Proteome profiling of human cutaneous leishmaniasis lesion

Abstract

In this study, we used proteomics and biological network analysis to evaluate the potential biological processes and components present in the identified proteins of biopsies from cutaneous leishmaniasis (CL) patients infected by Leishmania braziliensis in comparison with normal skin. We identified 59 proteins differently expressed in samples from infected and normal skin. Biological network analysis employing identified proteins showed the presence of networks that may be involved in the cell death mediated by cytotoxic T lymphocytes. After immunohistochemical analyses, the expression of caspase-9, caspase-3, and granzyme B was validated in the tissue and positively correlated with the lesion size in CL patients. In conclusion, this work identified differentially expressed proteins in the inflammatory site of CL, revealed enhanced expression of caspase-9, and highlighted mechanisms associated with the progression of tissue damage observed in lesions.

Figure 1

Images of representative 2D gels. The images are representative of six samples showing separation of proteins extracted from lesions of (a) cutaneous leishmaniasis (CL) patients and (b) normal skin. Directions of isoelectric focusing and SDS-PAGE are indicated on the top and on the side of the left gel image. Spots marked only in gel (a) indicate protein spots unique in the CL samples. Spots marked only in gel (b) show the protein spots unique in normal skin. Spots marked in gels (a, b) indicate protein spots differently regulated between the samples. The protein spots were identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. List of identified proteins is given in Table 1.

Figure 2

Schematic Venn diagram of the protein spots identified. The Venn diagram shows proteins unique to (a) cutaneous leishmaniasis (CL) patients, (b) normal skin, and (c) overlaps between biologic processes defined by the identified proteins between the samples. The diagram was built upon analysis of the identified proteins using a GoMiner tool. “Biologic Process” category was selected for the analysis of affected functions.

Figure 3

Network and canonical pathway built with 59 differentially expressed proteins. (a) Subnetwork modules associated with apoptosis extracted from the whole network using an MCODE tool. (b) Cytotoxic T lymphocyte–mediated apoptosis of target cell network (P<0.003) performed by Ingenuity Pathway analysis. The red color is an indication of the upregulated/unique proteins expressed in cutaneous leishmaniasis (CL) lesions, and green color indicates downregulated/unique proteins expressed in normal skin. Full and dashed lines represent direct and indirect interactions, respectively, between the proteins. Network shapes are represented in the legend.

Figure 4

Immunohistochemistry for caspase-9, caspase-3, and granzyme B in samples. Tissue sections of cutaneous leishmaniasis (CL) patients (n=8) were obtained and stained for (a) caspase-9, (c) caspase-3, and (e) granzyme B. Normal skin samples (n=3) were immunostained for (b) caspase-9, (d) caspase-3, and (f) granzyme B. (g) Isotype control is shown. All samples were counterstained with hematoxylin and examined by light microscopy. Scale bar=10 μm.

Figure 5

Correlation analysis between the protein expression in tissue from cutaneous leishmaniasis (CL) patients. Correlation analysis between the percentage of expression of (a) caspase-9 and granzyme B, (b) caspase-9 and caspase-3, and (c) caspase-3 and granzyme B in tissues from CL patients (n=8). Statistical comparisons were done using Spearman's (r²) rank test. P<0.05 was considered significant.

Figure 6

Correlation analysis between the protein expression and lesion size. Correlation analysis between the percentage of protein expression of (a) caspase-9, (b) caspase-3, and (c) granzyme B and lesion size of cutaneous leishmaniasis (CL) patients (n=8). Statistical comparisons were done using Spearman's (r²) rank test. P<0.05 was considered significant.