Microbiology

Staphylococcus Research

Kenneth Bayles, Ph.D.
My research has focused on the characterization of a novel regulatory system that controls the peptidoglycan hydrolase activity produced by bacteria. Using Staphylococcus aureus and Bacillus anthracis as model systems, we have identified two sets of genes that impact these processes. The first set (the cid and lrg genes) encode membrane-associated proteins that are proposed to function by controlling the access of the peptidoglycan hydrolases to their substrate (peptidoglycan) within the bacterial cell wall. The second set (lytSR and cidR) encodes regulatory proteins that modulate cid and lrg expression in response to physiological signals. Recent studies indicate that these signals include changes in proton motive force and the energy status of the cell. Research on this system has led to the proposal that it comprises the molecular components of bacterial programmed cell death (PCD) and that the cid and lrg gene products may be functionally homologous to the Bax/Bcl family of proteins that control apoptosis in eukaryotic cells.

Paul Fey, Ph.D.
The Fey laboratory studies Staphylococcus epidermidis as it is the preeminent cause of biomaterial-related infections and a significant cause of morbidity and mortality in hospital settings. In contrast to S. aureus, which produces an array of toxins and adherence factors, the most significant virulence factor associated with S. epidermidis biomaterial-related infections is the synthesis of large amounts of biofilm, which is largely composed of polysaccharide intercellular adhesin (PIA). PIA, which is synthesized by enzymes encoded by the four gene icaADBC operon, dramatically reduces the effectiveness of antibiotics and the host immune system. 

Enterovirus Research

The Enterovirus Research Group
The Enterovirus Research Group studies the biologies of human enteroviruses, which are a genus of viruses that can cause illness in humans that range from minor colds to more acute cases of myocarditis, meningitis, pancreatisis, and paralysis. The group consists of several well established scientists who have a mutual research interests in enterovirus biology, pathogenesis and immunology with a strong focus on the coxsackieviruses, and who are individually discussed along with research focuses below.

Steven Carson, Ph.D.
My interest in tissue factor and the initiation of blood coagulation began with my post-doctoral work at Yale Medical School (Carson and Konigsberg, 1980). I remain interested in blood coagulation, but most of my current research is focused on group B coxsackieviruses and their receptor(s) (Carson, Chapman, and Tracy, 1997; Carson, Chapman, Hafenstein, and Tracy, 2011) The group B coxsackieviruses can cause serious infections of the heart (myocarditis), pancreas (pancreatitis, and possibly diabetes), and brain (encephalitis), and can be particularly dangerous in babies. The medical significance and current research on these viral infections are described at the Enterovirus Research Group website at The University of Nebraska Medical Center and in Volume 323 of Current Topics in Microbiology and Immunology; Tracy, Oberste, Drescher, ed., Springer-Verlag, Berlin 2008. My current research investigates molecules that bind group B coxsackieviruses, their effects on virus viability, and how they alter the interaction of the viruses with the CAR (coxsackievirus and adenovirus receptor) and with cells (Organtini et al, 2014 and Carson, 2014). I am increasingly curious about selective pressures that drive these viruses to bind molecules other than the CAR (Carson, Chapman, Hafenstein, and Tracy, 2011).

Nora Chapman, Ph.D.
My research has been centered on the molecular biology of enteroviruses and their role in disease, particularly in heart disease. Enteroviruses are picornaviruses, small RNA viruses which are not enveloped and which have a positive strand genome (the genome is the mRNA for the virus). The genome encodes a large polyprotein, which is cleaved to produce viral proteins by viral encoded proteases. The activity of these proteases upon cellular proteins plays a role in the pathogenic effects of viral infection. The 5’ nontranslated region (5’NTR) of the viral genome includes an IRES (internal ribosome entry site) which allows the viral RNA to be translated for viral proteins despite the cleavage of translation initiation factors essential for most host cell protein translation. Much of my present research is on the role of defective enteroviruses in myocarditis and cardiomyopathy as well as determining the mechanism of persistence of this previously unreported natural form of the picornaviruses. My laboratory has demonstrated that the persistence of enteroviral RNA in human and murine myocarditic hearts is due to selection in the heart of variants with 5’ terminal deletions. These deleted genomes continue to replicate and produce infectious viral particles but have a greatly reduced yield of virus. We cloned of a third of the genome of the defective CVB2 that we isolated from heart tissue of a case of fatal myocarditis and demonstrated the defective function of the virus with the 5’nontranslated region cloned into our infectious CVB3 cDNA. This demonstrated that the findings that I have made on persistence of defective enteroviruses in the murine model of myocarditis and in cardiac cell culture are relevant for human disease. We have demonstrated that this selection for 5’ terminal deletions occurs in nondividing cells (such as in the fully differentiated heart) and is likely to involve host factors and viral structures important for initiation of positive strand replication. Demonstration of this type of defective replication and persistence is new for positive strand RNA viruses and may well demonstrate that we carry the consequences of these very common infections in hearts and other tissues for prolonged periods of time.

