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Research Projects - 2010

We anticipate having 10 students in our Summer Program in 2010. Eight of these will be supported by NSF REU funds; the other two by Department funds. Faculty who will be taking REU-funded students are indicated with an asterisk (*) and listed first.

NSF REU Mentors

Dr. John E. Butler, Professor*
Development of the Antibody Repertoire
Our laboratory is engaged in studies on the role of the ileal Peyers patches in the development of the antibody repertoire in late fetal and newborn piglets. There are two directions that the student’s project can take. First, the student would learn how to do immunohistochemistry in the Central Microscopy Core and would apply this method to determine the number of Bells bearing different isotypes in and around the follicles of the IPP in animals that are germfree versus those colonized with a bacterial exclusion flora. In addition the IPP in a surgically isolated loop of the lower ileum (no contact with bacteria or food antigens0 would be investigated by the same method. The second direction that can be taken is to study antibody repertoire development in these same tissue specimens using molecular biological methods. Specifically the relative transcription of different isotype could be quantified and /or the degree of repertoire diversification measured using an established cloning and hybridization procedure. It is often optimal to have two students who work together on the same general project but approach it with two different methods like those described above.

Dr. Craig D. Ellermeier, Assistant Professor*
Production and sensing of a novel antimicrobial peptide
Students in my laboratory will study one of two problems both of which focus on understanding the recent discovery of cannibalism in the model organism B. subtilis. We have identified a novel antimicrobial peptide produced by the sdpABC operon. We have recently found that SdpAB are required for increasing the activity of the antimicrobial toxin SdpC. Our evidence suggests other genes are required for the production of this toxin and one potential project would employ a genome wide transposon screen to identify genes required for toxin production. A second area of interest is characterizing a novel signal transduction system (SdpI-SdpR) which responds to the antimicrobial toxin SdpC. We have found that SdpR is sequestered to the membrane in an SdpC and SdpI dependent manner. Potential student projects include determining if SdpR and SdpI directly interact using bacterial two hybrid and performing a transposon screen for to identify other factors required for controlling SdpR activity.

Dr. Michael G. Feiss, Professor*
Genome recognition, processing and translocation in the large dsDNA viruses
The Feiss lab studies how dsDNA viruses recognize and process intracellular viral DNA, and the subsequent translocation of the DNA into the procapsid. One current project is to mutate the translocation enzyme of bacteriophage lambda to understand how ATP hydrolysis powers translocation. One possible undergraduate project would be to generate one or more novel mutant forms of the translocation enzyme, followed by asking about the effect of the mutation on mutant enzyme behavior in the DNA packaging process.

Dr. Alexander R. Horswill, Assistant Professor*
Acetate assimilation in Staphylococcus aureus
Staphylococcus aureus is known to secrete large amounts of acetate during aerobic growth. As the cells enter stationary phase, the acetate is taken up and used as source of carbon and energy. Published reports suggest that S. aureus senses acetate levels as a regulatory switch controlling multiple cellular events. How does the acetate assimilation process occur and how is it controlled? Through bioinformatic analysis, it is apparent that S. aureus has a chromosomal locus containing genes related to those from E. coli and Salmonella that control acetate uptake and assimilation. The goal of this project is to investigate these S. aureus genes and their encoded enzymes in order to understand the assimilation pathway. Further insight on this pathway may provide insight on the role of acetate as a regulatory switch.

Dr. Jon C.D. Houtman, Assistant Professor*
Characterizing the mechanism for the clustering of the adaptor protein LAT in human T cells
The undergraduate students in my laboratory will investigate the formation of large multiprotein signaling complexes and their function in human T cells. The activation of T cells is controlled by the formation of large protein clusters that occur at the adaptor protein LAT. These megadalton-sized clusters can contain up to 50 LAT molecules along with its various binding partners. We have shown that the formation of the LAT clusters is mediated by the interaction of LAT with the adaptor protein Grb2, a function that is controlled by the simultaneously binding of Grb2 with various enzymatic ligands. One potential project involves using quantitative biophysical tools, such as isothermal titration calorimetry and analytical ultracentrifugation, to further characterize the interaction of Grb2 with various ligands, including SOS1, c-Cbl and HPK1. Students will use what they learn to better understand the formation of LAT clusters and the clinically-relevant interaction of Grb2 with various ligands. Another potential project would be to examine the function of the clustering of LAT in modulating T cell activation. In this project, students would address the poorly characterized role that multiprotein clusters play in T cell biology and would have broad implications, since the activation of multiple receptors leads to the induction of similar signaling complexes.

