Microbial genetics; gene expression during bacterial differentiation, RNA polymerase-promoter interactions.
The work in our laboratory focuses on the control of gene expression during bacterial differentiation. Asa the bacterium Bacillus subtilis differentiates from the vegetative form into a dormant endospore, complex morphological and physiological changes occur that require the expression of many genes. During the process, new RNA polymerase sigma subunits appear (oF, oE, oG, oK), displacing one another and conferring on the RNA polymerase different specificities for the recognition of different classes of promoters. One focus of our laboratory is to elucidate the mechanisms that regulate sigma factor function. For example, the DNA binding protein SpoOA responds to environmental signals by activating the transcription of several key operons at the onset of sporulation. We are currently testing the model in which SpoOA, when bound to promoter DNA, interacts directly with the RNA polymerase sigma subunit. We are also studying an example of regulation of gene expression by a morphological cue. During sporulation B. subtilis divides into two compartments (forespore and mother cell) that follow different developmental paths. Forespore-specific transcription is initiated by oF-RNA polymerase, and results in the forespore-specific production of oG, which directs the subsequent forespore-specific transcription. However, oG does not become fully active until engulfment of the forespore is completed. We want to know how the activity of oG is coupled to this morphological change. We have shown that the anti-sigma factor SpoIIAB may play an important role, and now we are attempting to identify additional genes whose products regulate oG activity. The utilization of gene products during the assembly of the complex morphological structures of the spore is governed both by the order of their synthesis, and by the order of their assembly into these structures. It is not known how these two mechanisms are coordinated. Transcription of several genes encoding spore coat proteins is directed by oK, the last o of the cascade. However, premature synthesis of spore coat proteins does not result in the premature assembly of spore coat-like structures. We are attempting to elucidate the mechanisms that regulate the utilization of spore coat proteins.

Mechanisms of cell to cell signaling and quorum sensing in bacteria.
My lab is interested in the mechanisms of cell to cell signaling or quorum sensing in bacteria. Bacteria produce small chemical signals (pheromones or autoinducers) which regulate gene expression when they reach a critical concentration. We are using Escherichia coli and Proteus mirabilis as model systems to study this process.

Our studies in E. coli involve: (i) purification and structural characterization of extracellular signaling molecules, (ii) identification of genes involved in signal response, (iii) identification of genes involved in signal production, and (iv) the use of microarrays to identify genes activated/repressed by extracelluar signals.

In P. mirabilis, we are focused on the same general goals described above for E. coli. In addition, we are addressing the role of cell to cell signaling in biofilm formation and virulence gene expression via swarming motility.

Microbial physiology and genetics; biofilm development; post-transcriptional regulatory mechanisms.
My primary scientific interest is in the ways that microbes sense changes in the environment and respond by modifying their metabolism and behavior. Of greatest interest are responses that produce sweeping changes in phenotypic properties, mediated by the so-called "global regulatory systems" of bacteria. The global regulatory systems coordinate the expression of numerous genes that are distributed throughout the bacterial genome. A hallmark of these systems is that they are not individually isolated, but communicate in various ways with each other to form signaling networks, in which each participant system influences the others. A recent focus of our attention is the regulatory and biochemical mechanisms that guide the development of bacterial biofilms. Biofilms are communities of microbes attached to a surface or interface and enclosed in a polysaccharide matrix, which the microbes secrete. Biofilms block immune clearance, compromise antimicrobial therapies and are estimated to complicate over 60% of serious bacterial infections. An understanding of the workings of the global regulatory networks involved in biofilm formation and dispersal promises to offer practical solutions to numerous problems in medicine, agriculture, biotechnology and other industries, and environmental sciences.

