Lea (Spyres) Daniel: Research Interests

Bacterial Communication
My research interests are currently focused on interspecies and intraspecies communication in bacteria commonly referred to as quorum sensing.  Quorum sensing was originally discovered in Vibrio fischeri where it was determined that small molecules (N-acyl-L-homoserine lactones (AHLS)) called autoinducers were secreted into the environment.  These molecules diffuse freely into and out of the bacteria and once these molecules reach a critical threshold, or the culture reaches a critical density, the expression of genes responsive to this system are induced.  Since this original observation in the 1970s, different quorum sensing systems have been discovered in numerous gram positive and gram negative bacteria.  Many pathogens including Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella typhimurium have been suggested to control expression of certain virulence factors through quorum sensing mechanisms.  I am particularly interested in understanding and eventually modulating quorum sensing pathways as a novel means for treating bacterial infections focusing on the pathogen Clostridium difficile. 

Antibiotic Resistance and Clostridium difficile
The continued increase in antibiotic resistant bacteria has become progressively more evident with the recent spread of methicillin resistant strains of Staphylococcus aureus (MRSA).  Many other bacteria including Mycobacterium tuberculosis (tuberculosis) and Neisseriae gonhorrea (gonhorrea) are also developing resistance to antibiotics necessitating new strategies for treating these infections.  One such organism is C. difficile.  Like MRSA, C. difficile is mainly found in hospitals but has recently been spread in communities.  The incidence of C. difficile infections is increasing world-wide and the disease has become endemic in many elderly care centers.  As well as becoming more prevalent, C. difficile strains with increased virulence have also become more common.  The increase in the incidence and severity of C. difficile disease combined with an aging population and the ease of spread of the bacterium in the hospital environment makes research into the control of this disease eminent. 

Regulation of Toxin Production
Two large toxins produced by C. difficile are the primary cause of antibiotic associated diarrhea and the sometimes fatal psuedomembranous colitis.  These two large toxins enter into and modify mammalian cells contributing to the symptoms of these diseases.  Some individuals may be colonized but experience no signs of disease, while disease in other infected individuals may range from mild to fatal.  Since the toxins produced by C. difficile are responsible for the symptoms of disease, identifying the factors that influence toxin production could lead to ways to control disease without the use of traditional antibiotics.  Toxin production is currently believed to be regulated by complex interactions between the human immune system, normal flora of the colon, and C. difficile in colonized individuals.  The disease could be controlled, for example, by administering probiotics, or inhibitory molecules produced by normal flora or the human immune system. 

Disease Control
An under investigated area in C. difficile research is both interspecies and intraspecies interactions that regulate gene expression, specifically virulence factors such as toxin production.  I am exploring the importance of some of these interactions, starting with suppression of toxin production by common normal flora of the colon.  These experiments are simple to carry out and involve growing C. difficile in the presence or absence of extracts from normal flora, then using western blotting, Elisa, and real-time PCR to monitor the amount of toxin produced under the different conditions.  These inhibitor molecules will eventually be identified and isolated from the toxin suppressing normal flora then used to treat individuals who have succumbed to C. difficile associated disease.

Publications and Patents
1. Hegde V, Wang M, Mian IS, Spyres L, Deutsch WA The high binding affinity of human ribosomal protein S3 to 7,8 dihydro-8-oxoguanine is abrogated by a single amino acid change. DNA Repair 5(7):810-5 (2006)
2. Lea Spyres, Sally Gaddis, Ella Bedford, Stacey High, Nikki Liburd, K. Leslie Powell, Jeff Drake, Howard Thames, David Mitchell, Earl Walborg, Mahmoud Rouavhia, Aysegul Sahin, C. Marcelo Aldaz, Michael C. MacLeod Sensitive quantitation of p53-regulated gene expression in human epithelial cells and breast tumors by Q-RAGE. Anal Biochem. 345(2):284-95 (2005).
3. Lea M. Spyres, Jeremy Daniel, Amy Hensley, Maen Qa’Dan, William Ortiz-Leduc, and Jimmy D. Ballard  Mutational Analysis of the Enzymatic Domain of Clostridium difficile Toxin B Reveals Novel Inhibitors of the Wild-Type Toxin.  Infection and Immunity 71:3294-3301(2003).
4.  Ballard, J. D. and Spyres, L. M. (2003) Mutants of Clostridium difficile toxin B and methods of use.  US PATENT # 7,226,597.  Washington D.C.:  U.S. Patent and Trademark office.
5. Maen Qa’Dan, Matthew Ramsey, Jeremy Daniel, Lea M. Spyres, Barbara Safiejko-Mroczka, William Ortiz-Leduc, and Jimmy D. Ballard  Clostridium difficile toxin B activates dual caspase-dependent and caspase-independent apoptosis in intoxicated cells. Cellular Microbiology 4:425-434 (2002).
6. Lea M. Spyres, Maen Qa’Dan, Amy Meader, James J. Tomasek, Eric W. Howard, and Jimmy D. Ballard  Cytosolic Delivery and Characterization of the TcdB Glucosylating Domain by Using a Heterologous Protein Fusion. Infection and Immunity 69:599-601 (2001).
7. Maen Qa’dan, Lea M. Spyres, and Jimmy D. Ballard pH-Enhanced Cytopathic Effects of Clostridium sordellii Lethal Toxin. Infection and Immunity 69:5487-5493 (2001).
8. Maen Qa’Dan, Lea M. Spyres and Jimmy D. Ballard pH-Induced Conformational Changes in Clostridium difficile Toxin B. Infection and Immunity 68:2470-2474 (2000).