Dr. John R. Kirby, Associate Professor*
Genetics of behavior and development in Myxococcus xanthus
Students will investigate the role for signal transduction during development in the bacterial model organism, Myxococcus xanthus. These bacteria utilize chemosensory signaling pathways, homologous to chemotaxis systems, to regulate group behavior during predation and differentiation during sporulation. Students will use transposon mutagenesis to generate a bank of mutants and to identify those defective in predation on other bacterial species. A second project will focus on the isolation of bypass suppressors for known chemosensory mutants that are defective in predation on E. coli. The mutants will be used in assays to identify novel signaling molecules and signaling pathways required to regulate the complex behavior of cells within populations.

Dr. Linda L. McCarter, Professor*
Surface-induced differentiation of Vibrio parahaemolyticus

Students will investigate regulatory processes governing the regulation of swarming in the marine bacterium Vibrio parahaemolyticus. V. parahaemolyticus senses and responds to growth on surfaces by differentiating into a swarmer cell, which is adapted for movement over and colonization of surfaces. Students will contribute towards elucidating the scr regulatory network controlling swarming and sticking via the second messenger c-di-GMP. Potential projects include both classical and reverse genetic approaches, bioinformatics analyses, and physiological studies to identify candidate regulators and decipher their position in the swarming circuitry map. For example, candidate regulators will be identified by using reporter gene analyses coupled with transposon mutant suppressor analysis or by screening a Vibrio library for regulatory clones and DNA sequencing. Students will then use their DNA sequence to develop hypotheses about the role of a particular gene and design schemes to further test their hypotheses. This work will expand our understanding of a global signal integration system that allows the bacterium to make decisions important for surface colonization lifestyles.

Dr. Richard J. Roller, Professor*
Herpes simplex virus interactions with host cell membranes
Formation of a membrane vesicle by protein interactions is a process with relevance to many fundamental cellular processes including endocytosis and protein trafficking. Virus budding events provide an attractive system for studying membrane vesicle formation since they tend to occur at thigh level in infected cells and the proteins involved are encoded by easily manipulated genetic elements. Herpes simplex virus nuclear egress requires budding of virus particles into the nuclear membrane. Two virus proteins UL31 and UL34 are required for budding and their interaction is thought to promote membrane curvature in budding. We have isolated mutants in each protein that interfere with membrane curvature and the positions of these mutations identify domains of each protein that may be required for curvature. A summer REU student could express these putative wild type and mutant interaction domains as tagged fusion proteins separate from the remainder of each protein and do pull-down assays to test the hypotheses that these domains mediate a direct physical interaction, and that the mutations that disrupt membrane budding also disrupt that physical interaction.

Dr. George V. Stauffer, Professor*
Small regulatory RNA molecules
In Escherichia coli, the gcvB gene encodes a small non-translated regulatory RNA. The gcvB gene is activated by the GcvA protein in response to glycine and repressed by the GcvA + GcvR proteins in the absence of glycine. Recently, we showed that GcvB and GcvA play roles in the regulation of the hdeAB operon, encoding proteins involved in acid resistance. One potential project involves construction of hdeA-lacZ transcriptional fusions as well as a hdeA-lacZ translation fusion under control of the arabinose PBAD promoter. Students will use the fusions to learn whether the GcvB regulatory RNA and GcvA protein function at the level of transcription or post-transcriptionally. Putative GcvA binding sites have been identified in the hdeAB promoter region. A second potential project involves purifying His-tagged GcvA and running gel mobility shift assays with hdeAB promoter DNA to determine whether GcvA binds the promoter region, as well as change basepairs in the promoter region by directed mutagenesis that would alter binding. DNA sequencing will verify mutations. The students will learn how proteins interact with nucleic acids as well as form hypotheses as to how GcvA and GcvB control expression of the hdeAB operon.

