptoms, which mature into bradyzoite tissue cysts >1 week post-infection. In contrast to primary maternal infection which strongly affects the fetus, disease in the immunosuppressed patient population is primarily due to reactivation of dormant bradyzoite cysts residing in patient tissues and can lead to significant morbidity. Approximately 30% of the U.S. population is chronically infected with T. gondii, and harbor chemo- and immuno-resistant bradyzoite cysts within their tissues, particularly brain and muscle. Published studies have suggested that lifelong infection may produce psychological phenotypes in humans; however, there is no known treatment for the chronic bradyzoite stage of infection. Very recently, we have published a study of non-proprietary compounds active against intracellular Toxoplasma gondii tachyzoite growth; however, we have not established their mechanism of action. Moreover, the effect of these compounds on the bradyzoite cysts remains untested. Therefore, we propose to investigate the mechanism of action for these early lead anti-Toxoplasma compounds, and investigate the effect these compounds exert on bradyzoite cysts.

UNIVERSITY OF NEBRASKA AT KEARNEY

"Identification and Characterization of S-nitrosylated proteins in Borrelia burgdorferi"
Travis Bourret, Ph.D.

Abstract:  The Lyme disease spirochete Borrelia burgdorferi encounters a wide variety of environmental stresses during infection of both its arthropod hosts, ticks of the genus Ixodes, and its various mammalian hosts. Among the challenges faced by B. burgdorferi are potentially harmful Reactive oxygen species (ROS) (e.g. O2•-, H2O2 and OH•) and reactive nitrogen species (RNS) (e.g., NO, NO2•, N2O3 and ONOO-) produced by mammalian host phagocytes and neutrophils or within Ixodes scapularis midguts and salivary glands. While the effects of ROS on B. burgdorferi have been investigated by several groups, very little is known about the impact of RNS on B. burgdorferi pathogenesis. The focus of this proposal is to identify B. burgdorferi proteins that are S-nitrosylated by RNS and characterize the subsequent effects of S-nitrosylation on protein function, physiology, gene expression, and overall B. burgdorferi virulence.

WAYNE STATE COLLEGE

"Cloning, Over-Expression and Purification of Listeria monocytogenes InIA and InIB for Use in Drug Delivery"
Doug C. Christensen, Ph.D.
Collaborator: Andrew K. Benson, Ph.D. (UNL)

Abstract: The gene coding for inlA/B will be PCR amplified utilizing a primer set that allows for directional cloning with an Ek/LIC cloning vector (pET-46) [Novagen]. The gene will be inserted into the vector via sticky ends, stretches of non-coding nucleotides both upstream and downstream of the gene that do not anneal to the complimentary PCR generated strands but rather to complimentary sticky ends within the vector. This vector will incorporate a six Histadine-tag at the terminal end of the InlA/B genes. The His-tagged proteins can be purified in spin columns that contain filters with a high specificity for Histadine-rich polypeptide regions. Untagged proteins are eluted out in a series of washes then tagged proteins are eluted and collected. Although some proteins like GroES and GroEL still function properly with the attachment of a His-tag [3], the Histadine tail may change the conformation of the inlB protein, and may affect its ability to bind to target receptors. If the His-tag does affect the functional conformation of inlA/B, an exopeptidase should be able to cleave the His-tag and allow the protein to refold into its native conformation. The enzyme DAPase has successfully been utilized for His-tag removal, alone or with accessory enzymes Qcyclase and pGapase [1]. The vector we will use also contains an insert for ampicillin selection. Suspected insert-containing plasmid constructs will be subject to XbaI/XhoI dual digests which will be analyzed for desired restriction patterns. Promising colonies will be grown in LB broth containing ampicillin. These cells will be lysed and a protein purification columns will be used to isolate His-tagged internalin A/B. Analysis of protein isolation will via Western blotting utilizing His-probe primary antibody (Cat# SC-804, Santa Cruz Biotechnology). Isolated internalin A/B will first be exposed to various human cell lines (HeLa, 292-Kidney 293, CaCO, and THP-1 monocytes) for analysis of forced internalization through Western blot analysis and/or Epiflourscent microscopy. Successful internalization of the internalin would allow for trials of attaching various drugs and further analysis of internalin-drug internalization in human cell lines.

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