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Biochimie. 2019 Jun 15. pii: S0300-9084(19)30175-0. doi: 10.1016/j.biochi.2019.06.010. [Epub ahead of print]

Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis.

Author information

1
Université de Strasbourg, CNRS, Insect Models of Innate Immunity (M3I; UPR9022), 67084, Strasbourg, France; Department of Oral Immunity and Infectious Diseases, University of Louisville School of Dentistry, Louisville, KY, USA. Electronic address: florian.veillard@gmail.com.
2
Department of Oral Immunity and Infectious Diseases, University of Louisville School of Dentistry, Louisville, KY, USA; University of Information Technology and Management, Rzeszow, Poland.
3
Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland.
4
Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
5
Interdisciplinary Nanoscience Center (iNANO), and the Department of Molecular Biology and Genetics, Aarhus University, Aarhus, DK-8000, Denmark.
6
Department of Pathology and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
7
Department of Oral Immunity and Infectious Diseases, University of Louisville School of Dentistry, Louisville, KY, USA.
8
Discipline of Life Sciences, School of Dentistry, University of Sydney, Sydney, NSW, 2006, Australia; Institute of Dental Research, Westmead Centre for Oral Health, Sydney, NSW, 2145, Australia.
9
Department of Oral Immunity and Infectious Diseases, University of Louisville School of Dentistry, Louisville, KY, USA; Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. Electronic address: jan.potempa@icloud.com.

Abstract

Porphyromonas gingivalis uses a type IX secretion system (T9SS) to deliver more than 30 proteins to the bacterial surface using a conserved C-terminal domain (CTD) as an outer membrane translocation signal. On the surface, the CTD is cleaved and an anionic lipopolysaccharide (A-PLS) is attached by PorU sortase. Among T9SS cargo proteins are cysteine proteases, gingipains, which are secreted as inactive zymogens requiring removal of an inhibiting N-terminal prodomain (PD) for activation. Here, we have shown that the gingipain proRgpB isolated from the periplasm of a T9SS-deficient P. gingivalis strain was stable and did not undergo autocatalytic activation. Addition of purified, active RgpA or RgpB, but not Lys-specific Kgp, efficiently cleaved the PD of proRgpB but catalytic activity remained inhibited because of inhibition of the catalytic domain in trans by the PD. In contrast, active RgpB was generated from the zymogen, although at a slow rate, by gingipain-null P. gingivalis lysate or intact bacterial cell suspension. This activation was dependent on the presence of the PorU sortase. Interestingly, maturation of proRgpB with the catalytic cysteine residues mutated to Ala expressed in the ΔRgpA mutant strain was indistinguishable from that in the parental strain. Cumulatively, this suggests that PorU not only has sortase activity but is also engaged in activation of gingipain zymogens on the bacterial cell surface.

KEYWORDS:

Inhibition; Limited proteolysis; Periodontal disease; Posttranslational modification; Prodomain; Secretion; Sortase

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