Dr. David S. Weiss, Associate Professor*
Bacterial cell division
Students will investigate bacterial cell division using E. coli as a model organism. Our lab has recently identified three new cell division proteins that carry a C-terminal domain (called a SPOR domain) of ~70 a.a. that binds peptidoglycan. One potential project involves using a two-hybrid system to assay for protein-protein interactions involving the SPOR domain proteins and other division proteins. Students will use the information gained to formulate hypotheses about how the division proteins work together to accomplish the complex task of cytokinesis. In an alternative potential project, students will select for multi-copy suppressors that rescue the ability of certain SPOR protein mutants to form colonies on plates with 0.1% deoxycholate. DNA sequencing will be used to identify genes carried on the plasmids, and students will be asked to formulate hypotheses about how the genes rescue the mutants. This project has the potential to lead to the discovery on new cell division genes.

Dr. Timothy L. Yahr, Associate Professor*
Regulation of Pseudomonas aeruginosa type III secretion
Students will investigate gene regulation using the P. aeruginosa type III secretion system (T3SS) as a model. T3SS gene expression is controlled directly by the transcriptional activator ExsA. Recent data from our lab suggest that ExsA expression levels are controlled at the transcriptional, translational, and post-translational levels by several upstream regulatory pathways. One potential project in the lab would involve a genetic screen to identify genes that inhibit ExsA translation and provide a link between hypothesized regulatory mechanisms. A second project would involve purification of an RNA binding protein that is thought to promote ExsA translation through stabilization of the exsA mRNA or providing access of the mRNA to the ribosome. Students will formulate and test hypotheses through a combination of genetic and biochemical approaches.

 

Other Mentors

Dr. Michael A. Apicella, Professor
Biofilm formation in pathogenic Neisseria
The long-range goal of Dr. Apicella's research is to understand the factors involved in the pathogenesis of human pathogenic Neisseria and nontypeable Haemophilus influenzae infections in order to develop methods to inhibit these infectious processes either by vaccination or chemotherapy. These organisms are strict human pathogens and cause considerable disease worldwide. Dr. Apicella's research combines state of the art methodologies in molecular biology, cell biology, bioinformatics and macromolecular chemistry to study mechanisms involved in bacterial pathogenesis.

The studies of the Apicella laboratory on Neisseria gonorrhoeae have shown that this organism is unique since it utilizes different mechanisms of infection in men and in women. In men, the organism is able to infect the urethral epithelial cell by the binding of the terminal lactosamine on the gonococcal lipooligosaccharide (LOS) to the asialoglycoprotein receptor on the surface of the urethral epithelial cell. This initiates a process of clathrin-dependent receptor mediated endocytosis resulting in the internalization of the gonococcus. Studies in women have shown that infection in cervical epithelial cells is initiated by surface ruffling induced by the interaction of the gonococcus with the complement receptor 3 (CR3) receptor on the surface of the epithelial cell. This initiates an actin-dependent process that results in the internalization of gonococci into cervical epithelial cells. Present studies are focusing on the structures on the surface of the gonococcus that binds to CR3 and the cervical cell signaling process, which ensues. Recent studies in the Apicella lab indicate that the gonococcus can form a biofilm during infection in patients. Studies are now underway defining the nature of this structure at the biochemical and genetic level using gene chip array, mass spectrometry, NMR and classical carbohydrate methodologies.

The studies of the Apicella laboratory on nontypeable Haemophilus influenzae (NTHi) have shown that NTHi invades host cells by binding of the platelet-activating factor (PAF) receptor via LOS glycoforms containing phosphorylcholine (ChoP). The binding of the PAF receptor by NTHi initiates receptor coupling to a pertussis toxin-sensitive heterotrimeric G protein complex, resulting in a multifactorial host cell signal cascade and bacterial invasion. We are currently engaged in studies of NTHi biofilm formation in continuous flow chambers and during infection of airway epithelial cells. To date, these studies suggest that sialic acid plays an important role in biofilm development and gene regulation within the NTHi biofilm. We have initiated studies to determine how this organism acquires sialic acid from its environment since it cannot synthesize this sugar.

