Microbiology, immunology, infectious disease, corneal and tear physiology
Research in my laboratory focuses on the pathogenesis of bacterial infections of the cornea. The principal aim of my research is
to determine why patients who wear contact lenses are prone to infectious keratitis. These infections most often involve the bacterium
Pseudomonas aeruginosa and can lead to severe vision loss. The results of initial studies indicated that ocular flora is altered in
some contact lens wearers and that a large proportion of patients' lens cases are contaminated with bacteria during normal use.
However, neither of these phenomena entirely explain the pathogenesis of infection, since in general bacteria cannot infect a healthy
cornea, even when introduced in large numbers. This implies that there must be some form of compromise, in addition to bacterial
contamination of the eye before the infectious process can begin. For this reason we are interested in establishing how the healthy
cornea resists infection, how contact lens wear and other predisposing factors could compromise these defenses, and what bacterial
virulence factors are involved in initiating infection. This project involves the study of bacterial interaction with tear film factors
and the ocular surface, bacterial adherence to cornea and contact lenses, mechanisms of bacterial invasion and killing of corneal
epithelial cells, transcorneal migration of bacteria and disruption of normal host tissue physiology by bacteria.
It is widely believed that in the healthy eye infection is prevented because tear film factors are able to neutralize bacteria, and
that bacteria are not able to adhere to the cornea unless it is overtly injured. However, the results of our studies have demonstrated
that neither of these assumptions are entirely accurate. We have found that P. aeruginosa can survive for several hours in tears, and
that this microbe can adhere to uninjured cornea, under certain circumstances. Other findings are that ocular mucin and the cell surface
glycocalyx protect the cornea from infection by inhibiting bacterial adherence to underlying corneal epithelial cells, that contact lens
wear increases bacterial adherence to corneal epithelial cells, and that P. aeruginosa - which is thought to be an extracellular pathogen
- is in fact able to invade corneal epithelial cells. In addition to characterizing corneal defenses and the events that occur during
infection, we are also studying the molecular mechanisms involved in these processes with a view to developing therapeutic and/or
preventative measures.
Techniques that are being used to study bacterium/host interactions include adherence assays, bacterial invasion and cytotoxicity
assays, microscopy (including light, fluorescence, immunohistochemistry, scanning and transmission electron microscopy), various
biochemical assays and molecular genetics. Although much of this work is performed using primary epithelial cell cultures, we have
developed several other models for these studies. These include in vitro models for the study of bacterial interaction with human
corneal cells and whole cornea of rat, rabbit and mouse, and an in vivo model for infection in mice.
Selected Publications
Role of defensins in corneal epithelial barrier function against Pseudomonas aeruginosa traversal. Augustin DK, Heimer SR, Tam C, Li WY, Le Due JM, Evans DJ, Fleiszig SM. Infect Immun. 2011 Feb;79(2):595-605.
Factors Impacting Corneal Epithelial Barrier Function against Pseudomonas aeruginosa Traversal. Alarcon I, Tam C, Mun JJ, Ledue J, Evans DJ, Fleiszig SM. Invest Ophthalmol Vis Sci. 2011 Mar 14;52(3):1368-77.
The ADP-ribosylation domain of Pseudomonas aeruginosa ExoS is required for membrane bleb niche formation and bacterial survival within epithelial cells. Angus AA, Evans DJ, Barbieri JT, Fleiszig SM. Infect Immun. 2010 Nov;78(11):4500-10.
Pathogenesis of contact lens-associated microbial keratitis. Fleiszig SM, Evans DJ. Optom Vis Sci. 2010 Apr;87(4):225-32.
The impact of inoculation parameters on the pathogenesis of contact lens-related infectious keratitis. Tam C, Mun JJ, Evans DJ, Fleiszig SM. Invest Ophthalmol Vis Sci. 2010 Jun;51(6):3100-6.
Role of the corneal epithelial basement membrane in ocular defense against Pseudomonas aeruginosa. Alarcon I, Kwan L, Yu C, Evans DJ, Fleiszig SM. Infect Immun. 2009 Aug;77(8):3264-71.
Clearance of Pseudomonas aeruginosa from a healthy ocular surface involves surfactant protein D and is compromised by bacterial elastase in a murine null-infection model. Mun JJ, Tam C, Kowbel D, Hawgood S, Barnett MJ, Evans DJ, Fleiszig SM. Infect Immun. 2009 Jun;77(6):2392-8.
The role of twitching motility in Pseudomonas aeruginosa exit from and translocation of corneal epithelial cells. Alarcon I, Evans DJ, Fleiszig SM. Invest Ophthalmol Vis Sci. 2009 May;50(5):2237-44.
Pathogenic phenotype and genotype of Pseudomonas aeruginosa isolates from spontaneous canine ocular infections. Ledbetter EC, Mun JJ, Kowbel D, Fleiszig SM. Invest Ophthalmol Vis Sci. 2009 Feb;50(2):729-36.
Expression of surfactant protein D in human corneal epithelial cells is upregulated by Pseudomonas aeruginosa. Ni M, Tam C, Verma A, Ramphal R, Hawgood S, Evans DJ, Fleiszig SM. FEMS Immunol Med Microbiol. 2008 Nov;54(2):177-84.
Pseudomonas aeruginosa induces membrane blebs in epithelial cells, which are utilized as a niche for intracellular replication and motility. Angus AA, Lee AA, Augustin DK, Lee EJ, Evans DJ, Fleiszig SM. Infect Immun. 2008 May;76(5):1992-2001.
Mutation of the phospholipase catalytic domain of the Pseudomonas aeruginosa cytotoxin ExoU abolishes colonization promoting activity and reduces corneal disease severity. Tam C, Lewis SE, Li WY, Lee E, Evans DJ, Fleiszig SM. Exp Eye Res. 2007 Dec;85(6):799-805.
Life at the front: dissecting bacterial-host interactions at the ocular surface. Evans DJ, McNamara NA, Fleiszig SM. Ocul Surf. 2007 Jul;5(3):213-27. Review.
Human tear fluid protects against Pseudomonas aeruginosa keratitis in a murine experimental model. Kwong MS, Evans DJ, Ni M, Cowell BA, Fleiszig SM. Infect Immun. 2007 May;75(5):2325-32.
The Glenn A. Fry award lecture 2005. The pathogenesis of contact lens-related keratitis. Fleiszig SM. Optom Vis Sci. 2006 Dec;83(12):866-73.
Type III secretion-dependent modulation of innate immunity as one of multiple factors regulated by Pseudomonas aeruginosa RetS. Zolfaghar I, Evans DJ, Ronaghi R, Fleiszig SM. Infect Immun. 2006 Jul;74(7):3880-9.
Actin cytoskeleton disruption by ExoY and its effects on Pseudomonas aeruginosa invasion. Cowell BA, Evans DJ, Fleiszig SM. FEMS Microbiol Lett. 2005 Sep 1;250(1):71-6.
Links
Time-lapse video microscopy (Pseudomonas aeruginosa attacks on macrophages and corneal epithelial cells).
A feature article about our research is available on U.C. Berkeley's News and Events web site
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