Dr. Fleiszig’s research focuses on the pathogenesis of bacterial infection of the cornea. The long-term goals are to determine why patients who wear contact lenses are prone to infection, and to develop novel preventative/therapeutic strategies based on the knowledge acquired. The approach being utilized is to work towards developing an understanding of how P. aeruginosa, the bacterium most commonly isolated from contact lens related infections, interacts with the epithelial cells that line the surface of the cornea on which contact lenses are placed. P. aeurginosa causes sight threatening pathology in the eye and life threatening infections at other sites, including serious lung disease in people with cystic fibrosis or HIV, and serious skin infections in burns victims. Thus, this line of research could ultimately lead to new means to prevent or treat several types of disease.
Specific projects include:
1. Determining how the healthy corneal surface resists infection and how contact lens wear then compromises those defenses.
This long standing aim is focused on understanding the molecular factors that prevent bacterial penetration of the corneal epithelium when the eye is healthy, how the functionality of that defense system is modulated, and the bacterial factors that enable penetration when the system is compromised. Traditionally researchers have used “infection models” to study tissue interactions with bacteria. These involve deliberately compromising the tissue to induce enable susceptibility to infection. During the past few years, the Fleiszig lab has worked towards developing new models that do not bypass the need for bacteria to penetrate the epithelium, which is the event the laboratory seeks to understand. The Fleiszig laboratory has also successfully developed several in vivo and in vitro methods that together enable epithelial cell penetration by bacteria to be studied. In addition, novel imaging technologies are now enabling bacterial interactions with the epithelium to be imaged in living intact eyes.
Utilizing these technologies, in the past year the laboratory has been working towards determining virulence factors used by bacteria to traffic through the epithelium when it becomes susceptible. New data show that the type III secretion system, a toxin delivery system that bacteria use to inject proteins across host cell membranes, is critical for bacteria to penetrate the corneal epithelium.
In the past year, the laboratory has continued its efforts to develop therapies to prevent any type of infection of any body site based on eye derived molecules. The first year of the project revealed that human tear fluid modifies the biology of the corneal epithelial cells to enhance their resistance to microbes. In the past year, the laboratory worked on understanding the mechanisms by which tear fluid modifies epithelial cell immunity. Data show that tear fluid alters the expression of microRNAs, a rapid method by which gene expression can be modulated within tissues. Further, the laboratory has discovered that the corneal epithelial cells express a previously unknown class of antimicrobial peptides. The existence of these molecules helps to understand how epithelia resist infection, and could be used to developed new therapies to prevent or treat infection. Laboratory members who have contributed to this work are Drs. Connie Tam and James Mun, and UC Berkeley undergraduate student volunteers Gary Chan and Jong Hun Kim.
Another project in the laboratory is to understand the relationship between dry eye and susceptibility to infection. The knowledge gained is furthering our understanding of how the eye normally defends itself against infection, while also contributing to our knowledge about how dry eye impacts the biology of the ocular surface. New data show that dry eye in a healthy mouse does not predispose the eye to infection, and that this is related to upregulation of known defense factors.
This project is being conducted by Assistant Specialist Susan Heimer, who is also an Assistant Professor at Touro University-California College of Pharmacy. She is being assisted by undergraduate volunteer Kelsey Li-Chinn Liu.
2. Fundamental studies of bacterial/epithelial cell interactions using P. aeruginosa as a model organism.
The goal of the project is to determine the mechanisms used by invasive P. aeruginosa to survive inside corneal epithelial cells, and the relevance of intracellular survival to disease. Previously, the laboratory had found that ExoS, a protein encoded only by invasive P. aeruginosa strains, enables this capacity involving its ADP-r activity. New data show ExoY, another protein also secreted by invasive strains into host cells can also participate, but that it uses a completely different enzymatic (adenylate cyclase) activity. Other data reveal new insights into how the host responds to internalization by bacteria; for example that bacteria lacking the capacity to secrete these molecules are targetted to degradative compartments inside cells where they do not thrive.
3. Classification of P. aeruginosa strains and relationship to therapeutic response.
A project nearing completion is an offshoot of the SCUT (Steriods in Corneal Ulcer Therapy) study being conducted at the Proctor Foundation in collaboration with a number of other international organizations. The role of the Fleiszig Laboratory is to classify P. aeruginosa strains isolated from infections to determine if they are invasive or cytotoxic. The hypothesis is that invasive and cytotoxic strains, which differ in how they interact with cells, are associated with different treatment outcomes in patients, as previously found by the Fleiszig laboratory in a study utilizing mice. Lab members involved in this project include UC Berkeley School of Optometry students Chelsia Leong and Avanti Ghanekar, lab manager Arjay Clemente and Drs. Connie Tam and James Mun.