Dr. Gail A. Bishop, Professor
Optimizing the potential of B lymphocytes in cellular vaccines
The global human population is developing an increasing need for new and better vaccines, to combat both infectious and malignant disease. A limiting factor in human vaccine development has been the narrow selection of safe adjuvants, to increase the effectiveness of vaccines, and stimulate effective responses with fewer immunizations. Scientists have made tremendous advances in understanding how the components of adjuvants, distinct pathogen-associated molecular patterns (PAMPs), trigger specific receptors of the innate immune system. Of particular interest are ligands for receptors that recognize special features of viral or bacterial nucleic acids, as these ligands can be readily produced synthetically, without the safety concerns associated with purifying substances from large quantities of infectious microbes. This basic immunology project focuses on gaining a more complete understanding of how receptors for microbial nucleic acids interact with receptors of the adaptive immune system in the activation of B lymphocytes, with the long-term goal of applying this knowledge to better strategies in vaccine development. All effective vaccines in use today elicit a robust antibody response; in the case of anti-viral vaccines, this response is capable of virus neutralization. B cells are also now appreciated to play important roles in cytokine production and antigen presentation. The project will address questions considered key to understanding how innate and adaptive receptors stimulate B cell responses. What are the effects of interactive signals between adaptive and innate receptors on the function of B cells as antigen-presenting cells (APC)? We hypothesize that interactions between these receptor types will enhance the APC ability of B lymphocytes. This information will allow design of vaccines that maximize the efficacy of B cell activation, not only to produce antibodies and cytokines, but also to activate cellular responses as APC.

Dr. Steven Clegg, Professor
Molecular mechanisms of enterobacterial attachment to eucaryotic cells and tissues

The major focus of research in my laboratory is to investigate the mechanisms by which the enterobacteria Salmonella and Klebsiella adhere to, colonize, and grow on host tissues and surfaces. Both in vivo and in vitro models of infection using mice and biofilm chambers respectively are employed to achieve these goals. The bacterial genes responsible for these processes are identified by construction and characterization of mutants impaired in specific stages of pathogenesis. In addition, the molecular functions of gene products are investigated using a variety of techniques. Specific colonization factors have been identified for both these groups of bacteria and the regulatory network controlling the production of adherence and other surface factors is being elucidated. In addition, the pathoadaptive evolution of enterobacteria is being determined. In this case the role of allelic variants of virulence genes in facilitating infection of different hosts and/or host organ systems and tissues is being analyzed. By construction of specific mutants and manipulation of appropriate genes we are defining which determinants are subject to selection in order for bacteria to optimally grow on a variety of hosts.

Dr. Charles D. Cox, Professor
(a)The biochemistry and genetics of hemoglobin utilization by Pseudomonas aeruginosa
Pseudomonas aeruginosa is an opportunistic pathogen that causes hospital-related infections characterized by considerable tissue destruction and high mortality. Bacterial growth, which is essential for causing infections, depends upon the acquisition of iron from human tissue. The predominant iron-binding protein in serum, transferrin, inhibits bacterial growth because of the slow acquisition of iron from this protein. However, the release of minute amounts of human hemoglobin from erythrocytes stimulates rapid and luxurious growth. Another serum protein, haptoglobin, binds free hemoglobin preventing most bacteria from using hemoglobin. P. aeruginosa is not inhibited by the presence of haptoglobin. The biochemistry and genetics of iron and heme acquisition from hemoglobin in the presence of haptoglobin are the major topics of this research project. This information is being used to inhibit hemin acquisition from hemoglobin and stop bacterial growth during infections.

(b) The steep Lactobacillus species and corn wet milling
Corn steeping is a hydration process that prepares corn kernels for wet milling. A steep poorly defined, thermotolerant lactobacillus bacterium has been isolated from steep water. This bacterium, referred to as the steep lactobacillus, is difficult to grow in laboratory conditions. On the other hand, steep lactobacillus growth during corn steeping is luxurious and is associated with changes in the corn kernel, such as increased release of soluble compounds and increased starch release during milling. In addition, bacterial metabolites have made steep water an historical source of inexpensive, growth-promoting compounds. For that reason, steep water has been used as a growth medium in several pharmaceutical industries. The focus of this research project is to enhance the growth of steep lactobacillus to improve the steeping process and to increase the bacterial metabolites in steep water so that steep water can regain its historic status in the industrial synthesis of antibiotics.