G-protein-coupled receptor-mediated signal transduction and its role(s) in development.
G protein-coupled signaling plays a major role in regulating cell movement and cell behavior. These signals effect things from neuronal growth cone extension to immune cell function to embryonic cell movement to cancer metastasis. The Saxe lab focuses on the molecular mechanisms that underlie these signals. The model used is the cAMP signaling system in the eukaryotic microbe Dictyostelium. Extracellular cAMP signaling is known to effect changes in gene expression, morphogenetic cell movements and pattern formation in this organism. All of these effects are mediated through a family of four receptors that show temporal and spatial differences in distribution as well as differences in affinity for ligand. We have taken a genetic approach to defining the signaling pathways regulated by two of these receptors, cAR1 and cAR2. In particular we have isolated mutations that effect signaling between the receptors and the actin cytoskeleton. Among the genes identified is Scar (suppressor of cAR defect) which has revealed a widely conserved family of actin regulating proteins. Scar proteins are found in organisms from Dictyostelium to humans and play a critical role in regulating actin polymerization at the leading edge of motile cells and in endocytosis. Scar is related to WASp, the protein defective in the human immunodeficiency disease, Wiskott-Aldrich Syndrome. We are using a variety of molecular, genetic, biochemical, immunological and microscopic techniques to fully characterize the relationship between receptor signaling and Scar/WASp directed cell movement (e.g. chemotaxis). We have established that the basic mechanisms that regulate Scar/WASp function in Dictyostelium are virtually completely conserved in metazoan systems. We are extending our studies to these Scar/WASp regulators with the intent of providing fundamental information on receptor to actin cytoskeleton regulation.

Multidisciplinary antiviral research is aimed at discovering agents that could be used for the treatment HIV infections.
The major research emphasis of the Laboratory of Biochemical Pharmacology is in two medically important areas. First, the group focuses on the development of antiviral agents for the treatment of infections caused by human immunodeficiency viruses, and hepatitis viruses.

Work involves molecular modeling, synthetic, biochemical, pharmacological, and molecular approaches, including gene therapy and site directed mutagenesis. The main objective is to develop compounds for the prevention and treatment of these important diseases. Areas of particular interest include the characterization of drug-resistant virus variants and ways to overcome resistant viruses using combinations of drugs. Four compounds developed by this group have gone on to advanced clinical studies, and three have already been approved by the FDA for the treatment of HIV-1 infections.

The multidisciplinary antiviral research is aimed at discovering agents that could be used for the treatment HIV infections, and modalities aimed at preventing the development of drug-resistant viruses. Current research is in the fields of HIV, SIV, HBV, HCV, herpesviruses, and cryptosporidium. The ongoing work is primarily funded from a VA Merit Award; the NIH sponsored Emory University Center for AIDS Research (CFAR), several NIH grants.

Molecular mechanisms of bacterial virulence; control of gene expression in bacteria.
We are using molecular biological and microbial genetic techniques to study the molecular mechanisms of bacterial pathogenesis. Currently, we are focusing on two important human pathogens. We are studying the major virulence determinants of the group A streptococcus (S. pyogenes) and the regulation of their synthesis. We have developed the genetic tools (transposons, regulatable promoters) to ask about regulation and are using mouse models to assess virulence. The hope is that a greater understanding of the disease process will lead to improved approaches to prevention. The second pathogen we are currently investigating is enterotoxigenic Escherichia coli. We are studying the regulation of expression and the morphogenesis of the unique pili responsible for its attachment to the human gut. The pilus is composed of many copies of a single protein and we have found that it contains a minor protein at its tip. It is possible that this tip protein may be a good vaccine candidate. The proteins of the pilus and those needed for its assembly are different from those of other types of pili and we are working on improving our molecular understanding of this process. In addition, regulation of the synthesis of the proteins needed for these structures is complex: there is a silencing effect and the activator Rns, which is itself autoregulated, is needed for transcription. We are investigating the molecular details of this process, which appears to be a prototype for global regulation of virulence factors of many enteric pathogens.

