Pathogen Induced Changes in the Protein Profile of Human Tears from <em>Fusarium</em> Keratitis Patients

<div><p><em>Fusarium</em> is the major causative agent of fungal infections leading to corneal ulcer (keratitis) in Southern India and other tropical countries. Keratitis caused by <em>Fusarium</em> is a difficult disease to treat unless antifungal therapy is initiated during the early stages of infection. In this study tear proteins were prepared from keratitis patients classified based on the duration of infection. Among the patients recruited, early infection (n = 35), intermediate (n = 20), late (n = 11), samples from five patients in each group were pooled for analysis. Control samples were a pool of samples from 20 patients. Proteins were separated on difference gel electrophoresis (DIGE) and the differentially expressed proteins were quantified using DeCyder software analysis. The following differentially expressed proteins namely alpha-1-antitrypsin, haptoglobin α2 chain, zinc-alpha-2-glycoprotein, apolipoprotein, albumin, haptoglobin precursor - β chain, lactoferrin, lacrimal lipocalin precursor, cystatin SA III precursor, lacritin precursor were identified using mass spectrometry. Variation in the expression level of some of the proteins was confirmed using western blot analysis. This is the first report to show stage specific tear protein profile in fungal keratitis patients. Validation of this data using a much larger sample set could lead to clinical application of these findings.</p> </div

Identified tear proteins and their function.

a<p>MOWSE scores greater than the values given in the parenthesis are considered to be significant (<i>p</i><0.05). All proteins were also searched across multiple databases to confirm their identity.</p>b<p>Apparent/experimental molecular weight and pI of protein spot on 2-DE gels.</p>c<p>Theoretical molecular weight and p<i>I</i> of the identified protein in database.</p>d<p>Represents the peptides matched.</p>e<p>Sequence coverage (SC) represents the % aminoacid sequence covered in the protein by the matched peptides. 18R, 19M and 20L of α2 isoforms are labeled according to Gupta²²<i>et al.</i>, 2007.</p

3D and graphical representation of expression of up regulated proteins.

<p>Graphic views show the standardized log abundance of spot volume (y-axis) against the changes of proteins between the control and infected groups (x-axis) in all six gels. 3-D view of control and late stage infection sample spots is also shown.</p

Differentially Expressed Proteins in Tears of keratitis patients.

<p>Differentially Expressed Proteins in Tears of keratitis patients.</p

3D and graphical representation of expression of Haptoglobin α2 & β chain.

<p>Graphic views show the standardized log abundance of spot volume (y-axis) against the changes of proteins between the control and infected groups (x-axis) in all six gels. 3-D view of control and late stage infection sample spots is also shown.</p

Discrimination of differentially expressed proteins in six gels.

<p>The histograms were generated by DeCyder DIA software, plotting spot frequency (left y-axis) against log volume ratio (x-axis), and a normal distribution model (red line) was fitted to the main peak of the frequency histogram. A 2 fold variation in expression level was set as threshold. The threshold values are represented by vertical black lines in the histogram, and the differentially expressed spot features are shown as blue spots (volume decreased) and green spots(volume increased). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053018#pone-0053018-t002" target="_blank">Table 2</a> describes the details of spots which show variable expression.</p

The DIGE Experimental Design for Minimal Labeling with Cy Dyes used in this study.

<p>A total of 90 µg of labeled proteins were loaded on each gel for 2D electrophoresis; Tear sample from early, intermediate and late stage keratitis patients were used. Labeling, construction of pooled standards are described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053018#s4" target="_blank">materials and methods</a>. 250 pmoles of dye per 30 µg protein was used.</p

Unsupervised hierarchical of the expressed proteins.

<p>Log-transformed normalized protein spot volumes were used to perform unsupervised hierarchical cluster analysis. Green indicates decreased expression; red indicates increased expression. Patient groups; control and fusarium infected (Early, Intermediate and Late).</p

Immunoblot of tear proteins from control & keratitis patients separated on 2D PAGE.

<p>For 2-DE western blotting, 40 µg tear proteins was separated on pH 4–7 strip (7 cm), followed by second dimension separation on a 13.5% polyacrylamide gel. Tear proteins were transferred to nitrocellulose membranes for subsequent immunodetection with appropriate primary antibodies as described already.</p

Fluorescent protein profiles of a set of six gels.

<p>Experimental design is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053018#pone-0053018-t001" target="_blank">Table 1</a>. A&B represents <i>Fusarium</i> early keratitis; C&D represents <i>Fusarium</i> intermediate keratitis; E&F represents <i>Fusarium</i> late keratitis; A1–F1 represents control tears; A2–F2 represents the pooled internal standard. Each gel contained 90 µg of total protein separated by a pH 4–7 IPG strip in the first dimension and 12.5% polyacrylamide gel in the second dimension electrophoresis. Images were captured using a Typhoon Trio Variable Mode Imager.</p