INTRODUCTION: The Fleiszig Lab's research is focused on understanding pathogenesis of bacterial infection, using the cornea and the opportunistic pathogen Pseudomonas aeruginosa as model systems. Studies are aimed at understanding the molecular factors that prevent bacterial penetration of epithelia during health, how the functionality of that defense system is modulated, and the bacterial factors that enable penetration when the system is compromised. By studying the eye's defensive mechanisms and determining what microbial factors involved in initiating disease, we hope to develop therapeutics for preventing/treating infection on multiple surfaces of the body.
DETAIL: Our epithelial surfaces are normally resistant to infection. Therefore, researchers who study infectious disease in vivo commonly resort to use of models that deliberately compromise the target tissue (or otherwise bypass barriers) so that disease can be enabled and studied. These infection models have led to a plethora of important information about factors involved in pathology and/or its resolution when disease is initated. However, other models are needed to study barriers to infection, or early events that occur prior to disease initiation when it occurs in the absence of overt injury.
In our laboratory, we have developed novel in vivo and in vitro methods for studying defenses during health using the eye and the opportunistic bacterium Pseudomonas aeruginosa as models. We have also advanced imaging technologies that enable us to see into the living epithelium to observe what bacteria do and how the tissue responds in either resistant or susceptible states. Using these methods, and employing array/knockout/knockdown technologies, we have identified specific factors that modulate the ability of bacteria to penetrate the ocular surface epithelium. The data show that pathogen recognition systems are involved in resistance, and suggest that bacterial adaptation in vivo contributes to pathogenesis. Studies aimed at understanding early interactions between microbes and the ocular surface prior to disease initiation have potential for development of novel methods to prevent (rather than simply treat) infection of the eye or other sites.
The lab is working on two inter-related goals.
1. To determine how the healthy corneal surface resists infection and how contact lens wear then compromises those defenses.
a) The laboratory's long standing NIH (NEI) grant is aimed at 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. Barriers to our understanding in this area have included the lack of available in vivo models for studying these processes. Traditionally researchers have used "infection models" to study tissue interactions with bacteria. These involve deliberately compromising the tissue to induce enable susceptibility to infection, adding the microbe and then studying what happens next. Those who study corneal infection extensively use a scratch injury model that enables susceptibility by physically removing the epithelial barrier to expose the vulnerable stroma. Disease follows because direct placement of the microbe into the stroma stimulates a damaging inflammatory response.
This scratch injury model is problematic for the Fleiszig laboratory's research goals for two reasons. Firstly, it bypasses the need for bacteria to penetrate the epithelium, which is the event the laboratory seeks to understand. Secondly, the lab is interested in understanding the events that determine health when the cornea is resistant to infection, not events that occur when there is disease. Due to the lack of suitable model systems, most of our knowledge about epithelial interactions with bacteria and the potential role of the epithelia in defense have necessarily come from cell culture studies.
In the past 2 years, the laboratory has succeeded in developing multiple models (in vivo and in vitro) that enable epithelial cell penetration by bacteria to be studied. Together, these models provide the opportunity to do experiments either in the context of, or without, potentially confounding factors present in vivo, as is needed to test hypotheses. In the past year, the laboratory has also developed new imaging technologies that enable bacterial interactions with the epithelium to be imaged in living intact eyes without the need for any tissue dissection, staining, or other types of sample preparation/processing. A major equipment grant is currently pending at the NIH; if funded the new instrumentation will enable investigators at UC Berkeley and UCSF to non-invasively image living corneas of living animals. With access to that equipment, there is also potential for development of new methods for the diagnosis of human corneal disease.
b) A separate investigation to determine how the ocular surface retains its sterility is newly funded by a Grand Challenges Explorations Grant from the Gates Foundation. The long-term goal of this project is bold: To develop therapies to prevent any type of infection of any body site based on eye derived molecules.
2. Fundamental studies of bacterial/epithelial cell interactions using P. aeruginosa as a model organism.
This aspect of the research is funded by a new NIH grant from the NIAID. The goal of this project is to determine how P. aeruginosa survive inside corneal epithelial cells, and the necessity for that strategy in the disease process. The data to date show that corneal epithelial cells normally traffic bacteria to intracellular vesicles where they are killed, but P. aeruginosa possesses virulence factors that enable it to bypass this innate defense. Experimental approaches involving biochemical and genetic methods, combined with various images techniques, are being used to describe the details of the intimate interactions between P. aeruginosa and eukaryotic cells. The results will enlighten us about how bacteria cause disease and should also contribute to our understanding of epithelial cell biology.
P. aeruginosa causes sight threatening pathology in the eye and life threatening infections at other sites. These include serious lung disease in children with cystic fibrosis and in AIDS patients, and life theatening skin infections in burns victims. Thus, this line of research could ultimately lead to new means for treating multiple diseases.
In the News
A team of UC Berkeley vision scientists has found that small fragments of keratin protein in the eye play a key role in warding off pathogens.