Website: http://www.microbiology.emory.edu/scott/index.htm

Genetics of antibiotic resistance; antimicrobial peptides; transcriptional regulation of gene expression; mechanisms of bacterial pathogenesis.
We are interested in the molecular mechanisms of bacterial pathogenesis. In particular, research in our laboratory seeks to understand how Neisseria gonorrhoeae evades the antimicrobial action of host compounds that bathe mucosal surfaces and antibiotics. We have identified a gene cluster in gonococci that encodes four membrane proteins that form an efflux pump. This efflux pump exports structurally diverse antimicrobial compounds including drugs and detergent-like compounds (e.g., fatty acids and bile salts). We have discovered that expression of these genes is regulated by both cis- and trans-acting mechanisms. The trans-acting control element is a DNA-binding protein that behaves as a transcriptional repressor, while the cis-acting element is a 13 base pair inverted repeat sequence that lies within the promoter region of the repressor gene. This efflux pump operon is also subject to positive regulatory elements, such as a transcriptional activator protein termed MtrA. We are now in the process of determing the genetic and physiologic basis by which efflux pumps are over-produced in response to environmental signals. We are also interested in the mechanisms by which antibacterial peptides produced by white blood cells and certain epithelial cells that line mucosal surfaces exert their activity. We have studied the structure-function relationships of a number of peptides and have constructed mutant strains of Staphylococcus aureus that display decreased susceptibility to their killing activity. We are now characterizing the bacterial genes that seem to modulate bacterial susceptibility to these host-defensive peptides.

Molecular genetic analysis of Mycobacteria.
Tuberculosis and leprosy are important human disease that afflict more than 50 million individuals world-wide. The etiologic agents of these diseases are Mycobacterium tuberculosis and Mycobacterium leprae, respectively. Both of these mycobacteria are intracellular pathogens that grow within cells of the host immune system, primarily macrophages. Relatively little is known about the genes and gene products required for intracellular survival. Our research in this area concentrates on development and application of biophysical and genetic tools and strategies to identify mycobacterial genes that play roles in intracellular survival and replication. Current projects include using promoter-trap vectors and microarray hybridization approaches to identify differentially expressed genes.

We are also taking advantage of the recently published genome sequence of M. tuberculosis to direct studies to characterize gene expression in tubercle bacilli. Two-component global regulatory systems and sigma factors are being studied to elucidate details of the regulation of gene expression and characterize patterns of gene expression. The ultimate goal is to elucidate the mechanisms that underlie the transition from an active infection to a latent infection and from a latent infection to an active infection.

Pathogenesis of gamma-herpesviruses and development of lymphoma and other cancers.
The research in my lab focuses on 2 gamma-herpesviruses, Epstein-Barr virus (EBV) and murine gamma herpesvirus 68 (gHV68). A major property of all herpesviruses is their ability to persist for life in the infected individual. The gamma-herpesviruses are known to latently infect either B or T lymphocytes, and to be associated with the development of lymphoma and lymphoproliferative diseases. Our major interests are to understand: (i) how these viruses regulate viral gene expression during latency; (ii) how they modulate and avoid the host immune response; and (iii) how they switch from a latent infection to replication of the viral genome (referred to as reactivation), a process that is essential for propagation of these viruses to uninfected individuals. EBV is the etiologic agent of infectious mononucleosis and is closely associated with the development of Burkitt's lymphoma, nasopharyngeal carcinoma, 30-50% of Hodgkin's disease, and 50% of lymphomas that arise in immunosuppressed individuals (e.g., transplant patients and AIDS patients). Our research on EBV focuses on tissue culture models that recapitulate the various EBV genetic programs. The information gained from these studies is then employed to address the behavior of EBV in infected individuals. However, because there are no small animal models for studying EBV pathogenesis, we use gHV68 infection of mice to address specific issues of the host response to gamma-herpesvirus infection. The advantage of the latter model is that both the host and pathogen can be genetically manipulated to address fundamental aspects of host-pathogen interactions. gHV68 infection of mice causes several different chronic diseases in immunocompromised mice, including a severe vasculitis that affects the great elastic arteries and lymphoproliferative disease. We are currently identifying gHV68 genes involved in establishing and maintaining viral latency, as well as those involved in the development of chronic disease. In addition, we are actively characterizing the host response to viral infection to address how viral latency and persistent infection is controlled.

Website: http://www.emory.edu/MICROBIO/speck

Functions of the influenza hemagglutinin in host cell entry; influenza assembly.
The Steinhauer laboratory is primarily interested in influenza virus entry into host cells and the role of the hemagglutinin glycoprotein (HA) in this process. The work has a strong focus on structural and functional studies of the HA protein, particularly with regard to its receptor binding and membrane fusion properties. The work combines protein structure analysis and molecular virology techniques to address specific questions on how influenza viruses attach to cells, deliver their genomes, assemble at the end of the replication cycle, and evolve to evade host immune responses and the action of antiviral drugs. We are also attempting to exploit our knowledge of high resolution HA structures to design novel vaccines for influenza, and for other pathogens using influenza as a vector.

