Transcriptomic analysis reveals that mTOR pathway can be modulated in macrophage cells by the presence of cryptococcal cells

Cryptococcus neoformans and Cryptococcus gattii are the etiological agents of cryptococcosis, a high mortality disease. The development of such disease depends on the interaction of fungal cells with macrophages, in which they can reside and replicate. In order to dissect the molecular mechanisms by which cryptococcal cells modulate the activity of macrophages, a genome-scale comparative analysis of transcriptional changes in macrophages exposed to Cryptococcus spp. was conducted. Altered expression of nearly 40 genes was detected in macrophages exposed to cryptococcal cells. The major processes were associated with the mTOR pathway, whose associated genes exhibited decreased expression in macrophages incubated with cryptococcal cells. Phosphorylation of p70S6K and GSK-3β was also decreased in macrophages incubated with fungal cells. In this way, Cryptococci presence could drive the modulation of mTOR pathway in macrophages possibly to increase the survival of the pathogen.

Keywords:
Macrophage; mTOR pathway; Cryptococcus ; RNAseq; interatomic networks

Authorship

Alícia C. Piffer

Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Porto Alegre, RS, Brazil.Universidade Federal do Rio Grande do SulBrazilPorto Alegre, RS, BrazilUniversidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Porto Alegre, RS, Brazil.

Francine M. dos Santos

Marcos P. Thomé

Camila Diehl

Ane Wichine Acosta Garcia

http://orcid.org/0000-0002-4780-794X

Uriel Perin Kinskovski

Rafael de Oliveira Schneider

Alexandra Gerber

Laboratório Nacional de Computação Científica, Petrópolis, RJ, Brazil.Laboratório Nacional de Computação CientíficaBrazilPetrópolis, RJ, BrazilLaboratório Nacional de Computação Científica, Petrópolis, RJ, Brazil.

Bruno César Feltes

Universidade Federal do Rio Grande do Sul, Instituto de Informática, Porto Alegre, RS, Brazil.Universidade Federal do Rio Grande do SulBrazilPorto Alegre, RS, BrazilUniversidade Federal do Rio Grande do Sul, Instituto de Informática, Porto Alegre, RS, Brazil.

http://orcid.org/0000-0002-2825-8295

Augusto Schrank

http://orcid.org/0000-0002-5248-4620

Ana Tereza R. Vasconcelos

Guido Lenz

Lívia Kmetzsch

Marilene H. Vainstein

Charley C. Staats

http://orcid.org/0000-0002-2433-6903

Associate Editor:

Carlos R. Machado

Send correspondence to

Charley C. Staats. Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Biologia Celular e Molecular, Av. Bento Gonçalves, 9500, 91501-970, Porto Alegre, RS, Brazil. E-mail: staats@ufrgs.br.

Conflict of Interest

The authors declare that there is no conflict of interest that could be perceived as prejudicial to the impartiality of the reported research.

Author Contributions

ACP, AS, ATRV, GL, LK, MJV and CCS conceived, designed the study and reviewed draft of the manuscript; ACP, FMS, MPT, CD, AWAG, UPK performed the experiments; ACP, AG, BCF and CCS analyzed the data; AS, ATRV, GL, MHV provided reagents and equipments. All authors read and approved the final version.

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Figures

Figures (5)

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Figure 1 -
Bioprocesses potentially modulated by C. gattii presence in macrophage-like cells. (A-C) Enriched networks generated using the set of differentially expressed genes were clustered with MCODE. Each cluster was then analyzed for enrichment of bioprocesses using Bingo. The MCODE-generated subnetworks with the higher score are shown (left panel), as well the number of proteins present in each cluster and in the 4 bioprocesses with the highest number of associated proteins (right panel). Red: differentially expressed genes upregulated in macrophage cell line J774.16 in co-culture with fungus C. gattii compared to macrophage cell line without the fungi. Green: differentially expressed genes downregulated in macrophage cell line J774.16 in co-culture with C. gattii compared to macrophage cell line without co-incubation with yeast cells. Pink: Genes with marginal fold change (|log₂FC|< 0.58), in macrophage cell line J774.16 in co-culture with C. gattii compared to macrophage cell line without co-incubation with yeast cells. Grey: genes non-detected in RNA-seq. Blue: genes were detected in the RNA-seq, but did not present differential expression in macrophage cell line J774.16 in co-culture with C. neoformans compared to macrophage cell line without co-incubation with yeast cells.

Thumbnail

Figure 2 -
Bioprocesses potentially modulated by C. neoformans presence in macrophage-like cells. (A-C) Enriched networks generated using the set of differentially expressed genes were clustered with MCODE. Each cluster was then analyzed for enrichment of bioprocesses using Bingo. The MCODE-generated subnetworks with the higher score are show (left panel), as well the number of proteins present in each cluster and in the 4 bioprocesses with the highest number of associated proteins (right panel). Red: differentially expressed genes upregulated in macrophage cell line J774.16 in co-culture with fungus C. neoformans compared to macrophage cell line without the fungi. Green: differentially expressed genes downregulated in macrophage cell line J774.16 in co-culture with C. neoformans compared to macrophage cell line without co-incubation with yeast cells. Pink: Genes with marginal fold change (|log₂FC|< 0.58), in macrophage cell line J774.16 in co-culture with C. neoformans compared to macrophage cell line without co-incubation with yeast cells. Grey: genes non-detected in the RNA-seq. Blue: genes were detected in RNA-seq, but did not present differential expression in macrophage cell line J774.16 in co-culture with C. neoformans compared to macrophage cell line without co-incubation with yeast cells.

