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 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. My research focuses on the physiological function of CAR, the function of tissue factor in blood coagulation, and related cell biology. This research encompasses studies of protein structure-function relationships, lipid-protein interactions, ligand-receptor binding, enzyme kinetics, and cell biology.

The coxsackievirus and adenovirus receptor (CAR) is an integral membrane protein and a member of the immunoglobulin superfamily. It was identified due to its role in facilitating virus binding and infection, and was subsequently shown to mediate cell-cell adhesion. CAR localizes to cell-cell junctions, and is structurally related to Junctional Adhesion Molecules (JAMs). While interested in how CAR functions as a virus receptor, I am also interested in further characterization of its physiological function, including identification of natural ligands and cellular biochemical pathways in which it participates.

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 has had as its focus for the past 30 years, the biology of the group B coxsackieviruses (or CVB), a group of 6 human enteroviral serotypes (CVB1-6) that are common causes of serious human diseases. With there being at present on the order of 100 known different human enterovirus (HEV) serotypes - and this list is continually expanding - a need exists to understand the relationship of important HEV to diverse human diseases. The CVB are a superb model system to study pathogenesis as well as basic issues of HEV replication and evolution. The CVB can replicate in nearly all human and murine cell cultures as well as in mice, thus providing the investigator both in vitro and in vivo systems as tools. Various human diseases caused by CVB such as myocarditis (inflammation of the heart muscle), pancreatitis (pancreatic inflammatory disease), and type 1 (juvenile or insulin-dependent) diabetes (T1D), are able to be modeled in appropriate mouse models. Various populations of viruses have been characterized as particularly virulent (disease causing) or avirulent, with representative strains having been molecularly cloned as infectious cDNA copies of the viral genome. This laboratory continues to be instrumental in characterizing the CVB at the molecular, immunological, and pathological levels. A recent compendium dealing with these fascinating human viruses, with chapters by written by noted scientists in their field, was recently published [Group B Coxsackieviruses. Current Topics in Microbiology and Immunology, volume 323. S Tracy, KM Drescher, MS Oberste, editors. 2008].

Type 1 diabetes and CVB infections
The CVB have long been linked as causative agents of human type 1 diabetes (T1D). To facilitate study of the role of viruses as triggers of this disease, we defined two relevant models for understanding the role that HEV may play in human T1D development. Although the CVB replicate well in all mice, these viruses only induce T1D in the nonobese diabetic (NOD) mouse strain. The NOD mouse naturally develops autoimmune T1D on its own starting around 15 weeks of age. After a great deal of fruitless work trying to induce T1D in mice of diverse genetic backgrounds and finding we could only accomplish this in NOD mice, we feel that viral instigation of T1D is a dance with two partners: the host and the virus. The host with a genetically determined component - - autoimmune attack on its insulin-producing cells in the pancreatic islets - enables the virus replication in the same islet cells, resulting in rapid diminution of insulin-producing beta cells and T1D onset. The NOD mouse develops its autoimmune disease over time, such that the islets of young (3-5 week old) mice appear normal but as the mouse ages, more and more inflammatory cells appear in the islets and beta cell function declines. By 15 weeks of age or so, NOD mice are beginning to die of frank T1D. We showed that inoculation of young mice still without insulitis (inflammation of the islets) with CVB resulted in protection of these mice from developing T1D later in life. This was a fascinating finding and certainly news. Instead of causing T1D, the viruses halted the disease development. More mice failed to develop T1D which had received more virulent virus strains than mice inoculated with less virulent strains, suggesting that the virus load (possibly by antigen presentation) played a role in the extent of protection. The mechanism behind this observation has recently been explained by others, who have shown that upregulation of regulatory T cell populations can occur as a function of virus exposure, with the outcome that mice are protected from T1D development. We also showed that using the same viruses but inoculating older NOD mice with developing autoimmune disease, T1D onset rates could be rapidly increased. The difference between protection from T1D and promoting rapid onset of T1D, lay in the state of the host's autoimmune attack on its own islet cells. The virus is an opportunist, which takes advantage of susceptible host cells as they present themselves. Islets without inflammation are resistant to destructive CVB replication, while islets in older mice with ongoing inflammation, are highly susceptible to productive virus replication and resultant cell death. Thus, we postulate that in humans, human enterovirus (HEV) induced T1D can occur only in people with a genetic predisposition to developing T1D and further, only in those people with developing autoimmune inflammation of the pancreatic islets. This explains why despite these viruses being quite common, we are not all subject to developing T1D and why the disease is relatively rare. These findings are consistent with what we know of human history and ecology as well as the biology of these viruses and were recently reviewed (Tracy et al. [2010] Enteroviruses, type 1 diabetes and hygiene: a complex relationship. Rev Med Virol 20:106-116).

Deleting genomic sequence at the 5' terminus: persistence defined
The CVB cause acute infections: that is, the virus infects, sometimes induces a nasty disease, but is then cleared by the adaptive immune response and does not persist in the immune host. However in some cases, HEV have been observed to persist for long periods of time (weeks to months) in heart tissue of naturally-infected humans as well as in experimentally-inoculated mice. We recently discovered the novel mechanism by which the CVB accomplish this trick: the virus deletes variable lengths of sequence from the 5' terminus of the genome inward, resulting in a population of viruses which have deletions from 7-49 nucleotides in length. These deletions greatly slow virus replication, deleteriously affects positive strand viral RNA replication which in turn results in nearly equal positive/negative RNA strand ratios in infected cells and results in the detection, for the first time, of negative stranded viral RNA encapsidated in purified CVB virions. This observation indicates there may not be a specific RNA packaging signal but that viral RNA packaging is more dependent upon the local concentration of RNA when the virions begin to assemble. Recent results further demonstrate such deletions can occur in a CVB infection of human heart tissue, demonstrating clinical relevance. That we have observed these naturally occurring deletions arising only in quiescent primary cell cultures indicates there is a difference between classical cell lines, usually used to study HEV, and primary cultures of cells with defined lifetimes. The mechanism by which the deletions initially occur and propagate, has only recently been outlined, findings which resulted in being awarded an NIH research grant (Dr. Nora Chapman, principal investigator). Despite the fact that these terminal deletions result in a virus population that is profoundly inhibited in replication efficiency, they represent a novel reaction by the virus to finding itself in a hostile environment, host cells in which a key cellular protein is missing or very low in abundance. That such deletions occur may be a type of genetic memory, which results in the virus population reverting to a more primitive version of the enterovirus genome. In addition to helping to understand how HEV replicate in their host cells, these deletions may also point to a novel approach to understanding HEV evolution. 

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.