Comparative analysis of the tear protein profile in mycotic keratitis patients

Abstract

Purpose: Mycotic keratitis is a major cause of corneal blindness in India. A proper understanding of the pathogenesis may help in refining the existing treatment. The purpose of this study is to examine the total tear protein profile of fungal keratitis patients, which may have a bearing on pathogenesis and disease progression.

Methods: Tear samples were collected from culture positive fungal keratitis patients. Tears from the uninfected fellow eye and from other healthy individuals served as controls. Two-dimensional electrophoresis (2DE) was used for the separation of fractionated tear proteins, and selected protein spots, which showed differential expressions, were identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Wherever needed, tag sequencing of peptide fragments using post source decay (PSD) was done to confirm the identification.

Results: The glutaredoxin-related protein was expressed only in the tears of fungal keratitis patients. Six other normal tear proteins were present in both samples but with varied expression levels. Prolactin inducible protein and serum albumin precursor were upregulated in the infected samples. Cystatin S precursor, cystatin SN precursor, cystatin, and human tear lipocalin were downregulated in the infected samples.

Conclusions: Tears can be used as a clinical source to study the proteomic responses in patients with fungal keratitis. The glutaredoxin-related protein is known to be produced by Aspergillus during oxidative stress conditions, and the presence of this protein in the tears of patients with mycotic keratitis indicates that this pathogen undergoes stress-related gene expression during infection.

Figure 1

Representative 2DE gel map of tear proteins of normal subjects and fungal keratitis patients. Tear proteins (270 μg) were separated using an 18 cm pH 3–10 NL IPG strip in the first dimension and 12.5% SDS–PAGE followed by coomassie blue G-250 staining in the second dimension. The identified normal tear proteins, cystatin S precursor (spot 1), cystatin SN precursor (spot 5), cystatin (spot 29), human tear lipocalin (spot 49), prolactin inducible protein (spot 34), serum albumin (spot 44), and fungal protein such as the glutaredoxin-related protein (spot 54) are marked and compared between control tear samples, Fusarium keratitis tear samples, and Aspergillus keratitis tear samples.

Figure 2

Magnified 2DE map of spots which were differentially expressed in the tears of mycotic keratitis patients. Among six normal tear proteins identified, four proteins (cystatin S precursor [1], cystatin SN precursor [5], cystatin [29], and human tear lipocalin [49]) were downregulated and two proteins (prolactin inducible protein [34] and serum albumin precursor [44]) were upregulated in fungal keratitis” in “Among six normal tear proteins identified, four proteins (cystatin S precursor, cystatin SN precursor, cystatin, and human tear lipocalin) were downregulated and two proteins (prolactin inducible protein and serum albumin precursor) were upregulated in fungal keratitis.