Thumbnail

Figure 3 -
Fungal cells modulate the transcript levels of genes involved in the mTOR pathway. IFN-γ and LPS activated J774.16 cells were incubated for 2 h, 6 h and 24 h with C. neoformans or C. gattii. The measured quantity of the genes tested in each sample was normalized using the Ct values obtained for the Gapdh gene. Data are shown as the mean ± SEM from three experimental replicates of three biological replicates each. Columns denoted by asterisks display statistically significant differences when compared to control in each time point (A), or to the period of 2 h of co-incubation (B), as evaluated using ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Red asterisks refer to the comparison between means of C. gattii conditions, while green asterisks refer to the comparison between means of C. neoformans conditions.

Thumbnail

Figure 4 -
Fungal cells decreased the levels of the phosphorylated proteins p70S6K and GSK-3β. (A) Proteins were extracted from pre-activated J774.16 macrophage-like cells and pre-activated J774.16 macrophage-like cells after co-incubation for different times with C. gattii R265 or C. neoformans H99 and analyzed by western blotting. (B) Proteins were extracted from pre-activated J774.16 macrophage-like cells and pre-activated J774.16 macrophage-like cells after co-incubation for 24 hours with C. gattii R265, C. neoformans H99, purified R265 GXM and purified H99 GXM and subjected to western blotting. LC: loading charge.

Thumbnail

Figure 5 -
mTOR related targets of cryptococcal presence on macrophage cells. A partial mTOR pathway encompassing the analyzed genes and proteins is shown. Green labels represent proteins not evaluated. Red labels represent genes or proteins in which expression or activation are reduced by the presence of cryptococcal cells.

Figure 1 - Bioprocesses potentially modulated by C. gattii presence in macrophage-like cells. (A-C) Enriched networks generated using the set of differentially expressed genes were clustered with MCODE. Each cluster was then analyzed for enrichment of bioprocesses using Bingo. The MCODE-generated subnetworks with the higher score are shown (left panel), as well the number of proteins present in each cluster and in the 4 bioprocesses with the highest number of associated proteins (right panel). Red: differentially expressed genes upregulated in macrophage cell line J774.16 in co-culture with fungus C. gattii compared to macrophage cell line without the fungi. Green: differentially expressed genes downregulated in macrophage cell line J774.16 in co-culture with C. gattii compared to macrophage cell line without co-incubation with yeast cells. Pink: Genes with marginal fold change (|log₂FC|< 0.58), in macrophage cell line J774.16 in co-culture with C. gattii compared to macrophage cell line without co-incubation with yeast cells. Grey: genes non-detected in RNA-seq. Blue: genes were detected in the RNA-seq, but did not present differential expression in macrophage cell line J774.16 in co-culture with C. neoformans compared to macrophage cell line without co-incubation with yeast cells.

Figure 2 - Bioprocesses potentially modulated by C. neoformans presence in macrophage-like cells. (A-C) Enriched networks generated using the set of differentially expressed genes were clustered with MCODE. Each cluster was then analyzed for enrichment of bioprocesses using Bingo. The MCODE-generated subnetworks with the higher score are show (left panel), as well the number of proteins present in each cluster and in the 4 bioprocesses with the highest number of associated proteins (right panel). Red: differentially expressed genes upregulated in macrophage cell line J774.16 in co-culture with fungus C. neoformans compared to macrophage cell line without the fungi. Green: differentially expressed genes downregulated in macrophage cell line J774.16 in co-culture with C. neoformans compared to macrophage cell line without co-incubation with yeast cells. Pink: Genes with marginal fold change (|log₂FC|< 0.58), in macrophage cell line J774.16 in co-culture with C. neoformans compared to macrophage cell line without co-incubation with yeast cells. Grey: genes non-detected in the RNA-seq. Blue: genes were detected in RNA-seq, but did not present differential expression in macrophage cell line J774.16 in co-culture with C. neoformans compared to macrophage cell line without co-incubation with yeast cells.

Figure 3 - Fungal cells modulate the transcript levels of genes involved in the mTOR pathway. IFN-γ and LPS activated J774.16 cells were incubated for 2 h, 6 h and 24 h with C. neoformans or C. gattii. The measured quantity of the genes tested in each sample was normalized using the Ct values obtained for the Gapdh gene. Data are shown as the mean ± SEM from three experimental replicates of three biological replicates each. Columns denoted by asterisks display statistically significant differences when compared to control in each time point (A), or to the period of 2 h of co-incubation (B), as evaluated using ANOVA. , P < 0.05; , P < 0.01; , P < 0.001; ****, P < 0.0001. Red asterisks refer to the comparison between means of C. gattii conditions, while green asterisks refer to the comparison between means of C. neoformans conditions.

Figure 4 - Fungal cells decreased the levels of the phosphorylated proteins p70S6K and GSK-3β. (A) Proteins were extracted from pre-activated J774.16 macrophage-like cells and pre-activated J774.16 macrophage-like cells after co-incubation for different times with C. gattii R265 or C. neoformans H99 and analyzed by western blotting. (B) Proteins were extracted from pre-activated J774.16 macrophage-like cells and pre-activated J774.16 macrophage-like cells after co-incubation for 24 hours with C. gattii R265, C. neoformans H99, purified R265 GXM and purified H99 GXM and subjected to western blotting. LC: loading charge.

Figure 5 - mTOR related targets of cryptococcal presence on macrophage cells. A partial mTOR pathway encompassing the analyzed genes and proteins is shown. Green labels represent proteins not evaluated. Red labels represent genes or proteins in which expression or activation are reduced by the presence of cryptococcal cells.

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Transcriptomic analysis reveals that mTOR pathway can be modulated in macrophage cells by the presence of cryptococcal cells

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[1] Associate Editor:

Carlos R. Machado