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Impact of Leishmania infection on host macrophage nuclear physiology and nucleopore complex integrity.

Isnard A, Christian JG, Kodiha M, Stochaj U, McMaster WR, Olivier M - PLoS Pathog. (2015)

Bottom Line: Here we describe for the first time, using molecular biology and in-depth proteomic analyses, that GP63 alters the host macrophage nuclear envelope, and impacts on nuclear processes.Our results suggest that GP63 does not appear to use a classical nuclear localization signal common between Leishmania species for import, but degrades nucleoporins, and is responsible for nuclear transport alterations.In the nucleoplasm, GP63 activity accounts for the degradation and mislocalization of proteins involved amongst others in gene expression and in translation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Microbiology & Immunology, The Research Institute of the McGill University Health Centre, McGill University, Montréal, Quebec, Canada.

ABSTRACT
The protease GP63 is an important virulence factor of Leishmania parasites. We previously showed that GP63 reaches the perinuclear area of host macrophages and that it directly modifies nuclear translocation of the transcription factors NF-κB and AP-1. Here we describe for the first time, using molecular biology and in-depth proteomic analyses, that GP63 alters the host macrophage nuclear envelope, and impacts on nuclear processes. Our results suggest that GP63 does not appear to use a classical nuclear localization signal common between Leishmania species for import, but degrades nucleoporins, and is responsible for nuclear transport alterations. In the nucleoplasm, GP63 activity accounts for the degradation and mislocalization of proteins involved amongst others in gene expression and in translation. Collectively, our data indicates that Leishmania infection strongly affects nuclear physiology, suggesting that targeting of nuclear physiology may be a strategy beneficial for virulent Leishmania parasites.

No MeSH data available.


Related in: MedlinePlus

Quantitative proteomic analysis of macrophage nuclei after infection with L. major WT or L. major GP63-/-.(A) Comparison of nucleoplasmic proteins: L. major WT samples vs Nil samples. All proteins identified in Nil samples were represented according to their emPAI value (smallest to highest) as green diamonds. All proteins identified in L. major WT were represented as yellow triangles, according to the protein order of Nil samples. This allows visualizing which proteins are unique, smaller or higher in abundance in the Maj WT samples compared to the Nil ones. (B) Comparison of nucleoplasmic proteins: L. major GP63-/- samples vs Nil samples. Analysis was carried out as in A with L. major GP63-/- proteins identified represented as pink triangles. This allows visualizing which proteins are unique, smaller or higher in abundance in the Maj GP63-/- samples compared to the Nil ones. (C) Venn diagram of proteins identified in Nil, L. major WT and L. major GP63-/- samples. (D) Analysis of the changes in emPAI values of L. major WT and L. major GP63-/- samples. Displayed is the number of proteins (in %) of the L. major WT samples (yellow) and L. major GP63-/- samples (pink) according to the fold-change of their emPAI value in comparison to Nil samples. Blue lines correspond to -1.5x and +1.5x fold-change, which were considered as significant values. (E) Comparison of nucleoplasmic proteins: L. major WT samples vs L. major GP63-/- samples. All proteins identified in L. major GP63-/- (and Nil) samples were represented according to their emPAI value (smallest to highest) as pink diamonds. All proteins identified in L. major WT samples were represented as yellow triangles, according to the L. major GP63-/- sample protein order. This allows visualizing which proteins are unique, smaller or higher in abundance in the Maj WT samples compared to the Maj GP63-/- ones.
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ppat.1004776.g005: Quantitative proteomic analysis of macrophage nuclei after infection with L. major WT or L. major GP63-/-.(A) Comparison of nucleoplasmic proteins: L. major WT samples vs Nil samples. All proteins identified in Nil samples were represented according to their emPAI value (smallest to highest) as green diamonds. All proteins identified in L. major WT were represented as yellow triangles, according to the protein order of Nil samples. This allows visualizing which proteins are unique, smaller or higher in abundance in the Maj WT samples compared to the Nil ones. (B) Comparison of nucleoplasmic proteins: L. major GP63-/- samples vs Nil samples. Analysis was carried out as in A with L. major GP63-/- proteins identified represented as pink triangles. This allows visualizing which proteins are unique, smaller or higher in abundance in the Maj GP63-/- samples compared to the Nil ones. (C) Venn diagram of proteins identified in Nil, L. major WT and L. major GP63-/- samples. (D) Analysis of the changes in emPAI values of L. major WT and L. major GP63-/- samples. Displayed is the number of proteins (in %) of the L. major WT samples (yellow) and L. major GP63-/- samples (pink) according to the fold-change of their emPAI value in comparison to Nil samples. Blue lines correspond to -1.5x and +1.5x fold-change, which were considered as significant values. (E) Comparison of nucleoplasmic proteins: L. major WT samples vs L. major GP63-/- samples. All proteins identified in L. major GP63-/- (and Nil) samples were represented according to their emPAI value (smallest to highest) as pink diamonds. All proteins identified in L. major WT samples were represented as yellow triangles, according to the L. major GP63-/- sample protein order. This allows visualizing which proteins are unique, smaller or higher in abundance in the Maj WT samples compared to the Maj GP63-/- ones.