My laboratory also collaborates with Dr. Steven Tracy on detection of human enteroviruses in the pancreas and cardiac tissues. His work on type I diabetes has recently been reinforced by new findings of enteroviruses in the pancreas of type I diabetics (which although not a new finding triggered more interest in the role of these viruses in this disease). The expertise I acquired on detection of enteroviruses in hearts (design of RT-PCR methodology using databases of enterovirus sequences) has aided me in this collaboration. We also collaborate with Dr. Steven Carson on studies of the coxsackievirus receptor. This work is of great interest as the receptor, a part of tight junctions, may be more accessible under conditions of inflammation such as myocarditis or cardiomyopathy. Dr. Carson and I are presently defining variations and their functional consequences in a virus adapted to bind a co-receptor, decay accelerating factor. The interest stems from the fact that this alternative receptor may act as a cell signaling co-receptor in human isolates of coxsackievirus B and may be critical for access to the primary receptor, CAR, which is normally sequestered in tight junctions.

Steve Tracy, Ph.D.
My laboratory is broadly interested in the structure and function of the membrane receptor for coxsackieviruses and adenoviruses (CAR), and in molecular and cellular interactions in hemostasis. This research encompasses studies of protein structure-function relationships, lipid-protein interactions, ligand-receptor binding, enzyme kinetics, and cell biology. My current research investigates molecules that bind group B coxsackieviruses, their effects on virus viability, and how they alter the interaction of the viruses with the CAR and with cells. The coxsackievirus and adenovirus receptor (CAR) is an integral membrane protein and a member of the immunoglobulin superfamily. I am increasingly curious about selective pressures that drive these viruses to bind molecules other than the CAR (Carson, Chapman, Hafenstein, and Tracy, 2011).

Other Infectious Diseases Research

Steven Hinrichs, M.D.
Research projects in Dr. Hinrichs' laboratory are focused on three distinct areas of interest: molecular microbiology, regulation of gene expression and protein structure. A method has been developed for inhibiting tumor specific proteins that initiate cancer in certain types of tumors. The method incorporates knowledge regarding biochemical abnormalities in cells following changes in the chromosome, called translocations. These chromosomal events result in the production of new combinations of proteins that do not exist in normal cells. These combinations, termed chimeric proteins, are optimal targets for development of therapeutic molecules. A similar project is focused on identifying chemicals that could be used as antibodies. The drug target is primase, an essential enzyme that generates abort primers during DNP replication. Significant advances have been made using structural biochemistry and molecular modeling to develop lead compounds that were used to test and develop a model system. This work has lately evolved into a new project focused on inhibiting the activity of essential replication enzymes in bacteria. The project envisions the need for a new class of antibiotics in the future or for antibiotics that can be used to treat genetically engineered bacteria released by terrorists. Molecular modeling was used to suggest targets for mutagenesis and confirmation of function in vitro. The work involves collaboration with a wide range of scientists from electrical engineering to chemistry. A separate project is focused on the development of the next generation of molecular diagnostic tests for the detection and identification of infectious diseases.

