Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Apr 21;9(4):e95977.
doi: 10.1371/journal.pone.0095977. eCollection 2014.

Trypanosoma cruzi infection down-modulates the immunoproteasome biosynthesis and the MHC class I cell surface expression in HeLa cells

Ricardo Camargo  1 , Liliam O Faria  1 , Alexander Kloss  2 , Cecília B F Favali  1 , Ulrike Kuckelkorn  2 , Peter-Michael Kloetzel  2 , Cezar Martins de Sá  1 , Beatriz D Lima  1
Affiliations

Trypanosoma cruzi infection down-modulates the immunoproteasome biosynthesis and the MHC class I cell surface expression in HeLa cells

Ricardo Camargo et al. PLoS One. .

Abstract

Generally, Trypanosoma cruzi infection in human is persistent and tends to chronicity, suggesting that the parasite evade the immune surveillance by down regulating the intracellular antigen processing routes. Within the MHC class I pathway, the majority of antigenic peptides are generated by the proteasome. However, upon IFN-γ stimulation, the catalytic constitutive subunits of the proteasome are replaced by the subunits β1i/LMP2, β2i/MECL-1 and β5i/LMP7 to form the immunoproteasome. In this scenario, we analyzed whether the expression and activity of the constitutive and the immunoproteasome as well as the expression of other components of the MHC class I pathway are altered during the infection of HeLa cells with T. cruzi. By RT-PCR and two-dimensional gel electrophoresis analysis, we showed that the expression and composition of the constitutive proteasome is not affected by the parasite. In contrast, the biosynthesis of the β1i, β2i, β5i immunosubunits, PA28β, TAP1 and the MHC class I molecule as well as the proteasomal proteolytic activities were down-regulated in infected-IFN-γ-treated cell cultures. Taken together, our results provide evidence that the protozoan T. cruzi specifically modulates its infection through an unknown posttranscriptional mechanism that inhibits the expression of the MHC class I pathway components.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Analysis of mRNA levels and protein composition of HeLa constitutive proteasome during T. cruzi infection.
Semi-quantitative RT-PCR analysis of α1, α6, β1, β2 and β5 expression were done using total RNA from HeLa cells treated with IFN-γ and/or infected with T. cruzi. (A) The PCR products were analyzed by electrophoresis in 1.2% agarose gels stained with ethidium bromide. The reactions were carried as duplex-PCR, using GAPDH as internal control (arrows). (B) mRNA levels were determined by densitometry and plotted using the expression of GAPDH as normalizer. Each value represents the mean ± standard deviation of three individual experiments. (C) Two-dimensional gels of immunoprecipitated proteasomes from HeLa uninfected and infected with T. cruzi. HeLa cells in standard culture conditions were exposed or not to T. cruzi and cultured for 24 h. In the twenty-first hour of culture, cells were metabolically labeled with [35S]-methionine for three hours. Cell lysates (100 µg) were immunoprecipitated with anti-human proteasome antibodies and analyzed by two-dimensional electrophoresis. Panel D show the protein levels of proteasome α and β subunits quantified by densitometry. Each value represents the mean ± mean deviation of two independent experiments.
Figure 2
Figure 2. Analysis of immunoproteasome mRNA levels during T. cruzi infection.
Semi-quantitative RT-PCR analysis of β1i, β2i and β5i expression were done using total RNA from HeLa cells treated with IFN-γ and/or infected with T. cruzi. (A) The PCR products were analyzed by electrophoresis in 1.2% agarose gels stained with ethidium bromide. The reactions were carried as duplex-PCR, using GAPDH as internal control (arrows). (B) mRNA levels were determined by densitometry and plotted using the expression of GAPDH as normalizer. Each value represents the mean ± standard deviation of three individual experiments. The abundance of α1, β1i, β2i, β5i and PA28β mRNAs were also determined by real time RT-qPCR. The relative expression of the transcripts was calculated by normalization with GAPDH and HPRT1 housekeeping genes using the 2−ΔΔCt method. (C) The mRNA levels were plotted relatively to “IFN-γ” experimental condition (HeLa treated 24 h with IFN-γ). Each value represents the mean ± standard deviation of three independent experiments.
Figure 3
