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Bioinformatic and mass spectrometry identification of Anaplasma phagocytophilum proteins translocated into host cell nuclei.

Sinclair SH, Garcia-Garcia JC, Dumler JS - Front Microbiol (2015)

Bottom Line: The encoding genes were cloned to create GFP fusion protein-expressing clones that were transfected into HEK-293T cells.We confirmed nuclear translocation of six proteins: APH_0062, RplE, Hup, APH_0382, APH_0385, and APH_0455.Of the six, APH_0455 was identified as a type IV secretion substrate and is now under investigation as a potential nucleomodulin.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Cellular and Molecular Medicine, The Johns Hopkins University School of Medicine Baltimore, MD, USA ; Department of Pathology, The Johns Hopkins University School of Medicine Baltimore, MD, USA ; Department of Pathology, University of Maryland School of Medicine Baltimore, MD, USA ; Department of Microbiology and Immunology, University of Maryland School of Medicine Baltimore, MD, USA.

ABSTRACT
Obligate intracellular bacteria have an arsenal of proteins that alter host cells to establish and maintain a hospitable environment for replication. Anaplasma phagocytophilum secrets Ankyrin A (AnkA), via a type IV secretion system, which translocates to the nucleus of its host cell, human neutrophils. A. phagocytophilum-infected neutrophils have dramatically altered phenotypes in part explained by AnkA-induced transcriptional alterations. However, it is unlikely that AnkA is the sole effector to account for infection-induced transcriptional changes. We developed a simple method combining bioinformatics and iTRAQ protein profiling to identify potential bacterial-derived nuclear-translocated proteins that could impact transcriptional programming in host cells. This approach identified 50 A. phagocytophilum candidate genes or proteins. The encoding genes were cloned to create GFP fusion protein-expressing clones that were transfected into HEK-293T cells. We confirmed nuclear translocation of six proteins: APH_0062, RplE, Hup, APH_0382, APH_0385, and APH_0455. Of the six, APH_0455 was identified as a type IV secretion substrate and is now under investigation as a potential nucleomodulin. Additionally, application of this approach to other intracellular bacteria such as Mycobacterium tuberculosis, Chlamydia trachomatis and other intracellular bacteria identified multiple candidate genes to be investigated.

No MeSH data available.


Related in: MedlinePlus

Six A. phagocytophilum candidate genes were found to localize to the nucleus of HEK-293T cells. Candidate genes were fused to GFP and transfected into HEK-293T cells. Twenty four hours post-transfection, cells were stained with DAPI and imaged. Of the 42 GFP-fusion proteins created, six localized to the nucleus. APH_0805 is shown here as an example of a protein that did not localize to the nucleus.
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Figure 1: Six A. phagocytophilum candidate genes were found to localize to the nucleus of HEK-293T cells. Candidate genes were fused to GFP and transfected into HEK-293T cells. Twenty four hours post-transfection, cells were stained with DAPI and imaged. Of the 42 GFP-fusion proteins created, six localized to the nucleus. APH_0805 is shown here as an example of a protein that did not localize to the nucleus.

Mentions: As an inclusive screen, and because contamination of nuclear preparations could not be entirely excluded in iTRAQ studies, nuclear localization of proteins identified by bioinformatic methods or by iTRAQ mass spectrometry were confirmed by cloning the corresponding genes into a mammalian expression vector for expression as GFP fusion proteins. APH_0805 that was predicted to have nuclear localization yet lacked a predicted NLS and had a below-threshold Nuclear score was used as a non-translocating control. HEK-293T cells and PLB-985, a promyelocytic cell line, were transfected and examined for nuclear localization of the GFP-fusion proteins with Hoescht 33342 nuclear counterstaining. Six of the 42 proteins tested (36 from iTRAQ profiling, 7 from the bioinformatic screen), translocated to the nucleus: APH_0062 (hypothetical protein), RplE (50S ribosomal protein L5 [APH_0292]), Hup (DNA-binding protein HU [APH_0783]), and APH_0455, APH_0382, and APH_0385 (all HGE-14) (Figure 1 and Supplemental Figure 2). APH_0278 (tuf-1; elongation factor Tu) was not cloned, but instead the identical APH_1032 (tuf-2; elongation factor Tu) was used but did not enter the nucleus. Nine proteins were either unable to be cloned or cloning was not attempted, including: APH_0160 (putative thymidylate synthase, flavin-dependent, truncation, partial); APH_0196 (nitrogen assimilation regulatory protein); APH_0289 (ribosomal protein S17 [rpsQ]); APH_0820 (hypothetical protein); APH_0906 (hypothetical protein); APH_1023 (DNA-directed RNA polymerase, beta subunit [rpoC]); APH_1024 (DNA-directed RNA polymerase, beta subunit [rpoB]); APH_1034 (ribosomal protein S7 [rpsG]) and APH_1333 (transcription elongation factor GreA).