Peter Iwen, Ph.D.
Research activities in my lab include the development and optimization of molecular assays for the identification of microbial pathogens with an emphasis on fungi and bacteria (including the Mycobacterium species). Original work in my laboratory showed that the internal transcribe spacer region within the rDNA complex was a useful target for the identification of both the common and uncommon fungal and mycobacterial species. Subsequent work has continued on providing sequences of a wide variety of microbial species and using this information in collaboration with bioinformatics researchers to develop curated databases that can be used with different molecular platforms for identification purposes.

Guangshun Wang, Ph.D.
My research focuses on the identification, characterization, and engineering of novel antimicrobial agents based on structural, bioinformatics, and functional studies. Our ultimate goal is to develop novel compounds that curb pathogenic microbes, especially difficult-to-kill microbes such as methicillin-resistant Staphylococcus aureus (MRSA).

Naturally occurring antimicrobial peptides (AMPs) are universal effector molecules that directly eliminate invading pathogenic bacteria, fungi, viruses, and parasites. In mammals, including humans, such peptides may also modulate the adaptive immune systems. To date, more than 1600 AMPs have been identified in bacteria, fungi, plants, and animals. To better manage this information, my laboratory has established the Antimicrobial Peptide database (APD) as a tool for AMP naming, classification, search, statistical analysis, prediction, and design. Our database is also a useful resource for developing novel antimicrobial agents. These miniature proteins are capable of adopting a variety of three-dimensional structures, inspiring our design of natural mimics that benefit mankind. The objective of one of our NIH-funded projects is to develop AMPs into novel anti-HIV microbicides in collaboration with ImQuest BioSciences.

We are particularly interested in an in-depth understanding of the functional roles of human AMPs and their relationships with human diseases, including cancer. Recently, we have solved high-quality structures of human cathelicidin LL-37 and its important fragments by multidimensional nuclear magnetic resonance (NMR) spectroscopy. Dioctanoyl phosphatidylglycerol (D8PG) has been established as a new and unique membrane-mimetic model, which enables the detection of Phe-PG and Arg-PG interactions. Based on three-dimensional structures, we have identified the most potent region within LL-37 against MRSA, thereby identifying a useful template for designing novel therapeutic compounds against this superbug (US Patent 7,465,784). To elucidate the mechanism of action, we are utilizing a variety of biophysical and biochemical techniques. Our studies will lay the foundation for peptide engineering with a goal to overcome the hurdles (stability, toxicity, and production) on the way to the development of natural AMPs into novel therapeutics.

Another research direction of high interest to us is to engineer molecules that control protein-mediated signal transduction pathways essential for bacterial survival or infection in collaboration with colleagues in the Center for Staphylococcal Research. The lead compound will be optimized by combining NMR-based library screening with rational design based on three-dimensional structures of protein-protein complexes.

Prabagaran Narayanasamy, Ph.D.
The Narayanasamy Laboratory is working on pulmonary tuberculosis, caused by TB, is contagious to health, with over 2 billion people infected with latent TB and more likely to develop into active TB in presence of HIV infection. Gram-negative bacteria have intrinsic abilities to develop new mechanisms of resistance and can pass along genetic materials that allow the next generation of bacteria to become drug-resistant as well. Thus, there is a critical need for new antibiotics and drug delivery techniques to meet the needs of patients now and in the future. Our lab is working on asymmetric synthesis of drug compounds, drug discovery against drug resistant pathogens targeting MEP pathway, menaquinone pathway and iron metabolism. The property of cyclic diphosphate is also studied thoroughly. In addition we also use nanoparticle technology to target drug resistant pathogens and co-infection in macrophage. In addition we also discovered that Ga nanoparticle could be used as a single drug to treat HIV-TB co-infection. We will be optimizing the Ga nanoparticle to determine best results by in vitro and in vivo. Ultimately, these data could lead to the discovery of new drugs or techniques to treat drug resistant infections, with the potential that other human pathogens could be susceptible as well. In addition we also work in neurons development. Methyl glyoxal, a toxic material generated by glycolysis in brain, affects the survival of neurons. To protect the neurons we enhance the glyoxalase pathway to detoxify the methyl glyoxal. The successful flavanol antioxidants from our preliminary studies in cerebellar neurons will be tested for reducing aging and autism disorder in animal models.