Fusarium solani is a filamentous fungus that was the causative organism in an outbreak of keratitis in the USA in 2005/2006 that was traced to a lens care product. This outbreak caused an increased awareness of, and improved diagnostics for Fusarium, indicating that the prevalence of Fusarium keratitis is much higher than was previously estimated. Our findings show that Fusarium forms a biofilm on soft contact lenses, which protects the organisms from anti-mycotics, and also induces programmed cell death (apoptosis) in corneal epithelial cells. This mechanism may be important in penetration of hyphae through the epithelium to the stroma. Fusarium is also a major cause of microbial keratitis in rural southern USA, in southeast Asia and in many parts of the developing world, where spores (conidia) are common in the soil and in the air. Keratitis occurs as a result of agricultural accidents or other forms of trauma where dust or vegetable matter containing spores enter the corneal stroma and germinate. We developed a mouse model of Fusarium keratitis where mice develop severe corneal opacification and ulceration within 24h, and organisms are cleared within 48h following an intense neutrophil infiltration to the corneal stroma. In contrast, in immune deficient IL-1R1-/- and MyD88-/- mice, the organisms invade the anterior chamber and continue to replicate.
Aim 1 will examine the mechanism of IL-1R1/MyD88 in resident cells in the cornea and on the anti- fungal activity of neutrophils. Experiments will also examine the role of C-type lectins Dectin-1 and Dectin-2, and the role of Fusarium proteases that mediate penetration of hyphae through the corneal stroma and Descemet's membrane, and will examine the effect of these proteases in generating the neutrophil chemotactic peptide Pro-Gly-Pro by cleaving collagen fibrils.
Aim 2 will utilize Proteomics and targeted mutagenesis methods to identify biochemical pathways and Fusarium proteins associated with biofilm formation, and generate organisms in which key proteins in biofilm formation are disrupted. These strains will be examined for their ability to form biofilm, and to induce pro-apoptotic and immunomodulatory responses in corneal epithelial cells and Langerhans dendritic cells, and to induce contact lens associated keratitis. Experiments proposed in this aim will also identify key mediators in biofilm-induced apoptosis of corneal epithelial cells. Results of the proposed studies will greatly increase our understanding of the pathogenesis of this disease, and will identify novel targets for therapy and diagnosis. The common soil fungus Fusarium solani is a major cause of agriculture related and contact lens associated corneal infection and inflammation in southern, humid areas of the USA in the developing world, and was the causative organism in an outbreak of in the USA in 2005/2006 that was traced to a lens care product. Experiments outlined in this proposal will examine the molecular mechanisms by which resident and infiltrating cells in the cornea respond to the growing fungal hyphae to eliminate the organism and cause disease. Experiments will also identify Fusarium proteins that are responsible for biofilm formation on contact lenses, and together with the first set of studies will identify targets for interventional therapy for this disease.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY018612-05
Application #
8215702
Study Section
Anterior Eye Disease Study Section (AED)
Program Officer
Mckie, George Ann
Project Start
2008-02-01
Project End
2013-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
5
Fiscal Year
2012
Total Cost
$476,806
Indirect Cost
$173,108
Name
Case Western Reserve University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Ryan, Sean O; Leal Jr, Sixto M; Abbott, Derek W et al. (2014) Mgat2 ablation in the myeloid lineage leads to defective glycoantigen T cell responses. Glycobiology 24:262-71
Taylor, Patricia R; Roy, Sanhita; Leal Jr, Sixto M et al. (2014) Activation of neutrophils by autocrine IL-17A-IL-17RC interactions during fungal infection is regulated by IL-6, IL-23, RORýýt and dectin-2. Nat Immunol 15:143-51
Taylor, Patricia R; Leal Jr, Sixto M; Sun, Yan et al. (2014) Aspergillus and Fusarium corneal infections are regulated by Th17 cells and IL-17-producing neutrophils. J Immunol 192:3319-27
Chandra, Jyotsna; Pearlman, Eric; Ghannoum, Mahmoud A (2014) Animal models to investigate fungal biofilm formation. Methods Mol Biol 1147:141-57
Leal Jr, Sixto M; Roy, Sanhita; Vareechon, Chairut et al. (2013) Targeting iron acquisition blocks infection with the fungal pathogens Aspergillus fumigatus and Fusarium oxysporum. PLoS Pathog 9:e1003436
Carrion, Steven de Jesus; Leal Jr, Sixto M; Ghannoum, Mahmoud A et al. (2013) The RodA hydrophobin on Aspergillus fumigatus spores masks dectin-1- and dectin-2-dependent responses and enhances fungal survival in vivo. J Immunol 191:2581-8
Leal Jr, Sixto M; Pearlman, Eric (2012) The role of cytokines and pathogen recognition molecules in fungal keratitis - Insights from human disease and animal models. Cytokine 58:107-11
Karthikeyan, Rajapandian Sivaganesa; Leal Jr, Sixto M; Prajna, Namperumalsamy Venkatesh et al. (2011) Expression of innate and adaptive immune mediators in human corneal tissue infected with Aspergillus or fusarium. J Infect Dis 204:942-50
Sun, Yan; Chandra, Jyotsna; Mukherjee, Pranab et al. (2010) A murine model of contact lens-associated fusarium keratitis. Invest Ophthalmol Vis Sci 51:1511-6
Leal Jr, Sixto M; Cowden, Susan; Hsia, Yen-Cheng et al. (2010) Distinct roles for Dectin-1 and TLR4 in the pathogenesis of Aspergillus fumigatus keratitis. PLoS Pathog 6:e1000976

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