Mentions: With the criteria mentioned previously, we identified a total of 996 different proteins by LC-MS/MS analysis: 761 proteins in NP of uninfected cells, 653 proteins after infection with L. major WT, 643 proteins after L. major GP63-/- infection and 756 proteins in the case of L. mexicana infection. We considered a difference in emPAI values significant if the change was at least 1.5 fold, as frequencies analysis demonstrated that the majority of the proteins were under that range (Fig 5D and S4D). For an in-depth analysis of proteins affected by Leishmania infection we utilized the STRING-software to generate functional clusters of altered proteins, using gene ontology (GO) annotations. In two separate batches of analyses, we investigated proteins that exhibited changes in abundance in the NP in dependency of GP63 expression (Figs 5 and 6, S3, and S2 Dataset) and compared the differences of the nucleoplasmic protein content after infection with L. major WT and L. mexicana (Figs 7, S4, S5, and S3 Dataset).


Impact of Leishmania infection on host macrophage nuclear physiology and nucleopore complex integrity.

Isnard A, Christian JG, Kodiha M, Stochaj U, McMaster WR, Olivier M - PLoS Pathog. (2015)

Quantitative proteomic analysis of macrophage nuclei after infection with L. major WT or L. major GP63-/-.(A) Comparison of nucleoplasmic proteins: L. major WT samples vs Nil samples. All proteins identified in Nil samples were represented according to their emPAI value (smallest to highest) as green diamonds. All proteins identified in L. major WT were represented as yellow triangles, according to the protein order of Nil samples. This allows visualizing which proteins are unique, smaller or higher in abundance in the Maj WT samples compared to the Nil ones. (B) Comparison of nucleoplasmic proteins: L. major GP63-/- samples vs Nil samples. Analysis was carried out as in A with L. major GP63-/- proteins identified represented as pink triangles. This allows visualizing which proteins are unique, smaller or higher in abundance in the Maj GP63-/- samples compared to the Nil ones. (C) Venn diagram of proteins identified in Nil, L. major WT and L. major GP63-/- samples. (D) Analysis of the changes in emPAI values of L. major WT and L. major GP63-/- samples. Displayed is the number of proteins (in %) of the L. major WT samples (yellow) and L. major GP63-/- samples (pink) according to the fold-change of their emPAI value in comparison to Nil samples. Blue lines correspond to -1.5x and +1.5x fold-change, which were considered as significant values. (E) Comparison of nucleoplasmic proteins: L. major WT samples vs L. major GP63-/- samples. All proteins identified in L. major GP63-/- (and Nil) samples were represented according to their emPAI value (smallest to highest) as pink diamonds. All proteins identified in L. major WT samples were represented as yellow triangles, according to the L. major GP63-/- sample protein order. This allows visualizing which proteins are unique, smaller or higher in abundance in the Maj WT samples compared to the Maj GP63-/- ones.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4380401&req=5

