Format

Send to

Choose Destination
Cell Microbiol. 2018 May;20(5):e12825. doi: 10.1111/cmi.12825. Epub 2018 Feb 7.

A novel kinase function of a nucleoside-diphosphate-kinase homologue in Porphyromonas gingivalis is critical in subversion of host cell apoptosis by targeting heat-shock protein 27.

Author information

1
Division of Pulmonary, Critical Care and Sleep Medicine, University of Florida, Florida, USA.
2
Department of Oral Health Sciences, Medical University of South Carolina, Charleston, South Carolina, USA.
3
Department of Periodontology, University of Florida, Florida, USA.
4
Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina, USA.

Abstract

We have previously shown that a homologue of a conserved nucleoside-diphosphate-kinase (Ndk) family of multifunctional enzymes and secreted molecule in Porphyromonas gingivalis can modulate select host molecular pathways including downregulation of reactive-oxygen-species generation to promote bacterial survival in human gingival epithelial cells (GECs). In this study, we describe a novel kinase function for bacterial effector, P. gingivalis-Ndk, in abrogating epithelial cell death by phosphorylating heat-shock protein 27 (HSP27) in GECs. Infection by P. gingivalis was recently suggested to increase phosphorylation of HSP27 in cancer-epithelial cells; however, the mechanism and biological significance of antiapoptotic phospho-HSP27 during infection has never been characterised. Interestingly, using glutathione S-transferase-rNdk pull-down analysed by mass spectrometry, we identified HSP27 in GECs as a strong binder of P. gingivalis-Ndk and further verified using confocal microscopy and ELISA. Therefore, we hypothesised P. gingivalis-Ndk can phosphorylate HSP27 for inhibition of apoptosis in GECs. We further employed P. gingivalis-Ndk protein constructs and an isogenic P. gingivalis-ndk-deficient-mutant strain for functional examination. P. gingivalis-infected GECs displayed significantly increased phospho-HSP27 compared with ndk-deficient-strain during 24 hr infection. Phospho-HSP27 was significantly increased by transfection of GFP-tagged-Ndk into uninfected-GECs, and in vitro phosphorylation assays revealed direct phosphorylation of HSP27 at serines 78 and 82 by P. gingivalis-Ndk. Depletion of HSP27 via siRNA significantly reversed resistance against staurosporine-mediated-apoptosis during infection. Transfection of recombinant P. gingivalis-Ndk protein into GECs substantially decreased staurosporine-induced-apoptosis. Finally, ndk-deficient-mutant strain was unable to inhibit staurosporine-induced Cytochrome C release/Caspase-9 activation. Thus, we show for the first time the phosphorylation of HSP27 by a bacterial effector-P. gingivalis-Ndk-and a novel function of Ndks that is directly involved in inhibition of host cell apoptosis and the subsequent bacterial survival.

KEYWORDS:

apoptosis; bacterial secreted kinase; epithelial cells; human primary epithelial cells; infection; microbial-cell interaction; oral opportunistic pathogen; persistence; virulence

PMID:
29359393
PMCID:
PMC5893355
[Available on 2019-05-01]
DOI:
10.1111/cmi.12825
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Wiley
Loading ...
Support Center