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

TY - JOUR

T1 - Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis

AU - Veillard, Florian

AU - Sztukowska, Maryta

AU - Nowakowska, Zuzanna

AU - Mizgalska, Danuta

AU - Thøgersen, Ida B

AU - Enghild, Jan J

AU - Bogyo, Matthew

AU - Potempa, Barbara

AU - Nguyen, Ky-Anh

AU - Potempa, Jan

N1 - Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

PY - 2019/11

Y1 - 2019/11

N2 - 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.

AB - 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.

KW - Inhibition

KW - Limited proteolysis

KW - Periodontal disease

KW - Posttranslational modification

KW - Prodomain

KW - Secretion

KW - Sortase

U2 - 10.1016/j.biochi.2019.06.010

DO - 10.1016/j.biochi.2019.06.010

M3 - Journal article

C2 - 31212040

SN - 0300-9084

VL - 166

SP - 161

EP - 172

JO - Biochimie

JF - Biochimie

ER -