William M. Shafer, Ph.D.
Professor of Microbiology and Immunology
The Shafer Laboratory
Our research group is broadly concerned with how pathogenic bacteria escape the action of antimicrobials. These antimicrobials include classical antibiotics and those compounds such as those used by our innate defensive systems to combat infections, such as neutrophils (see the adjacent figure of a human neutrophil engulfing Neisseria gonorrhoeae). Studies on antibiotic resistance have also led us over the years to become involved in basic research on mechanisms of gene control in the pathogenic Neisseria and Staphylococcus aureus. Finally, we have been actively engaged in studies dealing with the role of antimicrobial peptides in innate immunity; the four major areas of study in our laboratory are described in detail below. Our research group, located at the Atlanta VA Medical Center, is currently supported by three NIH (RO1) grants and a VA Merit Award. Our group currently consists of a Senior Research Associate, four Postdoctoral Fellows, four graduate students, four Research Specialists and a Project Coordinator.
Use of Multi-Drug Efflux by Neisseria gonorrhoeae and Neisseria meningitidis
Bacteria use energy-dependent efflux systems to export antimicrobial agents that by-pass their protective outer barriers. These efflux systems are of particular interest to us because they are clinically relevant to the development of bacterial resistance to antibiotics. Our studies dealing with drug efflux pumps in gonococci and meningococci have also shown that they can recognize and export host-derived antimicrobial agents that are important in innate host defense, including antimicrobial peptides produced by phagocytic and or epithelial cells. The capacity of cis- and trans-acting factors to control efflux pump genes has been the central aspect of our work since 1994. We have identified regulons in gonococci that are modulated by four distinct DNA-binding proteins that repress (MtrR, FarR and MpeR) or activate (MtrA) efflux pump genes. As is described below, we will study how these genes contribute to gonococcal pathogenesis and or metabolism.
Pathogenic Mechanisms of Neisseria gonorrhoeae and Neisseria meningitidis
Gonococci and meningococci are strict human pathogens. Their ability to cause frequent (gonococci) or devastating (meningococcal meningitis or septicemia) disease represent novel challenges for investigators interested in virulence factors and mechanisms of microbial disease. Our studies dealing with mechanisms of pathogenesis have relied on ex vivo models such as human neutrophil or antimicrobial peptide killing assays and serum bactericidal assays. We have an established productive collaboration with Dr. Ann Jerse (www.usuhs.mil/mic/jerse.html), whose laboratory has developed a female mouse model for studying gonococcal infections. These models have been instrumental over the past years in helping us ascertain the function of certain gene products or microbial surface structures important in evading host defense mechanisms and will continue to be used (and expanded to cell culture technologies) as the number of genes suspected to encode virulence factors are identified.
Gene Control in Staphylococcus aureus
Staphylococcus aureus is an important pathogen in that it is responsible for greater than 50,000 deaths per year in the United States, a high number of hospital-acquired infections and can frequently express resistance to multiple antibiotics. Our interest in this pathogen focuses on how it responds to environmental stress conditions. In particular, given that it is a substantial problem for commercial and private areas in the food industry, we study the cold shock response in staphylococci. We have observed that this response and certain cold shock proteins (Csps) can have substantial effects on pigment production. Pigment production is a presumed virulence factor because it aids in the resistance of staphylococci to UV light, oxidative killing systems produced during infection and helps in providing resistance to desiccation. We have found that CspA can impact gene regulatory pathways involving the secondary sigma factor (SigB). The mechanism by which Csps control gene expression in staphylococci is under investigation.
Antimicrobial Peptides (AMPs) and Innate Immunity
Effective host defense against infectious microbes functions at several different structural and chemical layers. The capacity of AMPs to be "early responders" during microbial attack is now recognized as an important component of the overall efficacy of host defense. Our studies dealing with gonococci, meningococci and staphylococci and the efforts of others with different pathogens indicate that bacteria can alter levels of AMP-susceptibility. We want to understand if these changes impact virulence. Our studies concentrate on a subset of AMPs termed cathelicidins, which are often produced by phagocytic and epithelial cells. The sole human cathelicidin, LL-37, has both direct killing activity and immunomodulatory ability. Given the serious problem of multidrug resistant bacteria and the reduced productivity of pharmaceutical companies with respect to antibiotic development, we are also interested in the design and testing of novel AMPs that could be exploited as therapeutic agents.
MMG Graduate Students in Training in the Shafer Laboratory
Nazia Kamal (B.S. UC-Riverside). Nazia studies the regulation of a gene in Neisseria meningitidis that encodes a putative toxin that is expressed under iron-limiting conditions.
Paul Johnson (B.S. North Georgia College & State University). Paul studies the regulation of gene expression in Neisseria gonorrhoeae mediated by a putative transcriptional activator termed MtrA.
Alexandra Mercante (B.S. Louisiana State University). Alexandra studies the ability of a DNA-binding protein (MpeR) to control genes in Neisseria gonorrhoeae involved in antibiotic resistance and virulence.
Brea Duval (B.S. University of Florida). Brea studies the cold shock response in Staphylococcus aureus and how it is controlled by cold shock proteins.
The Shafer Laboratory celebrating in Cairns, Australia (September, 2006) during the 15th International Pathogenic Neisseria Conference.
(Left to right, Row 1, Alexandra Mercante [MMG grad student], Bill Shafer, Nazia Kamal [MMG grad student], Paul Johnson [MMG grad student]; Row 2, Jennie Rocco (post-doc) and Jason Folster (post-doc).
MMG Graduate Student Alumni
Claressa Lucas, PhD. June 1, 1992-October, 1996. Program in Microbiology and Molecular Genetics. Dr. Lucas was awarded a Predoctoral Minority Fellowship from the American Society for Microbiology (1995-1996). Dissertation: Physiology and Regulation of mtr-mediated antibacterial resistance in Neisseria gonorrhoeae. She is now a member of the Respiratory Disease Branch at the CDC
Lori Snyder, PhD. October 1, 1995-2001. Program in Microbiology and Molecular Genetics. Dissertation: Variation in the division cell wall synthesis gene clusters of the pathogenic Neisseria spp. Presently, Dr. Snyder is a postdoctoral fellow the University of Birmingham in the United Kingdom.
Wendy Veal, PhD. April 1, 1997-April 29, 2002. Program in Microbiology and Molecular Genetics. Dissertation: Genetic analysis of antibiotic hypersusceptibility in Neisseria gonorrhoeae. She is presently employed by the NIH in the regulatory affairs division of NIAID
Heather Alexander, PhD. October 8, 2003-February 20, 2004. Program in Microbiology and Molecular Genetics. Dissertation: Phase variation modulation and pathogenesis in Neisseria meningitidis. Due to the death of her advisor, Dr. Igor Stojiljkovic, Dr. G. Churchward and I assumed the duties of being Ms. Alexander's dissertation co-advisors. She received her Ph.D. degree in February, 2004 and is now a post-doctoral fellow in International Health with the FDA and CDC.
Andrew Rasmussen, B.S. October 8, 2003-July 25, 2005. Program in Microbiology and Molecular Genetics. Due to the death of his advisior, Dr. Igor Stojiljkovic, Dr. G. Churchward and I assumed the duties of being Mr. Rasmussen's dissertation co-advisors. He is currently a postdoctoral fellow at Yale University School of Medicine