Genetic basis and regulation of bacterial virulence components.
Our work is focused on genetic determinants of bacterial pathogenesis in Neisseria meningitidis and Streptococcus pneumoniae important causes of meningitis and bacteremia. These studies include the molecular mechanisms of attachment, colonization and invasion of human mucosal surfaces by pathogenic bacteria and conjugative transposons and role of transposons in bacterial virulence. We are also studying innate immunity and microbial pathogen interactions. We are examining the genetic, structural and pathogenic basis of meningococcal lipopoly(oligo)saccharide, meningococcal pili expression and meningococcal capsule and Toll-like receptor interactions with these virulence factors. A better understanding of how pathogenic bacteria cause disease is needed for new strategies for the design of vaccines that will protect against serious bacterial infections.

Regulatory mechanisms of gene expression; meningococcal pathogenesis.
The long-term goal of my research is to elucidate the regulatory mechanisms of virulence determinants in meningococcal pathogenesis and understand the signal transduction pathways by which meningococci sense and interact with the host. By directly studying these issues, my group continues to provide a fuller knowledge base for the development of vaccine strategies and therapeutic interventions. Currently, we focus on a novel two-component signal transduction system shown to be a global regulator mediating the expression of meningococcal virulence determinants including the structural modification of endotoxin, iron uptake and assimilation, and protein folding machinery. Using genetic, biochemical and molecular biological strategies we hope to provide not only a broad view of the regulatory scope of this important signal transduction system, but also a detailed understanding of both the molecular regulatory mechanisms and the interaction of this network with other regulatory control of gene expression. Furthermore, efforts are also focused on identifying the host signal that activates this two-component regulatory system.

Prevention and repair of DNA damage in prokaryotes; enzymes and their genetic regulation.
My major research interest is in DNA damage and repair in Escherichia coli. Its ease of genetic manipulation has enabled the identification, in our laboratory, of the biological roles of various repair endonucleases, several of which are almost universal in their distribution. There are currently two major projects.
The soxRS regulon. We found that a DNA repair enzyme, endonuclease IV, is induced by agents that generate superoxide. This led to our discovery of two new genes, soxR and soxS, that control an oxidative stress regulon of which endo IV is a part. soxR is a redox-sensitive transcriptional activator with an iron-sulfur center that is its sensor. We are now attacking the following problems: (a) Identification of other members of the regulon - we have been successful in making some good guesses based on protein function, but we now plan to switch to DNA array technology. (b) Identification of the SoxR reductases, the enzymes that keep the Fe-S center of SoxR in a reduced (transcriptionally inactive) form in uninduced cells. (c) Modification of SoxR to render it more soluble so that it can be used for X-ray crystallographic and NMR studies; our aim is to see how oxidation of the protein alters its structure and that of the DNA to which it is bound so that SoxR activates transcription.
Endonuclease V. This interesting enzyme cleaves DNA near any region where unpaired bases adjoin a bihelical region: base mismatches, deletion or substitution loops, hairpins, flaps, and pseudo-Y structures. Such simple, small protein of broad specificity has to be a primitive and universal DNA repair enzyme. In addition, it cleaves DNA containing hypoxanthine (deaminated adenine) and xanthine (deaminated guanine). We isolated a mutant and found as its only defect an unusual susceptibility to mutagenesis by nitrous acid, a deaminating agent. Nitrate and nitrite are preferred electron acceptors during anaerobic growth, and our hypothesis is that the enzyme evolved to repair the damage produced by the nitrosative by-products of anaerobic metobolism, just as the DNases evolved to repair the damage from the oxidative by-products of aerobic metabolism. We have found that nfi, the gene for Endo V, is induced by nitrite, low pH, acid permeants, nitrite, nutrient limitation, growth to late logarithmic phase, and oxygen limitation. Using an nfi-lacZ gene fusion, we hope to isolate regulatory mutants and identify the controlling genes. We are also further examining the mutator phenotype of nfi; preliminary results suggest that it is manifest during anaerobic growth under physiological conditions.