Bioinformatic and mass spectrometry identification of Anaplasma phagocytophilum proteins translocated into host cell nuclei.

Sinclair SH, Garcia-Garcia JC, Dumler JS - Front Microbiol (2015)

Six A. phagocytophilum candidate genes were found to localize to the nucleus of HEK-293T cells. Candidate genes were fused to GFP and transfected into HEK-293T cells. Twenty four hours post-transfection, cells were stained with DAPI and imaged. Of the 42 GFP-fusion proteins created, six localized to the nucleus. APH_0805 is shown here as an example of a protein that did not localize to the nucleus.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4319465&req=5

Figure 1: Six A. phagocytophilum candidate genes were found to localize to the nucleus of HEK-293T cells. Candidate genes were fused to GFP and transfected into HEK-293T cells. Twenty four hours post-transfection, cells were stained with DAPI and imaged. Of the 42 GFP-fusion proteins created, six localized to the nucleus. APH_0805 is shown here as an example of a protein that did not localize to the nucleus.
Mentions: As an inclusive screen, and because contamination of nuclear preparations could not be entirely excluded in iTRAQ studies, nuclear localization of proteins identified by bioinformatic methods or by iTRAQ mass spectrometry were confirmed by cloning the corresponding genes into a mammalian expression vector for expression as GFP fusion proteins. APH_0805 that was predicted to have nuclear localization yet lacked a predicted NLS and had a below-threshold Nuclear score was used as a non-translocating control. HEK-293T cells and PLB-985, a promyelocytic cell line, were transfected and examined for nuclear localization of the GFP-fusion proteins with Hoescht 33342 nuclear counterstaining. Six of the 42 proteins tested (36 from iTRAQ profiling, 7 from the bioinformatic screen), translocated to the nucleus: APH_0062 (hypothetical protein), RplE (50S ribosomal protein L5 [APH_0292]), Hup (DNA-binding protein HU [APH_0783]), and APH_0455, APH_0382, and APH_0385 (all HGE-14) (Figure 1 and Supplemental Figure 2). APH_0278 (tuf-1; elongation factor Tu) was not cloned, but instead the identical APH_1032 (tuf-2; elongation factor Tu) was used but did not enter the nucleus. Nine proteins were either unable to be cloned or cloning was not attempted, including: APH_0160 (putative thymidylate synthase, flavin-dependent, truncation, partial); APH_0196 (nitrogen assimilation regulatory protein); APH_0289 (ribosomal protein S17 [rpsQ]); APH_0820 (hypothetical protein); APH_0906 (hypothetical protein); APH_1023 (DNA-directed RNA polymerase, beta subunit [rpoC]); APH_1024 (DNA-directed RNA polymerase, beta subunit [rpoB]); APH_1034 (ribosomal protein S7 [rpsG]) and APH_1333 (transcription elongation factor GreA).

Bottom Line: The encoding genes were cloned to create GFP fusion protein-expressing clones that were transfected into HEK-293T cells.We confirmed nuclear translocation of six proteins: APH_0062, RplE, Hup, APH_0382, APH_0385, and APH_0455.Of the six, APH_0455 was identified as a type IV secretion substrate and is now under investigation as a potential nucleomodulin.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Cellular and Molecular Medicine, The Johns Hopkins University School of Medicine Baltimore, MD, USA ; Department of Pathology, The Johns Hopkins University School of Medicine Baltimore, MD, USA ; Department of Pathology, University of Maryland School of Medicine Baltimore, MD, USA ; Department of Microbiology and Immunology, University of Maryland School of Medicine Baltimore, MD, USA.

ABSTRACT
Obligate intracellular bacteria have an arsenal of proteins that alter host cells to establish and maintain a hospitable environment for replication. Anaplasma phagocytophilum secrets Ankyrin A (AnkA), via a type IV secretion system, which translocates to the nucleus of its host cell, human neutrophils. A. phagocytophilum-infected neutrophils have dramatically altered phenotypes in part explained by AnkA-induced transcriptional alterations. However, it is unlikely that AnkA is the sole effector to account for infection-induced transcriptional changes. We developed a simple method combining bioinformatics and iTRAQ protein profiling to identify potential bacterial-derived nuclear-translocated proteins that could impact transcriptional programming in host cells. This approach identified 50 A. phagocytophilum candidate genes or proteins. The encoding genes were cloned to create GFP fusion protein-expressing clones that were transfected into HEK-293T cells. We confirmed nuclear translocation of six proteins: APH_0062, RplE, Hup, APH_0382, APH_0385, and APH_0455. Of the six, APH_0455 was identified as a type IV secretion substrate and is now under investigation as a potential nucleomodulin. Additionally, application of this approach to other intracellular bacteria such as Mycobacterium tuberculosis, Chlamydia trachomatis and other intracellular bacteria identified multiple candidate genes to be investigated.

No MeSH data available.


Related in: MedlinePlus