Dr. John T. Harty, Professor
T cell responses to infection

Dr. Bradley D. Jones, Associate Professor
Host cell invasion by Salmonella typhimurium
Our laboratory is interested in the invasive phenotype of pathogenic Salmonella and how these bacteria regulate this important virulence property. To invade, these bacteria inject effector proteins into targeted host cells, which results in a dramatic rearrangement of the cellular membrane and bacterial uptake. Salmonella tightly regulates the expression of the invasive phenotype through a series of regulatory proteins that sense and respond to environmental stimuli. We have a established a series of assays that measure the expression of various Salmonella invasion proteins and regulators with the goal of identifying ways to disrupt the Salmonella invasive phenotype. We are currently screening a small cyclic peptide library for clones that produce inhibitory peptides that disrupt expression of Salmonella invasion genes. Clones that are identified are rigorously screened for specificity and site of action. Multiple projects are underway to characterize these peptides and to define their mechanisms of action on the Salmonella invasion pathway. Furthermore, we have initiated projects to apply this system to other aspects of Salmonella pathogenesis including adherence and colonization phenotypes. Undergraduate projects include learning molecular genetic techniques, such as mutagenesis, cloning, sequencing and genetic mapping experiments, which will allow characterization of cyclic peptides function and site of action. In addition, students may learn tissue culture techniques and a variety of cell invasion assays.

Dr. Al J. Klingelhutz, Associate Professor
Human papillomavirus and the development of cancer
Human papillomaviruses (HPVs) are small DNA viruses that infect cutaneous and mucosal epithelium. Certain HPV subtypes play a role in the development of cervical and oral cancers. The life cycle of HPV is closely associated with the differentiation program of the epithelial cells that it infects. Two genes of HPV, E6 and E7, have been shown to be involved in the development of cancer. When expressed in human cells, E6 and E7 inactivate two important tumor suppressor proteins, p53 and pRb. E6 also activates an enzyme called telomerase, which adds telomeric repeats to the ends of chromosomes. Altogether, these interactions confer immortality, a necessary trait of malignancy. HPV E6 also has other functions, such as activation of signaling pathways, that are likely to be involved in tumorigenic progression. A project for a summer student would involve the use of HPV E6 mutants to dissect what specific functions of E6 are necessary for various aspects of cellular transformation. This project would entail learning and using molecular biology and cellular biology techniques. The studies have the potential of leading to a better understanding of how cancer develops and potentially to better treatment strategies.

Dr. Wendy J. Maury, Associate Professor
Regulation of enveloped virus entry and pathogenisis
A goal of our research is to understand how enveloped viral glycoproteins bind to and enter permissive cells. An appreciation of the cellular attachment factors, receptors and subsequent internalization pathways used by a virus to enter cells provide an avenue for the development of antiviral therapies. To this end, my laboratory studies two related lentiviruses, human immunodeficiency virus (HIV-1) and equine infectious anemia virus (EIAV) as well as the filoviruses, Ebola and Marburg. Our recent studies have helped to elucidate the endosomal pathway used by EIAV and filoviruses for entry and identified Ebola glycoprotein amino acids that are critical for Ebola virus internalization. Several different approaches are currently being developed within the lab to block the entry of these viruses into target
cells.