Molecular mechanism of melanoma and prostate cancer metastasis and development of viral vaccines.
UC18 (MEL-CAM/CD146), has been postulated to play an important pathogenic role in metastatic melanoma progression. To study its role in mediating metastasis, we have used RT-PCR to amplify and clone the human MUC18 cDNA gene and used RACE-RT-PCR to amplify and clone the mouse MUC18 cDNA gene. We have produced both recombinant proteins in a bacteria GST expression system and purified them for making polyclonal antibodies in chickens. We have also cloned these genes into a mammalian expression vector. We have transfected the expressible mouse cDNA gene into a murine melanoma cell line that does not express MUC18 and obtained G418-resistant clones that expressed high levels of MUC18. We tested the effect of expression of MUC18 on induction of lung metastasis in syngeneic mice by injection of these MUC18-high-expression clones via i.v. and via s.c. routes. We found that these high-expression clones induced efficient lung nodule formation (metastasis) via only the i.v. route, suggesting that MUC18 is important for metastasis. Surprisingly, we found the expression of MUC18 in three mouse melanoma cell lines have tumor suppression effect. We are in the process of studying the mechanism of induction of metastasis by MUC18 in vivo. We are also trying to identify the heterophilic ligand(s) and co-factors of MUC18. We also studied the expression of MUC18 in prostate cancer cell lines and tissues. We found that human MUC18 only expressed in metastatic prostate cancer cell lines, but not in the non-metastatic cancer cell line. Human MUC18 was not expressed in the normal prostatic acinar epithelial cells, or in BPH. But it was highly expressed in precancerous acinar epithelial cells of PIN and in the prostate cancer tissues as well as in metastatic lesions in lung and lympn node. Thus the level of MUC18 expression appeared to increase with increasing pathological grades. We have proposed that MUC18 may also mediate metastasis of prostate cancers. To test this hypothesis, we injected orthotopically(into the prostate gland)the MUC18- exprssing human prostate cancer LNCaP cells in nude mice. We found that increasing MUC18 expression increased the tumor take and metastasis of the cells from prostate gland to various organs (seminal vesicles, ureter, kidney,and peri-aortic lymph nodes. We thus provided evidence that MUC18 also plays an important role in causing metastasis of the human prostate cacner LNCaP cells in xenograft model. Currently we are also collaborating with Dr. Leland Chung’s group on his bone metastasis xenograft model, and with Drs. Chris Gregory and Thomas Pretlow on their CWR22 xenographft model. We are also collaborating with Dr. Norman Greenberg on his TRAMP model and with Dr. Jeff Gordon on his neuroendocrine cells-derived prostate carcinoma transgenic mouse model. We are also collaborating with the members of the Emory Prostate Cancer Center on prostate cancer metastasis. We have also cloned the genomic copies of these genes that contain the 5'-flanking transcription regulatory sequences for studying transcription factors and signal transduction mediators that regulate their expression in normal versus cancer cells. We are also collaborating with other Emory faculty on developing cancer vaccines.

Immunology; role of macrophage in the immune response to intracellular pathogens.
Our laboratory is currently involved in studying the immune response to microbial antigens and the molecular basis of bacterial pathogenesis. Attempts to understand the mechanism by which macrophages process and present antigens to T lymphocytes and how protective immunity to pathogens is achieved are major goals. Experimental approaches include the genetic analysis of bacterial virulence, the analysis of monoclonal T and B cell hybridomas, the use of flow cytometry to study the cytokine expression and cell markers of lymphocytes and macrophages, and the use of synthetic peptides to define antigenic epitopes. We are involved in defining the function and activation requirements of gamma/delta T cells that are present at front lines of defense in epithelial tissues. We are also interested in the regulation of cytokine production such as IL-1, IL-2, IL-4, IL-6, IL-7, IL-10 IL-12, gamma interferon and TNF. IL-12 is especially interesting in that it can act as a potent adjuvant as well as a positive regulator of differentiation of TH1-type T cells intimately involved in immunity to intracellular pathogens. Ultimate goals include the rational design of vaccines effective for prevention of microbial infection. Microbial models include Listeria monocytogenes, Salmonella typhimurium and Mycobacterium tuberculosis. In summary, mechanistic studies of lymphocyte and macrophage function using the modern tools of immunology, cell biology, and genetics form the basis of our research and graduate student training.