ppat.1004776.g005: Quantitative proteomic analysis of macrophage nuclei after infection with L. major WT or L. major GP63-/-.(A) Comparison of nucleoplasmic proteins: L. major WT samples vs Nil samples. All proteins identified in Nil samples were represented according to their emPAI value (smallest to highest) as green diamonds. All proteins identified in L. major WT were represented as yellow triangles, according to the protein order of Nil samples. This allows visualizing which proteins are unique, smaller or higher in abundance in the Maj WT samples compared to the Nil ones. (B) Comparison of nucleoplasmic proteins: L. major GP63-/- samples vs Nil samples. Analysis was carried out as in A with L. major GP63-/- proteins identified represented as pink triangles. This allows visualizing which proteins are unique, smaller or higher in abundance in the Maj GP63-/- samples compared to the Nil ones. (C) Venn diagram of proteins identified in Nil, L. major WT and L. major GP63-/- samples. (D) Analysis of the changes in emPAI values of L. major WT and L. major GP63-/- samples. Displayed is the number of proteins (in %) of the L. major WT samples (yellow) and L. major GP63-/- samples (pink) according to the fold-change of their emPAI value in comparison to Nil samples. Blue lines correspond to -1.5x and +1.5x fold-change, which were considered as significant values. (E) Comparison of nucleoplasmic proteins: L. major WT samples vs L. major GP63-/- samples. All proteins identified in L. major GP63-/- (and Nil) samples were represented according to their emPAI value (smallest to highest) as pink diamonds. All proteins identified in L. major WT samples were represented as yellow triangles, according to the L. major GP63-/- sample protein order. This allows visualizing which proteins are unique, smaller or higher in abundance in the Maj WT samples compared to the Maj GP63-/- ones.
Mentions: With the criteria mentioned previously, we identified a total of 996 different proteins by LC-MS/MS analysis: 761 proteins in NP of uninfected cells, 653 proteins after infection with L. major WT, 643 proteins after L. major GP63-/- infection and 756 proteins in the case of L. mexicana infection. We considered a difference in emPAI values significant if the change was at least 1.5 fold, as frequencies analysis demonstrated that the majority of the proteins were under that range (Fig 5D and S4D). For an in-depth analysis of proteins affected by Leishmania infection we utilized the STRING-software to generate functional clusters of altered proteins, using gene ontology (GO) annotations. In two separate batches of analyses, we investigated proteins that exhibited changes in abundance in the NP in dependency of GP63 expression (Figs 5 and 6, S3, and S2 Dataset) and compared the differences of the nucleoplasmic protein content after infection with L. major WT and L. mexicana (Figs 7, S4, S5, and S3 Dataset).

Bottom Line: Here we describe for the first time, using molecular biology and in-depth proteomic analyses, that GP63 alters the host macrophage nuclear envelope, and impacts on nuclear processes.Our results suggest that GP63 does not appear to use a classical nuclear localization signal common between Leishmania species for import, but degrades nucleoporins, and is responsible for nuclear transport alterations.In the nucleoplasm, GP63 activity accounts for the degradation and mislocalization of proteins involved amongst others in gene expression and in translation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Microbiology & Immunology, The Research Institute of the McGill University Health Centre, McGill University, Montréal, Quebec, Canada.

ABSTRACT
The protease GP63 is an important virulence factor of Leishmania parasites. We previously showed that GP63 reaches the perinuclear area of host macrophages and that it directly modifies nuclear translocation of the transcription factors NF-κB and AP-1. Here we describe for the first time, using molecular biology and in-depth proteomic analyses, that GP63 alters the host macrophage nuclear envelope, and impacts on nuclear processes. Our results suggest that GP63 does not appear to use a classical nuclear localization signal common between Leishmania species for import, but degrades nucleoporins, and is responsible for nuclear transport alterations. In the nucleoplasm, GP63 activity accounts for the degradation and mislocalization of proteins involved amongst others in gene expression and in translation. Collectively, our data indicates that Leishmania infection strongly affects nuclear physiology, suggesting that targeting of nuclear physiology may be a strategy beneficial for virulent Leishmania parasites.

No MeSH data available.


Related in: MedlinePlus