Dr. C. Martin Stoltzfus, Professor
HIV mRNA Splicing
In our lab, we study HIV-1, an example of a "complex" retrovirus and the cause of AIDS disease. We are interested in post-transcriptional events and are particularly interested in how the viral RNA is spliced, transported and packaged into virus particles. There are a number of antiviral drugs that are being successfully used to treat HIV disease that that act at different stages of the virus life cycle. The downside of this is that virus mutates and develops resistance. For this reason, it is important to develop a broader arsenal of antiviral drugs to hit virus at different steps of the life cycle. Understanding how HIV-1 regulates its RNA splicing is only beginning to be understood. Further understanding of this step may lead to development of novel approaches to inhibit virus replication. We have discovered that the RNA splicing of HIV-1 is regulated by interaction with cellular factors binding to cis elements on the viral genome. We have localized these cis elements and have identified several host factors that bind to these elements. So far, we have studied these elements in the region of the Tat, Vif and Vpr mRNA acceptor splice sites. Tat is a small basic protein that acts as a transactivator during virus infection. Vpr is a protein that causes a cell cycle arrest in infected cells. Vif is a protein that neutralizes a cellular antiviral protein.As a summer project, we propose to use a method called antisense inhibition to alter HIV-1 alternative splicing and in this way inhibit virus replication. We will first determine by in vitro splicing assays which oligonucleotides are effective. We will then introduce these oligos into cells and determine the effects on HIV-1 splicing and on virus replication. Techniques to be used include Western blotting, DNA transfection, RT-PCR, and polyacrylamide gel electrophoresis, HIV infectivity.

Dr. Steven M. Varga, Associate Professor
Viral Immunology of RSV
Respiratory virus infections represent a significant health problem and economic burden throughout the world. Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection worldwide. Virtually all children are infected with RSV by the age of 2 and 1-2% of all infected children require hospitalization. It is believed that RSV infection accounts for 130,000 hospitalizations and 4,500 deaths annually in the United States alone. A RSV vaccine developed in the 1960’s caused augmented disease and increased mortality among some of the infant vaccine recipients upon a subsequent natural infection with RSV. In addition, RSV infection of children has been implicated in the development of asthma later in life. Despite the significant health and economic burden RSV causes, a safe and effective RSV vaccine has yet to be developed. In order for a more safe and effective RSV vaccine to be developed, a better understanding of the immune response must first be established. Our laboratory uses a mouse model to mimic the RSV vaccine-enhanced disease observed in the children from the vaccine trial to gain a better understanding of the mechanisms leading to the inappropriate immune response that developed in these vaccinated children. Our work will aid in our understanding of the factors that contribute to RSV vaccine-enhanced disease and provide novel and valuable information necessary for the development of a safe and effective RSV vaccine or immune intervention.

Dr. Mary E. Wilson, Professor
Pathogenesis of infection with the parasitic protozoan, Leishmania
Dr. Wilson's research focuses on the molecular and immunobiology of infection with the protozoan parasite, Leishmania chagasi, a cause of the disease visceral leishmaniasis. The lab examines interactions between the parasite and mammalian host that result in either survival or eradication of the parasite. Leishmania are obligate intracellular parasites of macrophages. The abundant parasite surface glycoprotein MSP is important for initial parasite attachment to macrophage receptors, evasion of complement killing, and intracellular survival. GP63 abundance parallels the virulence of the parasite. The lab studies molecular mechanisms regulating MSP expression in the parasite life stages. Leishmania are phagocytosed through caveolae on the macrophage surface, generating large membrane ruffles in the macrophage. Using confocal and electron microscopy the lab is investigating the routes through which the parasite moves into the infected cell, and how these relate to intracellular survival. Phagocytosis causes dramatic changes in macrophage gene expression, which is characterized with methods such as DNA microarray and Rnase protection assay. Macrophages down-modulate expression of several genes involved in microbicidal responses, and up-regulate expression of genes inhibiting inflammation, upon leishmania phagocytosis. The roles of several inhibitory factors (e.g. transforming growth factor-b, IL-10) in innate and adaptive immune responses to the parasite are under investigation.

Humans living in endemic areas develop widely divergent outcomes of L. chagasi infection, ranging from spontaneous cure to progressive, fatal visceral leishmaniasis. In collaboration with a professor in northeast Brazil, Dr. Wilson’s group is examining the hypothesis that polymorphic alleles at immune response genes contribute to an individual’s susceptibility to develop different outcomes after infection. Other studies are addressing recombinant parasite T cell antigens that could be used in antileishmanial vaccine development. The above studies require the use of basic molecular biology, protein biochemistry, immunology, and genetics techniques.