Regulation of Leishmania (L.) amazonensis protein expression by host T cell dependent responses: differential expression of oligopeptidase B, tryparedoxin peroxidase and HSP70 isoforms in amastigotes isolated from BALB/c and BALB/c nude mice

Abstract

Leishmaniasis is an important disease that affects 12 million people in 88 countries, with 2 million new cases every year. Leishmania amazonensis is an important agent in Brazil, leading to clinical forms varying from localized (LCL) to diffuse cutaneous leishmaniasis (DCL). One interesting issue rarely analyzed is how host immune response affects Leishmania phenotype and virulence. Aiming to study the effect of host immune system on Leishmania proteins we compared proteomes of amastigotes isolated from BALB/c and BALB/c nude mice. The athymic nude mice may resemble patients with diffuse cutaneous leishmaniasis, considered T-cell hyposensitive or anergic to Leishmania's antigens. This work is the first to compare modifications in amastigotes' proteomes driven by host immune response. Among the 44 differentially expressed spots, there were proteins related to oxidative/nitrosative stress and proteases. Some correspond to known Leishmania virulence factors such as OPB and tryparedoxin peroxidase. Specific isoforms of these two proteins were increased in parasites from nude mice, suggesting that T cells probably restrain their posttranslational modifications in BALB/c mice. On the other hand, an isoform of HSP70 was increased in amastigotes from BALB/c mice. We believe our study may allow identification of potential virulence factors and ways of regulating their expression.

Fig 1. Footpad lesion sizes (difference between infected and non-infected footpads) in mm of BALB/c (n = 6) and BALB/c nude (n = 5) mice infected with L. amazonensis, measured weekly for 13 weeks.

Fig 2. Comparison of lymphoid, myeloid and dendritic cell populations in spleens (A), popliteal lymph nodes (B) and footpads (C) of infected and non infected BALB/c and BALB/c nude mice by flow cytometry.

Results of one experiment with 3 animals of each for control condition and four of each for infection. Statistical analyses by ANOVA followed by Tukey´s multiple comparison test for all comparisons except for lymphoid and dendritic cells in footpads (Fig. 2C left), where t test was employed. * = p<0.05

Fig 3. Representative 2D gel images.

A. Negative (black and white) image of a pool of all samples labeled with Cy2, highlighting the spots corresponding to differentially expressed HSP70, OPB and TXNPx isoforms and the 3D images of the spots marked with arrows. B. Representative image of one experiment with amastigote samples from BALB/c and BALB/c nude labeled with Cy5 (red) and Cy3 (green), respectively

Fig 4. Western blot for OPB expression in 5 pairs of lesion derived amastigotes from BALB/c and BALB/c nude mice.

A. Western blot image showing OPB (upper band) and GAPDH (lower band) in soluble extracts from BALB/c derived amastigotes (1–5) and BALB/c nude derived amastigotes (6–10). B. Expression of OPB relative to GAPDH in the 10 samples. Statistical analysis by t-test. * = p<0.05

Fig 5. Western blot for HSP70 expression in 5 pairs of lesion derived amastigotes from BALB/c and BALB/c nude mice.

A. Western blot image showing HSP70 (upper band) and GAPDH (lower band) in soluble extracts from BALB/c derived amastigotes (1–5) and BALB/c nude derived amastigotes (6–10). B. Expression of HSP70 relative to GAPDH in the 10 samples Statistical analysis by t-test. * = p<0.05

Fig 6. Western blot for tryparedoxin peroxidase (TXNPx) expression in 5 pairs of lesion derived amastigotes from BALB/c and BALB/c nude mice.

A. Western blot image showing tryparedoxin peroxidase (upper band) and GAPDH (lower band) in soluble extracts from BALB/c derived amastigotes (1–5) and BALB/c nude derived amastigotes (6–10). B. Expression of tryparedoxin peroxidase relative to GAPDH in the 10 samples. Statistical analysis by t-test. * = p<0.05

Fig 7. 2D Western blot for TXNPx in amastigotes from BALB/c and BALB/c nude mice.

A. Western blot image showing three isoforms (1, 2, 3) in soluble extracts from BALB/c nude (upper image) and BALB/c (lower image) derived amastigotes. B. Relative abundance of each isoform after normalization (considering the sum of three isoforms similar in the two samples). C. Western blot image showing labeling of the isoforms with anti-phospho S, T, Y pooled antibodies in the membranes shown in A.

Fig 8. Immunohistochemistry for OPB, TXNPx and HSP70 in infected and non infected (small pictures) footpads from BALB/c and BALB/c nude mice.