Figure 3. Analysis of immunoproteasome protein expression during T. cruzi infection.
(A) Lysates (25–50 µg) of HeLa cells treated with IFN-γ and/or infected with T. cruzi were analyzed by western blot using anti-immunoproteasome subunits antibodies as indicated. (B) Protein levels were determined by densitometry and plotted using the expression of α6 subunit as experimental normalizer. Infection was confirmed using anti-tubulin antibody. (C) Western blot analysis of PA28β expression during different times of infection. HeLa cells were pre-treated with IFN-γ for 24 h and then infected with T. cruzi for 24, 48 and 72 h. (D) Protein levels were plotted using the expression of α1 as experimental normalizer. All values in this figure represent mean ± standard deviation of three individual experiments.
Figure 4
Figure 4. Effect of T. cruzi infection on proteasome proteolytic activities.
Proteolytic assays were performed using extracts of HeLa cells treated with IFN-γ and/or T. cruzi-infected combined with luminogenic substrates specific for chymotrypsin-, trypsin- and caspase-like proteasome activities. The specific activity of the proteasome was estimated, for each treatment and substrate, through samples treated with the proteasome inhibitor MG-132. Mean of luminescence intensities were plotted relatively to the control. Prior to the catalytic assays, cell lysates (20 µg/lane) were analyzed by SDS-PAGE 13% stained with coomassie blue (A). Gels were used as loading control. (B) Chymotrypsin- (C) trypsin- and (D) caspase-like assays. Each value represents the mean ± standard deviation of three individual experiments and each sample was analyzed in triplicates.
Figure 5
Figure 5. Quantification of mRNA and protein expression of TAP1 and MHC class I molecule during T. cruzi infection.
(A) The abundance of TAP1, β2M and HLA mRNAs were determined by real-time RT-qPCR using the total RNA from HeLa cells treated with IFN-γ and/or infected with T. cruzi. The relative expression of the transcripts was calculated by normalization with GAPDH and HPRT1 housekeeping genes using the 2−ΔΔCt method. The mRNA levels were plotted relatively to “IFN-γ” experimental condition (HeLa treated 24 h with IFN-γ). Each value represents the mean ± standard deviation of three independent experiments. (B) Lysates (25–50 µg) of HeLa cells treated with IFN-γ and/or infected with T. cruzi were analyzed by western blot using human anti-TAP1 and anti-MHC class I antibodies as indicated. Infection was confirmed using anti-tubulin antibody. (C) Protein levels were determined by densitometry and plotted using the expression of α6 subunit as experimental normalizer. Each value represents the mean ± standard deviation of three independent experiments.
Figure 6
Figure 6. Effect of T. cruzi infection on MHC class I cell surface expression.
(A) Immunofluorescence microscopy of HeLa cells treated with IFN-γ and/or T. cruzi-infected stained with human anti-MHC class I antibody (yellow) and DAPI (blue). The arrows highlight the infected cells that clearly had the MHC class I expression down-modulated by T. cruzi infection. Bars  = 25 µm. To precisely quantify the MHC class I cell surface expression, samples used in microscopy were analyzed by flow cytometry. (B) The protein expression was determined as median fluorescence intensity (MFI). Each value represents the mean ± standard deviation of three independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. WHO – World Health Organization (2014) Fact Sheet N°340 – Chagas disease (American trypanosomiasis). Available: http://www.who.int/mediacentre/factsheets/fs340/en/index.html. Accessed 12 January 2014.
    1. Brener Z (1973) Biology of Trypanosoma cruzi . Annu Rev Microbiol 27: 347–382. - PubMed
    1. Dutra WO, Gollob KJ (2008) Current concepts in immunoregulation and pathology of human Chagas disease. Curr Opin Infect Dis 21: 287–292. - PMC - PubMed
    1. Burleigh BA, Andrews NW (1995) The mechanisms of Trypanosoma cruzi invasion of mammalian cells. Annu Rev Microbiol 49: 175–200. - PubMed
    1. Ley V, Robbins ES, Nussenzweig V, Andrews NW (1990) The exit of Trypanosoma cruzi from the phagosome is inhibited by raising the pH of acidic compartments. J Exp Med 171: 401–413. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

This work was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Distrito Federal (FAP-DF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.